UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Identification of Mg2+/Ca2+-sensing system in mouse distal convoluted tubule cells and characterization… Bapty, Brian Walter 1999

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-ubc_1999-388506.pdf [ 7.66MB ]
Metadata
JSON: 831-1.0089210.json
JSON-LD: 831-1.0089210-ld.json
RDF/XML (Pretty): 831-1.0089210-rdf.xml
RDF/JSON: 831-1.0089210-rdf.json
Turtle: 831-1.0089210-turtle.txt
N-Triples: 831-1.0089210-rdf-ntriples.txt
Original Record: 831-1.0089210-source.json
Full Text
831-1.0089210-fulltext.txt
Citation
831-1.0089210.ris

Full Text

IDENTIFICATION OF A M g /Ca -SENSING SYSTEM IN MOUSE DISTAL CONVOLUTED TUBULE CELLS AND CHARACTERIZATION OF ITS ROLE IN HORMONE-STIMULATED MAGNESIUM TRANSPORT 2+  2+  by BRIAN WALTER BAPTY  B.Sc, The University of British Columbia, 1992 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILIOSOPHY in THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF MEDICINE EXPERIMENTAL MEDICINE, NEPHROLOGY  We accept this thesis as conforming \to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA April 30, 1999  In  presenting  degree  at  this  the  thesis  in  partial  fulfilment  of  University  of  British  Columbia,  I agree  freely available for copying  of  department publication  this or of  reference  thesis by  this  for  his thesis  and study. scholarly  or for  her  Department  of  The University of British Vancouver, Canada  Date  DE-6 (2/88)  Columbia  purposes  gain  shall  requirements that  agree  may  representatives.  financial  permission.  1 further  the  It not  be is  that  the  Library  permission  granted  by  understood be  for  allowed  an  advanced  shall make for  the that  without  it  extensive  head  of  my  copying  or  my  written  ii  ABSTRACT Distal convoluted tubule cells of the kidney play a crucial role in the determination of the final urinary magnesium excretion. In addition to responding to changesin local divalent cation concentrations, magnesium absorption in these cells is also under the influence from a number of hormones. Immortalized mouse distal convoluted tubule cells (MDCT) retain a number of the properties  ofin vivo  distal convoluted tubule cells.  This study was performed to identify polyvalent-cation sensing mechanisms in M D C T cells and to describe the co-ordinated response to hormones that control magnesium reabsorption. The presence of Ca -sensing 2+  receptor (Ca -SR) was confirmed by sequencing a M D C T R T - P C R product that had greater than 92% 2+  identity with the published rat clone. Western blot analysis demonstrated the presence of the Ca^-SR protein. Microfluorescence studies and analysis of hormone-induced c A M P generation, were used to determine changes in the cell's physiological response to stimulation of the polyvalent cation-sensing receptor. M D C T cells respond to extracellular Mg " ', C a , G d , and neomycin with a transient increase in 2  to M g  2 +  1  2+  3+  [Ca ]j.  and C a , M D C T cells can differentiate between small concentration changes in M g 2+  2 +  2 +  With respect  and C a , even 2+  in the presence of an excess of the other cation. Evidence is provided for a separate mechanism that responds to extracellular M g . Parathyroid hormone (PTH), arginine vasopressin (AVP), calcitonin (CT), glucagon, 2 +  and prostaglandin E , stimulate c A M P generation and increased the M g  2 +  2  uptake rate in M D C T cells.  Aldosterone potentiates this hormonal stimulation. Hormone-stimulated Mg " ' uptake and c A M P generation 2  1  are inhibited with stimulation of the M g 7 Ca -SR. Pre-treatment of M D C T cells with l,25,(OH) D (16 2  2+  2  hr, 10-7 M ) increased M g  2 +  uptake by 78 ±4%. Stimulation of the cation-sensing mechanism did not inhibit  l,25,(OH) D stimulated M g 2  3  3  2 +  transport. Accordingly, the polyvalent cation-sensing receptor present in  M D C T cells has differential effects on hormone mediated transport. We conclude further still that a specific Mg -sensing mechanism is present in M D C T cells and that this mechanism inhibits hormone mediated M g 2+  2 +  uptake in these cells. We infer from these studies that a cation-sensing mechanism is located in the distal convoluted tubule and plays a role in the control of distal M g  2 +  absorption.  I  iii TABLE OF CONTENTS  ABSTRACT  ii  TABLE OF CONTENTS  iii  LIST OF FIGURES  vii  LIST OF TABLES  ix  LIST OF ABBREVIATIONS  x  ACKNOWLEDGEMENTS  xii  GENERAL INTRODUCTION  1  I  The Calcium-Sensing Receptor and its Localization Along the Nephron  1  II  Function of the Ca -SR  3  III  Genetic disorders arising from mutations in the Ca -SR 2+  6  IV  Summary Magnesium and Calcium Renal Handling  6  2+  V  Objectives of the present study  8  VI  Experimental Rational  9  Chapter 1. IDENTIFICATION, AND FUNCTION, OF Ca -SR IN MDCT CELLS 2+  10  I  Background  10  II  Materials and Methods  10  II. 1  Materials  10  II.2  Methods  10  0.1  Cell Culture  10  0.2  RNA preparation  11  0.3  Reverse Transcription-polymerase chain reaction analysis  12  0.4  Southern Hybridization  13  0.5  Western blots  13  0.6  Determination of [Ca  2+  ]i  14  IV  0.7  Determination of [Mg  0.8  cAMP Determination  16  0.9  Creating and Screening a cDNA Library  16  2+  15  ]i  0.10 Xenopus oocyte isolation, preparation, and injection  III  16  0.11 Statistical analysis  17  Results  17  0.1  RT-PCR analysis of Ca -SR mRNA in MDCT cells  18  0.2  Western Analysis of the Ca -SR in MDCT cells  18  0.3  Intracellular Ca signaling in MDCT cells by Ca -SR activation  18  0.4  Ca -SR activation inhibits hormone-stimulated cAMP release  29  IV  2+  2+  2+  2+  2+  Discussion  29  Chapter 2: ATTEMPTS TO ISOLATE A SPECIFIC Mg -SENSING RECEPTOR 2+  32  I  Background  32  III  Results  32  0.1  Intracellular Ca signaling in MDCK, but not HEK cells, in 32 response to M g 2+  2+  0  IV  0.2  Expression of the Palaemon serratus Mg response in Xenopus oocytes  0.3  A Ca -SR cDNA, or related transcript, was not identified 33 by screening a Palaemon serratus oocyte or MDCK cDNA library  2+  33  2+  Discussion  Chapter 3. CHARACTERIZATION OF MAGNESIUM UPTAKE INFLUENCED BY PEPTIDE HORMONES ACTING THROUGH G-PROTEIN LINKED RECEPTORS.  34 37  I  Background  37  III  Results  39  0.1  Glucagon, arginine vasopressin, parathyroid hormone, and 39 calcitonin stimulate cAMP release in Mg -depleted MDCT cells 2+  0.2  Mg uptake in MDCT cells.  40  0.3  Glucagon stimulates Mg uptake in MDCT cells.  41  0.4  A VP stimulates Mg uptake in MDCT cells  46  0.5  Calcitonin and PTH have similar effects on Mg uptake as 46 glucagon and AVP  0.6  Glucagon- and AVP-stimulated Mg entry does not require 46 protein synthesis  0.7  cAMP stimulates Mg uptake in MDCT cells  48  0.8  The effect of protein kinase A and protein kinase C inhibition on hormone-stimulated Mg uptake.  50  2+  2+  2+  2+  2+  2+  2+  0.9 IV  Glucagon and calcitonin stimulated Mg uptake is inhibited 53 with phospholipase-C inhibition 2+  Discussion  53  Chapter 4. THE INHIBITION OF HORMONE STIMULATED Mg UPTAKE WITH ACTIVATION OF THE POLYVALENT CATION-SENSING RECEPTOR 2+  57  I  Background  57  III  Results  57  0.1  Activation of the Mg 7Ca -sensing mechanism inhibits hormone-stimulated cAMP release  57  0.2  Activation of Mg 7Ca -sensing diminishes hormonestimulated Mg uptake into Mg -depleted MDCT cells.  58  Activation of Mg 7Ca -sensing does not affect cAMP stimulation of Mg uptake  64  2  2  2+  2+  2+  0.3  2+  2  2+  2+  0.4  Activation of Mg /Ca -sensing does not inhibit amiloride- 67 stimulated Mg uptake 2+  2+  2+  IV  Discussion  67  Chapter 5. MINERAL CORTICOID HORMONES EFFECT Mg UPTAKE IN MDCT CELLS 2+  70  VI  I  Background  70  III  Results  71  0.1  Aldosterone potentiates hormone-stimulated Mg uptake  71  0.2  Aldosterone potentiates hormone-stimulated cAMP accumulation in MDCT cells.  75  0.3  Aldosterone potentiates hormone-stimulated Mg uptake and cAMP generation through de novo protein synthesis  75  0.4  Stimulation of the Ca -SR inhibits Aldosterone stimulated Mg uptake  81  2+  2+  2+  2+  IV  Discussion  87.  Chapter 6. 1,25-DIHYDROXYVITAMIN D STIMULATES Mg UPTAKE INTO MOUSE DISTAL CONVOLUTED TUBULE CELLS  82  2+  3  I  Background  82  III  Results  83  0.1  1,25(OH) D stimulates Mg entry in MDCT cells  83  0.2  1,25(OH) D and PTH stimulate Mg uptake by separate pathways  84  0.3  Stimulation of the Ca -SR does not inhibit l,25(OH) D stimulated Mg uptake.  89  2+  2  3  2+  2  3  2+  2  3  2+  IV  Discussion  91  GENERAL CONCLUSIONS  92  GENERAL DISCUSSION  92  FUTURE RESEARCH  105  BIBLIOGRAPHY  106  Vll  LIST OF FIGURES  Fig.  1  Model of polyvalent cation sensing receptor  2  Fig.  2  Summary of calcium and magnesium absorption along the nephron  4  Fig.  3  Agarose gel of RT-PCR products from MDCT cells  19  Fig.  4  Nucleotide sequence of MDCT RT-PCR product  20  Fig.  5  Western analysis of the Ca -SR  22  Fig.  6  External polyvalent cations stimulate transient cytosolic calcium increase 22  Fig.  7  MDCT Ca -SR is sensitive to small changes in [Mg ] and [Ca ] in the presence of elevated divalent cation concentrations  27  Fig.  8  The polyvalent cation sensing receptor of MDCT cells responds sensitively to [Mg ] and [Ca ]  27  2+  2+  2+  2+  0  2+  D  2+  Fig.  9  Repetitive stimulation of MDCT-Ca -SR with [Mg ] and [Ca ] .  Fig.  10  External polyvalent cations transiently increase cytosolic Ca in MDCK 35 but not HEK 293 cells  Fig.  11  Extracellular Mg (15 mM), but not Ca , evokes increases in CI" currents in Xenopus oocytes injected with 50 ng prawn mRNA  Fig.  12  Glucagon and arginine vasopressin (AVP) stimulate cAMP accumulation 43 in MDCT cells  Fig.  13  Glucagon and arginine vasopressin (AVP) stimulate Mg uptake in Mg -depleted mouse distal convoluted tubule (MDCT) cells  44 45  2+  2+  28  2+  0  0  2+  2+  36  2+  2+  2+  Fig.  14  Concentration-dependence of glucagon-stimulation of Mg entry in MDCT cells  Fig.  15  Concentration-dependence of AVP stimulation of Mg uptake in MDCT 46 cells  Fig.  16  Summary of hormone induced stimulation of Mg uptake and cAMP generation in MDCT cells  49  Fig.  17  Exogenous cAMP stimulates Mg entry in MDCT cells  51  Fig.  18  The phospholipase inhibitor U73122 inhibits hormone stimulated Mg uptake  2+  2+  2+  2+  2+  52  Fig.  19  Concentration-dependence of extracellular Mg or Ca inhibition of glucagon-stimulated cAMP  60  Fig.  20  Activation of the Mg /Ca -sensing mechanism diminishes hormonestimulated Mg uptake into MDCT cells  62  Summary of the effects of Mg /Ca -sensing mechanism activation on hormone-stimulated Mg uptake  63  2+  2+  2+  2+  2+  Fig.  21  2+  2+  2+  Fig.  22  Glucagon-stimulation of Mg uptake is dependent on the concentration of65 extracellular Mg used to determine d([Mg ];)/dt 2+  2+  Fig.  23  2+  Activation of Mg 7Ca sensing does not alter exogenous cAMPmediated Mg uptake 2  66  2+  2+  Fig.  24  Activation of Mg 7Ca sensing does not alter amiloride-stimulated Mg uptake  Fig.  25  Aldosterone potentiates glucagon- and AVP- stimulated Mg uptake in 72 Mg -depleted mouse distal convoluted tubule (MDCT) cells  2  2+  2+  68  2+  2+  Fig.  26  Aldosterone potentiates glucagon-stimulated Mg entry in MDCT cells 73  Fig.  27  Aldosterone potentiates AVP-stimulated Mg uptake in MDCT cells  74  Fig.  28  Aldosterone potentiation of hormone-stimulated Mg entry is through dihydropyridine-sensitive pathways  78  Fig.  29  Aldosterone potentiates glucagon-stimulated intracellular cAMP accumulation in a concentration-dependent manner  79  Fig  30  Aldosterone and glucagon stimulated Mg uptake are inhibited by stimulation of the Ca -SR  80 85  2+  2+  2+  2+  2+  Fig.  31  1,25(OH) D stimulates Mg uptake in Mg -depleted (MDCT) cells  Fig.  32  Concentration-dependence of l,25(OH) D -stimulation of Mg entry in 86 MDCT cells  Fig.  33  Summary of l,25(OH) D -stimulated Mg uptake in MDCT cells  87  Fig.  34  PTH stimulates cAMP formation and Mg entry into MDCT cells  88  Fig.  35  Summary of the effects of activation of Mg /Ca -sensing on hormonestimulated Mg uptake  90  Model of the inhibitory effects of Mg 7Ca " sensing on hormonestimulated Mg uptake in the DCT  104  2+  2  2+  3  2+  2  3  2+  2  3  2+  2+  2+  2+  Fig.  36'  2  2+  2+  ix LIST O F T A B L E S  Table  1  Polyvalent cations stimulate cytosolic Ca transients in MDCT cells  23  Table  2  Activation of Ca -SR inhibits hormone-stimulated cAMP release  30  Table  3  Glucagon and AVP stimulate cAMP release in magnesium-depleted MDCT cells  41  Table  4  Hormone-stimulated Mg uptake in MDCT cells is dihydropyridine sensitive  42  Table  5  Role of protein synthesis on glucagon stimulated Mg uptake in MDCT cells  48  Table  6  Glucagon and AVP act, in part, through protein a kinase A-mediated pathway  50  Table  7  Activation of Mg /Ca -sensing inhibits hormone-stimulated cAMP accumulation  59  Table  8  Aldosterone potentiates hormone-stimulation of cAMP accumulation in MDCT cells  70  Table  9  Role of protein synthesis on glucagon-stimulated Mg uptake and cAMP release in MDCT cells  77  Table  10  1,25(OH) D does not stimulate Mg uptake through a protein kinase A pathway  89  Table  11  Comparison of data obtained from MDCT cells and data from intact distal 103 tubules  2+  2+  2+  2+  2+  2+  2+  2+  2  3  X  LIST O F  General  Ca -SR  Ca -sensing receptor outside the parathyroid gland  Mg -SR  putative Mg -sensing receptor  Mg 7Ca -SR  Mg and Ca sensing system where one or both may be present  Casr  Ca -sensing receptor in the parathyroid gland as defined by Brown et al (4)  total RNA, RNA  total ribonucleic acid isolated from a cell  mRNA  RNA isolated containing a poly-adenosine tail  cDNA  single strand deoxy-ribonucelic acid complementary to mRNA  PCR  polymerase chain reaction  RT-PCR  reverse transcribed polymerase chain reaction; amplificationfromcDNA strand  2+  2+  2+  2+  2  2+  2+  Mg ,Ca , CI", ....  ionic (unbound) state of specific element  [Mg ],, [Ca ] , [Cl-]i  concentration of specified ion within the cell  2+  2+  2+  i  [Mg ] ,[Ca ] , [Cl"] ,  concentration of specified ion outside the cell  TAL  thick ascending limb  DCT  distal convoluted tubule  CNT  connecting tubule  CCD  cortical collecting duct  mCD  medullary collecting duct  TPTXed  thyroparathyroid ectomized  MDCT  mouse distal convoluted tubule  2+  2+  0  Cell lines  2+  2+  2+  Anatomical  ABBREVIATIONS:  0  0  XI  hormones  messengers  MDCK  Madin Darby canine kidney  AtT-20  mouse pituitary cell line  HEK  human embryonic kidney  CHO  Chinese hamster ovary  Aldo  aldosterone  AVP  arginine vasopressin  CT  calcitonin  Glu  glucagon  PTH  parathyroid hormone (bovine PTH, 1-34)  GDP  guanosine diphosphate  GTP  guanosine triphosphate  cAMP  cyclic adenosine monophosphate  IP , PIP  inositol tri-phosphate  G-protein  heterotrimeric GTP/GDP binding protein, contains a,B, and y subunitis  G,  Gai (inhibitory) subunit released from G-protein  G  Gas (stimulatory) subunit released from the G-protein  3  2  s  Go  any of a number of other identified type of Ga subunit  20-HETE  20-hydroxyeicosatetraenioc acid  PLA  phospholipase A  2  2  PKA  protein kinase A  PKC  protein kinase C  PLC  phospholipase C  toxins, blockers, Neo  neomycin  and agonists  Rp-cAMP  cyclic adenosine monophosphate Rp-isomer  PMA  Phorbol 12-myristate 14-acetate  IBMX  3-isobutylyl-l-methylxanthine  Xll  ACKNOWLEDGMENT:  I would like to thank Dr.Peter A. Freidman for providing the MDCT cell line. Dr Allen Speigel and Dr.Paul Goldsmith( Metabolic Diseases Branch, NIDDK/NIH, Bethesda, MD, USA) for the ADD antibody. Dr's Marie and Henrie Goudeau for the prawn oocytes, and Lucie Canaff and Dr. Geoffrey Hendy (Departments of Medicine, Physiology and Human Genetics, McGill University and Royal Victorian Hospital, Montreal Quebec,) for Western blot contributions and independent PCR data corroboration. In addition I would like to thank Dr. Gary Quamme for his guidance, patience, and support throughout this work. I am grateful also for the efforts and friendship of Dr. Long-Jun Dai, Gordon Ritchie, and Don Huysmans.  1 General Introduction The epithelial cells of the kidney are intrinsically responsive to their changing environment and yet are under hormonal control. In the epithelium of the nephron, where the apical membrane is in contact with the tubular fluid and the basolateral membrane is in contact with the blood, the cells are in contact with two different environments. The body's nutrient requirements are coordinated as dictated by hormonal signals, in the face of a changing environment. Accordingly the epithelium must detect these changes and respond appropriately. One class of extracellular sensors that cells can use for detecting changes in their environment, and hormonal signals from other cells, are G-protein coupled receptors. I.  The Ca -Sensing receptor (Ca -SR) and its Localization Along the Nephron. 2+  2+  The G-protein coupled receptor superfamily of proteins are seven predicted hydrophobic trans membrane segments. With associated G-protein alpha subunits they have the ability to stimulate the exchange of bound GDP for GTP in response to agonist binding [ 1 ]. The superfamily is subdivided into six families (AF) which are again subdivided into groups. Families of receptors contain 20% or more sequence identity over the predicted transmembrane segments. Sub-groups share common biochemical properties. Only families A, B, and C occur in vertebrates with family C further restricted to mammals [2,3]. Families D, E, and F are restricted to fungi and slime molds [2,3]. The Ca -SR, which responds to polyvalent cations such as magnesium, gadolinium and neomycin, 2+  was first cloned by Brown et al.frombovine parathyroid gland where it is involved with the control of parathyroid hormone (PTH) secretion [4]. This is a family C receptor is related to metabotropic receptors and a newly identified multigene family of pheromone receptors [5]. The Ca -SR contains three major domains: 2+  1) a large extracellular amino-terminal domain consisting of 613 amino acids which is thought to possess the cation binding sites, 2) a 250 amino acid domain with seven predicted membrane spanning segments characteristic of the superfamily of G protein-coupled receptors, and 3) a carboxyl  2  3 terminal domain of222 amino acids that likely resides within the cytoplasm and is involved with intracellular signalling processes [4] (Fig. 1). There is evidence that extracellular polyvalent cations bind to the extracellular domain initiating a number of intracellular signals [5,6,7,8]. The human receptor is coded for by a single gene located at chromosome 3 q 13.3 -q21 [9]. The Ca 2+  SR found in the parathyroid gland has been shown to be present in bovine, rat, mouse, and human kidneys [4,10,11,12,13]. The Ca -SR has been localized along the nephron with immuno-fluorescence, in situ 2+  hybridization, Northern analysis, as well as RT-PCR techniques [10-16]. Relative abundance, apical or basolateral orientation, and mechanism of action vary along the nephron (Fig. 2) [14]. Using RT-PCR the Ca -SR receptor has been identified in the glomerulus [11,17]. Immuno2+  fluorescence, however, failed to support this finding in rats, suggesting very low expression in these cells [ 15]. In the rat the Ca -SR is present on the luminal border of the proximal convoluted and straight tubule with 2+  decreasing intensity from the Sf to SIII segments [15]. The highest expression of the Ca -SRis detected at 2+  the basolateral border of cortical thick ascending limb (TAL) cells and to a lesser extent in the medullary TAL [15,16]. Basolaterally located Ca -SR was also detected in the macula densa, distal convoluted tubule 2+  (DCT) and type A intercalated cells [15]. Within the DCT cells, Riccardie/ a/.[10,15] have also reported some sub-membranous apical staining suggesting the presence of the Ca -SR in intracellular vesicles. In the 2+  inner medullary collecting duct Ca -SR expression was localized to the apical membrane as in the proximal 2+  tubule [10,15]. The mechanism by which the Ca -SR affects transport in the kidney has not been fully 2+  characterized. //. Function of the Ca -SR: 2+  The function of the Ca -SR is to induce a suitable cellular response to changes in extracelluler 2+  divalent cation concentrations. The complete nature of this response is not uniform and is still undefined in many of the tissues which express the Ca -SR. When the bovine Casr (Casr used specifically for bovine 2+  parathyroid Ca -SR) was expressed mXenopus leavis oocytes the binding of agonist resulted in activation 2+  4  Proximal convoluted tubule  Loop of Henle  Dstal tubule  Basolateral  Fig.  2.  Calcium and magnesium absorption (as a percent of that filtered at the glomerulus) and distribution of the calcium sensing receptors along the nephron.Insert indicated apical or basolateral presentation on the cell surface.  of phospholipase C and mobilization of intracellular Ca , and the opening of Ca -activated CI" channels [4]. 2+  2+  This was the physiological assay used to test for expression and the method by which the receptor was originally cloned by Brown and colleagues [4]. To date, expression studies of Ca -SR transfected into human embryonic kidney cells (HEK) have 2+  implicated a number of second messengers including Q, G , G,, G-protein stimulated phospholipase C, A, and q  D, as well as some down-stream metabolites such as arachadonic acid. [6,7]. Similar responses to this was also seen when the Ca -SR was expressed in Chinese hamster ovary (CHO) cells [18]. When expressed 2+  in HEK cells there is evidence of a negative feedback whereby PKC phosphorylates Thr of the Ca -SR 888  leading to a diminished response to Ca  2+ 0  2+  [19]. In these studies, as well as expression studies in Xenopus  oocytes, the hallmark of Ca -SR stimulation an increase or and oscillation of [Ca ];. In vivo, however, 2+  2+  stimulation of the Ca -SR must be linked to appropriate physiological response from the cell. There are no 2+  endogenous Ca -SR's in HEK, CHO cells, or Xenopus oocytes. 2+  Characterization of the native Ca -SR's response to agonists in three microdissected regions of the 2+  kidney has generated some physiological data. Previous to the cloning of the Ca -SR, Mathias and Brown 2+  noted that an elevation of [Ca ] decreased PTH-dependant cAMP production in dispersed rat proximal 2+  0  tubule cells [8] and increasing extracellular polyvalent cations in bovine parathyroid cells lead to an increase in [Ca  2+  ]i.  In the TAL, Takaichie? al. noted that increasing Ca results in diminished hormone-dependant 2+  0  cAMP production [20]. Cloning of the Ca -SR helped to explain these observations. In TAL cells, where 2+  the highest expression of Ca -SR in the kidney is identified, two theories have been suggested to link 2+  stimulation of the Ca -SR with reduction in cation reabsorption. Wange? al. have provided compelling 2+  evidence that 20-hydroxyeicosatetraenicioc acid (20-HETE) , the phospholipase A (PLA ) liberated 2  2  arachadonic acid break-down product, directly inhibits the 70pS Kchannel [7]. This apically located channel +  contributes significantly to K recycling and maintenance of the lumen positive environment which is the +  driving force for the movement of calcium and magnesium. In contrast, using isolated perfused tubules, Celeste et al., suggest the Ca -SR acts to inhibit CI" conductance through an inhibition of the cAMP z+  pathway which does not involve protein kinase C (PKC), PLA , or metabolic derivatives [21]. This CI" 2  current is associated with maintenance of the transepithelial voltage gradient. The characterization of a Ca !+  inhibitable (Type 6) adenylate cyclase in the rat TAL [23] lends some support for this theory. In isolated rat inner medullary collecting ducts stimulation of the apical Ca -SR leads to a reduction 2+  in vasopressin-elicited water permeability [10]. In this case the Ca -SR has been co-localized with Ca 2+  2+  dependant PKC's as well as gprotein alpha subunits G /G,,, and G . The physiologically important affect is i3  a reduction in insertion of aquaporin 2 into the apical membrane likely through the effect of Q on membrane 3  trafficking [10]. This leads to diminished water uptake and a more dilute urine. Sands and Hebert et al postulate that diuresis induced by hypercalciuria and hypermagnesuria in this segment inhibits renal stone formation [10].  ///. Genetic disorders arising from mutations in the Ca -SR: z+  Some insight into the role of the Ca -SR can be inferredfromfamilial diseases arisingfrommutations 2+  of the Ca -sensing receptor. Two disorders arisingfrominactivating mutations in the Ca -SR have been 2+  2+  identified. Familial hypocalciuric hypercalcemia (FHH, also known as FBH,and FBHH), an autosomal dominant heterozygous inactivating mutation with 100% penetrance, and neonatal severe hyperparathyroism (NSHPT, also know as NHPT), the homozygous form of FHH [24-33,and reviewed in 34,35]. FHH is generally an asymptomatic disorder, exhibiting mild hypercalcemia, mild hypermagnesemia, relative hypocalciuria, and inappropriately normal levels of PTH [34,35]. In contrast, NSHPT is life threatening without parathyroidectomy and is characterized by very high serum Ca concentrations, hypotonia, skeletal 2+  7 demineralization, respiratory distress, and parathyroid hyperplasia [34,35]. With disruption of the Ca -SR 2+  gene, Ho et al. developed a transgenic mouse model for FHH and NSHPT which closely mirrors the human clinical condition of both disorders [26]. Of significant note in this study is that the elevation of serum Mg  2+  was not significantly different in the heterozygous and homozygous mouse indicating additional mechanisms must also be present for control of serum Mg . 2+  Familial hypocalcemic hypercalciuria is a dominant, heterozygous, gain-of-function mutation in the Casr gene [31,32]. Patients demonstrate mild to severe symptoms similar to hypoparathyroidism with the exception of hypercalciuria even at low serum calcium concentrations [34,35]. The hypercalciuria is the result of the defective cation-sensing receptor in the kidney. Both activating and inactivating mutations cluster around the first third of the amino terminal, the region associated with Ca binding, or within the 2+  transmembrane domain, likely resulting in defective G-protein coupling [29,32]. These observations have provided insight into the importance and function of the receptor in the kidney with respect to calcium balance and offer new questions as to the contibuting role it may play in magnesium balance.  IV. Summary of Renal Magnesium and Calcium Handling  Control of body magnesium homeostasis principally resides within the kidney nephron. The proximal tubule reabsorbs 5-15%, the thick ascending limb of the loop of Henle absorbs 70-80% passively through the paracellular pathway, and the distal tubule actively reclaims some 5-10% of the filtered magnesium. Although the distal tubule normally reabsorbs up to 10% of the magnesium filtered through the glomerulus this amount is significant as it represents 60-70% of the magnesium delivered to this segment from the loop of Henle [36] (Fig.2). As there is little magnesium reabsorption beyond the distal tubule in the collecting ducts, the tubule segments comprising this portion of the nephron play  8 an important role in determining the final urinary excretion of magnesium and are, therefore, the focus of current research. The majority of filtered calcium (60-70%) is passively reabsorbed in the proximal tubule across the paracellular pathway down its concentration gradient as salt and water are removed [37]. About 20% of the filtered calcium is absorbed in the cortical TAL segment [37]. The relatively high luminal positive voltage across the TAL epithelial cells drives cations through the paracellular pathway. About 10% of the filtered Ca is extracted in the distal tubule (Fig. 2). Calcium absorption within the distal tubule is thought to be trans 2+  cellular and active in nature[37]. The distal tubule is composed of the distal convoluted tubule (DCT), connecting tubule (CNT), cortical connecting duct (CCD), and medullary connecting duct (CD)[38]. f  Little is known about the cellular mechanisms of absorption in the distal convoluted tubule because this segment is difficult to isolate for in vivo microperfusion studies. Accordingly, isolated cell lines have been used to study functional responses. We have used an immortalized mouse distal convoluted tubule (MDCT) cell line to characterize cation transport in this nephron segment. The MDCT cell line possesses amilorideinhibitable Na conductance, chlorothiazide-sensitive NaCl cotransport, and hormone stimulated magnesium +  and calcium transport [39,40,41]. Accordingly, this established cell line has proven to be very useful in describing functions of the intact tubule. V. Objectives:  1. To identify and characterize extracellular Mg /Ca sensing in an immortalized distal tubule cell line. 2+  2+  2. To determine the function of extracellular Mg 7Ca sensing on hormone actions within mouse distal 2  convoluted tubule cells.  2+  9  VI. Experimental Rational  The MDCT cell line has proven to be a very useful model of the in vivo distal convoluted tubule. Preliminary experiments indicated that an extracellular polyvalent cation-sensing receptor is present in MDCT cells. My experiments showed that this polyvalent cation-sensing receptor present in MDCT cells is more sensitive to extracellular Mg than Ca . Mg -sensing receptors have been reported in the number of 2+  2+  0  2+  0  tissues including prawn oocytes so that they may be a good source in which to identify such receptors. Activation of the Ca -sensing receptor did not inhibit basal Mg uptake in MDCT cells but preliminary 2+  2+  evidence suggested that it influenced hormone-dependent cAMP formation. Accordingly, we characterized hormone (PTH, calcitonin, glucagon, AVP) actions within the MDCT cells. Finally, the actions of the Ca  2+  -SR were determined on hormone-stimulated Mg uptake in MDCT cells. The steroid hormones, 2+  mineralocorticoids and vitamin D metabolites, were shown to stimulate Mg entry rates by different 2+  intracellular signaling pathways. Accordingly, it was of interest to examine Ca -SR actions on these 2+  hormones. These studies clearly demonstrate the importance of polyvalent cation-sensing receptor(s) in the MDCT cell line and by inference in the intact distal convoluted tubule.  10  Chapter 1. Identification of a kidney Ca -SR in MDCT Cells. 2+  I. Background The distal convoluted tubule plays an important role in salt conservation. This segment reabsorbs as much as 10% of the filtered Mg and Ca that are under control of many hormones including PTH, 2+  2+  calcitonin, glucagon, and vasopressin [37]. Accordingly, isolated cell lines have been used to study functional responses. An immortalized mouse distal convoluted cell line (MDCT) has been extensively used to characterize cation transport in this nephron segment [42,43 ]. In the initial study, we show that a polyvalent cation-sensing mechanism is present in MDCT cells that responds sensitively and equipotently to extracellular Mg and Ca . The presence of polyvalent cation-sensitive receptors in this established cell line will facilitate 2+  2+  study of their function in control of magnesium and calcium absorption within the DCT [44].  II. Materials and Methods. Includes materials and methods used throughout the present study. II. 1 .Materials.  Basal Dulbecco's minimal essential medium (DMEM) and Ham's F-12 media were purchased from GIBCO. Customized magnesium-free media was purchased from Stem Cell Technologies Inc. (Vancouver, BC). Fetal calf serum was from Flow Laboratories (McLean, VA). Mag-fura-2/AM was obtained from Molecular Probes (Eugene, OR). Hormones, vitamin-D, and other chemicals were purchased from Sigma (St.Louis Mo.) unless otherwise mentioned. II.2. Methods:  II.2.1 Cell Culture:  The MCDT cell line was generated by Pizzoniae? al. [42]. Cortical thick ascending limb and distal convoluted tubule cells were isolated from mice and established in primary culture. Transformation was  11  accomplished with chimeric adenovirus 12-simian virus 40 (AD12/SV40)[42]. The MDCT cell line was cultured in Dulbecco's modified Eagle's medium (DMEM)/ Ham's F-12,1:1, media supplemented with 10% fetal calf serum, 1 mM glucose, 5 mM L-glutamine, 50 U/ml penicillin, and 50 U/ml streptomycin in a humidified environment of 5% C0 and 95% air at 37°C. For the hybridization studies, MDCT cells were 2  grown to confluence on plastic supports and harvested by scraping into buffered solution. For the fluorescent studies confluent cells were washed three times with phosphate buffeted saline (PBS) containing 5mM ethylene glycol-bis(B-aminoenolyl ether) N,N,N',N',- tetraacetic acid (EGTA), trypsinized, and seeded on glass coverslips. Aliquots of harvested cells were allowed to settle onto sterile glass coverslips in 100-mm Coming tissue culture dishes, and the cells were grown to confluence over one or two days in media supplemented as described above.  II.2.2. RNA preparation: Total RNA was extracted from confluent MDCT cells using TRIzol (GIBCO BRL). Briefly, cells from a 150 cm flask were rinsed in 5 ml cold PBS, centrifuged and lysed with a 5 min incubation in TRIzol 2  reagent. Then, 0.4 ml chloroform was added and the mixture shaken vigorously for 15 seconds. After two minutes at room temperature the mixture was centrifuged at 12,000 X g for 15 minutes and the upper aqueous phase containing the extracted RNA removed to a separated tube. RNA was precipitated from the aqueous phase with addition of an equal volume of isopropanol and centrifuged at 12,000 X g for 10 minutes at 4 C. 0  The RNA precipitate was washed twice with 75% ice-cold ethanol, dried under vacuum, and taken up in diethyl-pyrocarbonate (DEPC)-treated water. Ten micrograms of RNA were incubated with RNase free DNase (134 U) in the presence of 5 mM MgCl at 37°C for 10 minutes. The DNase was heat inactivated 2  with a five minute incubation at 99°C. isolated RNA was stored in 70%) ethanol at -80°C until use. For isolation of mouse cortical kidney RNA an adult mouse was euthanised with cervical dislocation and the  12 kidney was immediately removed and put on ice. While on ice the kidney cortex was dissected then placed in liquid nitrogen. The sample was pulverized in liquid nitrogen before TRIzol extraction. Messenger RNA used for the creation of the cDNA library was derived from the total RNA using the Poly-ATtract™ system for mRNA isolationfromtotal RNA (Promega Madison, WI.) according to kit instructions. II.2.3. Reverse Transcription-polymerase chain reaction analysis.  Reverse-transcription-polymerase chain reaction (RT-PCR) was carried out as follows. One,ug total RNA was taken up to 12/il in DEPC treated water, heated to 70°C then quickly chilled on ice. Four/il 5x first strand buffer (GIBCO-BRL), 2 fA 0.1 M DTT, 1 fA 10 mM dNTP mix, and 1 (A (50 fuM) random hexamers were added at room temperature and incubated for 5 min. The temperature was raised to 42°C and \/A (200 units) of Superscript II RNase FT reverse transcriptase (GIBOCO-BRL) was added and the reaction allowed to proceed for 90 min at 42 °C. The reaction was heat inactivated at 70 °C over 15min. Five microliters of the RT reaction mixture was electrophoresed in 1 % agarose gels, stained with ethidium bromide, to check for quantity and quality of cDNA. One jA of the cDNA reaction mixture, or for the negative control 1 pi\ (0.5  jug//u\)  non-reverse  transcribed RNA, was used as the template for PCR. The 50/A PCR mix included 0.5 fA Taq Polymerase (Perkin-Elmer-Cetus), 5 fA 10* PCR buffer, 2.5 (A (50 mM) MgCl ,2/A 10 mM dNTP mix, 36.25fA distilled 2  H 0, 1.5 /^l of each primer (15 mM). A cDNAfragmentof 509 bp corresponding to the mRNA sequence 2  encoding the majority of the transmembrane-spanning segment was amplified using the primer pair, sense 5'-GTTCCGAAACACACCTATCGTCAAG-3' and antisense 5'-TGAACTTGGCTTCGTTGAAGTTCTC3'. The PCR amplification consisted of 32 cycles of denaturation at 94°C for 40 seconds, annealing at 55 °C for 30 seconds, and polymerization at 72° C for 45 seconds, using a Gene Amp PCR system thermocycler Model 9600 (Perkin-Elmer-Cetus, Branchburg, Nj, USA).  13 Sequence analysis of PCR-amplified Ca -SR product was performed after subcloning the amplified 2+  fragment into pCRII TA-cloning vector (Invitrogen, San Diego, Ca. USA) according to kit instruction. The insert was sequenced on an Applied Biosystems Model 373A gene sequencing system.  11.2.4. Southern Hybridization:  In a screw cap vial, lOOng of full length human kidney Ca -sensing receptor DNA was made up 2+  to 9/A, boiled for 5 minutes, spun briefly, and stored on ice. To this was added 2/il 10 X Klenow buffer, 2 fA d(A,T,G,)TP (10 mM), 1 fA random hexamer (100 pmoVfA), 1^1 Klenow, 5 /A P-dCTP (50 /j.Ci). The 32  reaction was carried out at 37 °C for 30 minutes. Unincorporated nucleotide is removed by a Sephadex G-50 spin column, equilibrated with 10 mM Tris/HCl, 1 mM EDTA (pFf 7.6), and the probe collected. The probe was boiled for 5 minutes before use. The PCR products were separated by agarose gel (1% in IX TAE) electrophoresis and transferred by downward alkaline capillary transfer to GeneScreen. The blot was crosslinked in a UV Stratalinker (Stratagene San Diego, Ca, USA.). Membranes were pre-hybridized for 1 hour at 55°C in 30 ml hybridization buffer (0.3 g fatty acid free BSA, 10.5 ml sodium phosphate buffer, 9.0 ml de-ionized formamide, 10.5 ml 20% SDS). For hybridization, the pre-hybridization solution was removed and fresh buffer, with boiled probe was added. Hybridization proceeded overnight in a 50 °C hybridization carousel. Membranes were washed 2 X 10 minutes in 250 ml 150 mM NaP/0.1% SDS. Washing was continued at 55°C with intermittent monitoring of membrane counts. When counts were sufficiently reduced the membranes were removed and exposed overnight at -70 °C on Kodak X-OMAT film (Eastman Kodak, Rochester NY, USA) .  11.2.5. Western blots:  Cells were lysed in triple detergent buffer [50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.02% NaN , 3  14 0.1%SDS, 1 mM EDTA, 100Mg/mlPMSF,2^1/mlleupeptin,2Aig/mlaprotonin,0.15NP^0,0.5% sodium deoxycholate) for five minutes on ice. The cell lysates were spun at 1200 X g, two minutes, 4°C and the supernatants stored at -80°C. Aliquotes were electrophoresed through 8% SDS-polyacrylamide gels and blotted onto polyvinylidene difluoride (PVDF) membranes (BioRad). Membranes were rinsed in TBS-Tween 20 (10 mM Tris-HCL, pH 8.0,150 mMNaCl, 0.05% Tween 20), blocked with 5% dried milk powder in TBSTween 20 for 2 hours and incubated in Ca -SR antiserum. A mouse monoclonal antibody (ADD) raised 2+  against the peptide comprising residues 214 to 236 of the Ca -SR was used [45]. This antibody, as well as 2+  the peptide against which it was raised, was provided by Drs. P.K. Goldsmith and A.M. Spiegel (NIH, Bethesda, MD, USA) and K.V. Rodgers (NPS Pharmaceuticals, UT, USA) and has been extensively characterized with respect to specificity for the Ca -SR [45]. As the control, immuno-blotting was carried 2+  out as above with the antiserum pre-absorbed for one hour with the peptide (10/ig/ml) against which it was raised.  II.2.6. Determination of [Ca  2+  Epifluorescence microscopy was used to monitor changes in the fiira-2 fluorescence of the MDCT cells. Glass cover slips, with cells loaded with fura-2, were mounted in a plexiglass chamber containing 50(kl buffer. The chamber was mounted on an inverted Nikon Diaphot-TMD microscope, with a Fluor xlOO objective, and fluorescence was monitored under oil immersion within a single cell over the course of study. Fluorescence was recorded at 1-s intervals using a dual-excitation wavelength spectrofluorometer (Deltascan, Photon Technologies, Princeton, NJ) with excitation at 3 3 5 and 385 nm (chopper speed set at 100 Hz) and emission at 505 nm. All experiments were performed at 23 °C. Solution changes were made without an interruption in recording. The intracellular Ca concentration [Ca ]j was calculated, assuming a dissociation constant of 224 2+  2+  15 nM for the Fura-2 Ca complex after correction for extracellular fura-2 and autofluorescence. The final 2+  concentration is as described by Gynkyewicz et al [46] and Malgaroli et al [47] based on the equation: [Ca], =K (R-R /R -R)Sf2/Sb2 d  min  max  Where K is the disassociation constant; Rmin is the fluorescence ratio at the excitation wavelengths 335/385 d  nm for uncomplexed fura-2 (zero calcium); Rmax is the ratio of fluorescence at 335/385 nm for fura-2 saturated with Ca ; Sf2 and Sb2 are the fluorescence intensities at 385 nm with zero Ca and excess Ci , 2+  2+  +  respectfully; R is the ratio of fluorescence at wave lengths 335/385 of the sample to be measured. Dye content and instrument sensitivity are free to change between ratio measurements as they cancel. R, Rmin, and Rmax must be measured without changing instrumentation so a bias does not occur during an experiment. Fura-2/AM and mag-fura-2-AM are hydrophobic and thus pass easily into cells across the plasma membrane. Once inside the cell cytosolic esterases cleave the acetoxymethyl groups from the dye rendering a highly charged compound which is incapable of crossing the cell membrane. The dyes must be allowed to de-esterify as incomplete de-esterification of the dye interferes with measurements. Loaded cells were, therefore, washed twice with buffered salt solution (145 mM NaCl, 4.0 mM KC1, 1 mM CaCl , 0.5 mM 2  MgCl , 0.8 mM Na HP0 „ 0.1 mM KH P0 , 5 mM glucose, and 20 mM HEPES-Tris, pH 7.4 (depending 2  2  4  2  4  on the experiment divalent cation may be removed). The cells were incubated a further 20 min to ensure complete de-esterification and finally washed once withfreshbuffer solution. In all experiments involving [Ca ]; analysis, single traces are shown, but similar results were obtained 2+  in at least four separate experimentsfromindependent cell preparations.  II.2.7 Determination of [Mg ] 2+  i  Coverslips were mounted into a perfusion chamber, and [Mg ]| determined with the use of the 2+  Mg -sensitive fluorescent dye, mag-fura-2 [48]. The cell-permeant acetoxymethyl ester (AM) form of the 2+  16 dye was dissolved in dimethyl sulfoxide (DMSO) to a stock concentration of 5 mM and then diluted to 5 uM mag-fura-2/AM in media for 20 min at 19 °C. The free [Mg ] was calculated from the ratio of the fluorescence at the two excitation wavelengths 2+  ;  as described using a dissociation constant (K ) of 1.4 mM for the mag-fura-2*Mg complex [48]. The 2+  d  minimum (R, ) and maximum (R^J ratios were determined for the cells at the end of each experiment using nin  20 uM digitonin. R,„ for mag-fura-2 was determined by the addition of 50 mM MgCl in the absence of Ca , 2+  ax  2  and R^,, was obtained by removal of Mg and addition of 100 mM EDTA, pH 7.2. 2+  11.2.8. cAMP measurements  3',5'- Cyclic adenosine monophosphate (cAMP) was determined in confluent MDCT cell monolayers cultured in 24-well plates in DMEM-Ham F-12 media without serum. After addition of various hormones, MDCT cells were incubated at 37°C for 5 min in the presence of 0.1 mM isobutylmethylxanthine (IBMX). The cAMP was extracted with 5% trichloroacetic acid that was removed with diethyl ether acidified with 0.1 N HC1. The aqueous phase was dried, dissolved in Tris-EDTA buffer, and cAMP measured with a radioimmunoassay kit (Diagnostic Products Corperation, Los Angeles, Ca, USA). 11.2.9. cDNA Library  cDNA libraries were constructed using Superscript™ Lambda System for cDNA Synthesis andA, Cloning (GibcoBRL) according to kit instructions.  11.2.10. Xenopus oocyte isolation, preparation, and injection  Ovarian lobes were surgically removed from adult female Xenopus laevis toads anesthetized with 5.7 mM tricaine buffered to pFf 7.4 with Tris base. Portions of ovarian lobes were incubated for 120 min in Ca free buffer (5mM HEPES, 83 mM NaCl, 2 mM KC1, and 1.06 mM MgCl containing 0.2% collagenase 2+  2  17 type II). Stage 5 or 6 oocytes were separated manually and washed extensively and stored overnight in buffer (96 mM NaCl, 2 mM KC1, 1.8 mM CaCl , 1.0 mM Na HP0 , 2.5 mM Na-Pyruvate, 5.0 mM HEPES, 0.8 2  2  4  mM MgCl , titrated to 7.4 pH with NaOH, lO/45/ml Penicillin/Streptomycin). 2  Oocytes were injected using a WPI Nanolitre injector (A203XV4) with a glass pipette (5-7/im). Standard injected volumes were 50 nl of water (negative control) or 50 nl mRNA (l/ug/fA) in water, oocytes were then incubated 2-3 days at 19° C in buffer. Chloride currents were measured using standard two electrode voltage clamp techniques. Glass microelectrodes had a tip resistance of 1.5-3 MQ when filled with 3 M KC1. Oocytes were clamped to a potential of -60 mV using a GeneClamp 500 amplifier (Axon Instruments). Current traces were recorded directly to disk. The oocytes are recorded while in (<500ul) storage buffer without antibiotics and pyruvate. /  External Ca and Mg are added as experimentally required with buffer changes via total replacement. 2+  2+  II. 2.11. Statistical analysis  Representative tracings of fluorescence intensities are given. Significance was determined by Tukey's analysis of variance or Student's Mest where appropriate. A probability of P < 0.05 was taken to be statistically significant. All results are means ± SE unless otherwise indicated. III. Results  In a preliminary study, with Mg -depleted MDCT cells, we noted that 10 mM [Ca \ was without 2+  2+  effect on hormone-stimulated Mg refill, as determined with mag-fura-2, over 10-30 min of fluorescence 2+  determinations [49]. However, it was of interest in these studies that the addition of either [Ca ] or [Mg ] 2+  2+  0  0  resulted in rapid and transient intracellular increase in Ca concentration which prompted us to examine this 2+  cell line further for the expression of extracellular polyvalent cation-sensing receptors [44]. In the present studies, a functional polyvalent cation-sensitive receptor was shown, by two methods, to be expressed in MDCT cells [44]. First, the Ca -SR sequence was obtained from RT-PCR amplified products of MDCT cell 2+  18 mRNA, and Western analysis showed that the message was expressed in protein form. Second, Ca -SR2+  stimulated intracellular Ca concentration ([Ca ];) release was determined by microfluorescence. 2+  III. 1  2+  RT-PCR analysis ofCa -SR mRNA in MDCT cells. 2+  The primer set used here amplified a product from MDCT cell cDNA of 509-bp corresponding to a portion of the mRNA encoding the majority of the transmembrane spanning region of the Ca -SR (Fig. 3). 2+  This was confirmed by subcloning and sequencing of the product (Fig. 4). As positive controls a similar PCR product was also obtained from AtT-20 and TT cell RNA - these cell lines have previously been reported to express the Ca -SR [50,51,52]. As negative control, under the identical conditions of reverse transcription 2+  and amplification, no PCR product was obtained from RNA of COS-7, MC3T3-E1 on NIH3T3 cells, all previously reported to be negative for the Ca -SR [53]. The nucleotide sequence of the MDCT RT-PCR 2+  product shared greater than 92% identity with the corresponding rat Ca -SR sequence [13] (Fig. 4). 2+  111.2  Western Analysis of the Ca -SR in MDCT cells. 2+  Western analysis of cell extracts from MDCT cells revealed a similar pattern of bands to that seen in parathyroid gland and kidney preparations(Fig. 5). The predominant species representing non-glycosylated and glycosylated forms ranged from 120-160 kDa with some additional higher molecular weight aggregates. The immunostaining of the bands was specifically abolished by pre-absorption of the antibody with peptide (data not shown). Extracts of COS-7 kidney cells, mouse MC3T3-E1 osteoblast-like cells and mouse NIH3T3 fibroblasts were negative for specific Ca -SR staining (Fig. 5). 2+  111.3  Intracellular Ca signalling in MDCT cells by Ca -SR activation. 2+  2+  In parathyroid cells the Ca -SR has been shown to be coupled to a G-protein which, upon activation, 2+  q  stimulates PLC activity leading to inositol 1,4,5- trisphosphate (IP) generation and in turn intracellular Ci 3  release [54-59]. To determine if the Ca -SR expressed in the MDCT cells is associated with similiar 2+  intracellular signalling, we determined [Ca ]j in response to extracellular polyvalent cations (Fig. 6). 2+  +  19  RT-PCR analysis of Ca -SR mRNA expression. Complementary DNA was synthesized from total RNA and used as template for PCR amplification. PCR products (4 pi) after either 24 (lanes 1 -7) or 32 (lanes 8-13) cycles were electrophoresed on an ethidium-bromidestained 1% agarose-TBE gel. Lane M, 1 kb ladder; lanes 1 and 8, MDCT; lanes 2 and 9, AtT-20; lanes 3 and 10, TT; lanes 4 and 11, COS-7; lanes 5 and 12, MC3T3-E1; lanes 6 and 13, NIH3T3; lane 7, -RT. After Southern blotting the PCR product was hybridized to a Plabelled Ca -SR DNA probe (data not shown) [44]. 2+  32  2+  20  1 1  TTC phe  CGA arg  AAC asn  ACA thr  CCT pro  ATC ile  GTC val  AAG lys  GCC ala  ACC thr  AAC asn  CGA arg  GAG glu  40 14  CTG leu  TCC ser  TAC tyr  CTC leu  CTG leu  CTC leu  TTC phe  TCA ser  CTC leu  CTC leu  TGC eye  TGC cys  TTC phe  79 27  TCC ser  AGC ser  TCC ser  CTG leu  TTC phe  TTC phe  ATT ile  GGG giy  GAG glu  CCC pro  CAG gin  GAC gin  TGG trp  118 40  ACC thr  TGC cys  CGC arg  CTG leu  CGA arg  CAG gin  CCC pro  GCC ala  TTC phe  GGC giy  ATC ile  AGC ser  TTC phe  157 53  GTG val  CTT leu  TGT cys  ATC ile  TCG ser  TGC cys  ATC ile  TTG leu  GTG val  AAG lys  ACC thr  A AT asn  CGA arg  196 66  GTC val  CTC leu  CTG leu  GTA val  TTT phe  GAG glu  GCC ala  AAA lys  ATA ile  CCC pro  ACC thr  AGC ser  TTC phe  235 79  CAC his  CGG arg  AAG lys  TGG trp  TGG trp  GGA g'y  CTC leu  AAC asn  CTG leu  CAG gin  TTC phe  CTG leu  CTG leu  274 92  GTT val  TTC phe  CTC leu  TGC cys  ACC thr  TTC phe  ATG met  CAG gin  ATT ile  GTC val  ATC ile  TGC cys  ATC ile  313 105  ATC ile  TGG trp  CTC leu  TAC tyr  ACG thr  GCA ala  CCC pro  CCC pro  TCC ser  AGC ser  TAC tyr  CGC arg  AAC asn  352 118  CAC his  GAG glu  CTG leu  GAA glu  GAC asp  GAA glu  ATC ile  TTC phe  ATC ile  ACG thr  TGC cys  CAT his  GAG glu  391 131  GGC g'y  TCA ser  CTC leu  ATG met  GCG ala  CTC leu  GGC giy  TCC ser  CTG leu  ATC ile  GGC g'y  TAC tyr  ACC thr  430 144  TGC cys  CTC leu  CTG leu  GCT ala  GCC ala  ATC ile  TGC cys  TTC phe  TTC phe  TTT phe  GCC ala  TTC phe  AAG lys  469 157  TCT ser  CGG arg  AAG lys  CTG leu  CCA pro  GAG gin  AAC asn  TTC phe  AAC asn  GAA glu  GCC ala  AAG lys  TTC phe  Fig.4.  Nucleotide sequence ofthe MDCT cell RT-PCR product shown in Fig.3. Primer sequences are underlined (with one nucleotide missing from each primer at the 5' and 3' ends of the actual PCR product). The sequence is greater than 92% homologous to the corresponding portion ofthe rat Ca -SR [44]. 2+  21  Fig. 5.  Western analysis of the Ca -SR. Cell or parathyroid gland extracts (10 pg protein each) were subjected to SDS-PAGE on a 4-12% gradient gel. The blot was stained with Ca^-SR mouse monoclonal antibody (ADD [21]). Lane 1, MDCT cells; lane 2, rat parathyroid; lane 3, COS-7 cells; lane 4, MC3T3-E1; lane 5, NIH3T3 cells. The bands were demonstrated to be specific after staining the blot with the same antiserum pre-incubated with the peptide against which it was raised (data not shown) [44]. 2+  22  Fig. 6.  External polyvalent cations transiently increase cytosolic Ca in MDCT cells. MDCT cells were cultured in DMEM/Ham's F-12 (1:1) with 10% fetal calf serum containing 0.6 mM magnesium and 1.5 mM calcium. The cells were loaded with fura-2 for 30 min in a buffer solution containing (in mM): MgCl 0.5, CaCl 1.0, NaCl 145, KC1 4.0, Ka HP0 0.8, KH P0 0.2, glucose 5, and HEPES-Tris 20, pH 7.4. In order to test the effect of [Mg ] , the buffer solution was changed to one containing the above but without MgCL This bathing solution was replaced 1-2 min later with one containing 0.5 mM MgCl . To test [Ca ] , the cells were initially bathed with the above solutions containing no CaCl . This was replaced with one containing 1.0 mM CaCl . The complete buffer solution, containing 0.5 mM MgCl 0.5, and 1.0 mM CaCl , was used to determine the effects of external 0.1 mM GdCl and 0.01 mM neomycin. Again, these were done through bath solution changes. Tracings are representative of 4-10 separate experiments [44]. 2+  2  2  2  4  2+  2  4  0  2+  2  0  2  2  2  3  2  23 MDCT cells were cultured in normal media containing 0.5 mM magnesium and 1.0 mM calcium. The cells were loaded with fura-2 for 30 min in buffer solutions containing 0.5 mM magnesium and 1.0 mM calcium; the extracellular fura-2/AM was removed with 2 x washes of nominally calcium-and magnesiumfree buffer solutions and [Ca ] was quantified by fluorescence. The polyvalent cations (in mM): MgCl 0.5, 2+  f  2  CaCl 1.0, GdCf 0.1, or neomycin 0.01, were added in buffer as indicated in Fig. 6. The cations were 2  changed by complete replacement of bathing solutions. [Mg ] was tested in the presence of normal [Ci ] 2+  +  0  0  in the buffer and the effects of [Ca ] were determined in the presence of normal [Mg L Addition of 2+  2+  0  external [Mg ] increased [Ca ] from basal levels of 107±23 nM to peak concentrations of 1046 ±121 nM, 2+  2+  0  i  n = 6, p<0.05 (Table 1). [Ca? ] resulted in an increase in [Ca ^ to 732±92 nM, Gd to 1637±161 nM; and +  2  3+  0  neomycin to 1073±155 nMfrombasal levels (Table 1). Table l.Polyvalent cations stimulate cytosolic Ca transients in MDCT cells. 2+  Concentration  Response  Basal [Ca ]j /  A[Ca ]i /  mM  (pos/total)  nM  nM  2+  0.5  (6/9)  107±23(6)  1046±121*(6)  Ca  2+  1.0  (10/13)  92±15(10)  732±92*(10)  Gd  3+  0.1  (5/8)  93±13(5)  1637±161*(5)  0.01  (7/11)  107±8(7)  1073±155*(7)  Extracellular Cation  Mg  Neomycin  2+  n  2+  Table 1 Legend:  Normal MDCT cells were loaded with fura-2 and MgClj, CaCl , GdCl or neomycin added to buffer solutions at the indicated concentrations as given in legend to Fig.3. A[Ca ] was the maximal change in cytosolic Ca from basal Ca levels in those cells that responded. The cells which responded to the addition of the polyvalent cations is indicated as afractionof the total cells studied (pos/total). ()" is the number of separate observations and * indicates significance, p<0.05frombasal [Ca ] [44]. Replacement of bathing solution 2  3  2+  2+  (  2+  2+  ;  24 with isosmolar salt solutions, containing either NaCl or KC1, did not elicit cytosolic Ca transients. Of 108 2+  individual cells studied in this thesis and other reports, a total of 24 cells failed to respond to extracellular polyvalent cations. There were no differences in the number of non-responsive cells observed for each of the tested extracellular cations (Table 1). This appeared to be an all-or-none response as subsequent additions of very high concentrations of extracellular cations did not stimulate [Ci ] in those cells that initially +  {  did not respond. The reason for the non-responsiveness in some cells is not known. In all cases, the change in cytosolic Ca in the positive cells was transient, returning to near basal levels within 1 -2 min. The majority 2+  of the cytosolic Ca transients were monophasic. Moreover, the responses were not dependent on the 2+  presence of extracellular Ca indicating that the rise in [Ca ] was from intracellular sources. The profile 2+  2+  i  of the Ca -SR-mediated rise in cytosolic Ca is different than the sustained rise in [Ca^]; observed following 2+  2+  the addition of PTH or calcitonin to MDCT cells [40,41,60]. There was no change in the mean cytosolic Mg" 4  concentration, 0.52±0.02 mM, n = 5, during these manipulations as determined with the Mg -sensitive 2+  fluorescent probe, mag-fura-2 (data not shown). These results support the notion that the MDCT cell line possesses a functional Ca -SR which elicits a transient increase in cytosolic Ca in response to extracellular 2+  2+  polyvalent cations. The relative potencies of extracellular cations in stimulating Ca -SR-sensitive cytosolic Ca 2+  2+  signalling for many renal and extra-renal cells have been reported to be in the order of 3-5 mM for [Ca ] 2+  0  and about 5-20 mM for [Mg ^ [4,12,13,56,58,59-61]. Figure 7 summarizes the changes in MDCT cytosolic 2  Ca , in response to [Mg ] and [Ca ] , in buffer solutions containing normal MgC|, 0.5 mM, and CaCl , 2+  2+  2+  0  0  2  1.0 mM, concentrations. The addition of either 0.2 mM [Mg ] or 0.2 mM [Ca ] induced significant 2+  2+  0  0  increases in cytosolic Ca levels. Changing the bathing solution but not the [Mg ] concentration, 0.5 mM, 2+  2+  0  had no effect on [Ca ] (Fig.7, Panel A). Replacement of bathing solutions containing 0.7 mM MgCl (total) 2+  ;  2  for normal buffer solutions (0.5 mM MgCl ) resulted in an increase in [Ca ] from basal levels of 102±12 to 2+  2  ;  25 728± 130 nM, n = 4. Rapid replacement of normal buffer solutionswith one containing 1.2 mM CaCl (total) 2  resulted in an increase in [Ca ] to 542±73 nM, n = 4 (Fig. 7, Panel B). Note, replacement of the normal 2+  s  bathing solution containing 1.0 mM [Ca?] did not elicit a response. Accordingly, the polyvalent cation-sensing +  0  mechanism in MDCT cells is sensitive to changes in both [Mg ] and [Ca ] within the physiological ranges 2+  2+  0  0  of divalent cations. There may be other receptors in the MDCT cell which are sensitive to [Mg ] in the 2+  0  physiological range. The Ca -SR has been proposed to be a Mg -sensing receptor in addition to its role in 2+  2+  Ca metabolism [5,62]. There is some evidence of different binding sites for each polyvalent cation so that 2+  the receptor may respond in a selective manner to either [Mg ] and [Ca ] [62]. To test whether the 2+  2+  Q  0  MDCT Ca SR is responsive to [Mg ] independently of [Ca ] , we determined the response to Mg in 2+  2+  2+  2+  0  0  0  the presence of high concentrations of [Ca ] (Fig. 8). MDCT cells were bathed in buffer solution containing 2+  0  5 mM CaCl , no magnesium and challenged with 0.2 mM Mg . The addition of 0.2 mM [Mg ] stimulated 2+  2  2+  0  0  a significant rise in [Ca ] in the presence of 5 mM [Cd ] (Fig. 8, Panel A). To determine the sensitivity 2+  +  s  0  of the Ca -SR to [Ca ] in the presence of a background of high concentrations of Mg , MDCT cells were 2+  2+  2+  0  0  immersed in a buffer solution containing 5.0 mM MgCl , zero calcium and 0.2 mM CaCl was added (Fig.8, 2  2  Panel B). The addition of 0.2 mM CaCl led to significant increases in [Ca ]j. The polyvalent cation2+  2  sensitive mechanism present in MDCT cells is responsive to small changes in [Mg^] or [Ca ] in the +  2+  0  0  presence of large background concentrations of extracellular calcium and magnesium, respectively. These results suggest that the Ca -SR responds independently and equipotently to [Ca ] and [Mg ] or that there 2+  2+  2+  0  0  are separate receptors for the two cations. The term Mg 7Ca -SR will, therefore, be used to indicate the 2  response to polyvalent cations in MDCT cells.  2+  26  Fig. 7.  The calcium sensing receptor of MDCT cells responds sensitively to [Mg ] and [Ca ]. MDCT cells were loaded with fura-2 according to the methods given in the legend to Fig. 6, but the loading and test buffer solutions contained (in mM): MgCl 0.5, CaCl 1.0, NaCl 145, KC14.0, Na HP04 0.8, KH P0 0.2, glucose 5, and HEPES-Tris 20 (pH 7.4). MgCl (A) or CaCl (B) were added by changing buffer solutions containing the concentrations shown. Tracings are representative of 4 to 5 separate experiments [44]. 2+  2  2  2  2  4  2+  2  2  27  DE059602  Fig. 8.  The Ca -SR of MDCT cells is sensitive to small changes in [Mg ] and [Ca ] in the presence of elevated divalent cation concentrations. The MDCT cells were loaded with fura-2 as given in legend to Fig. 6. The bathing solution was changed to one containing either 5.0 mM CaCl (panel A) or 5.0 mM MgCl (panel B). About 5 min later, the MDCT cells were challenged with bathing solutions containing either 0.2 mM [Mg ] (panel A) or 0.2 mM [Ca ] (panel B), in the presence of 5.0 mM CaCl or 5.0 mM MgCl , respectively. Tracings are of one experiment but are representative of 4 separate experiments [44]. 2+  2+  2+  0  2  0  2  2+  0  2+  0  2  2  28  Fig. 9.  Repetitive stimulation of MDCT-Mg 7Ca -SR with [M£ ] and [Ca ] . MDCT cells were loaded with fura-2 in buffer solution (given in legend to Fig.4) containing no magnesium or calcium. Buffer solution containing [Mg ] 0.5 mM or [Ca ] 1.0 mM, were changed where indicated. The given [Mg ] and [Ca ] concentrations are final concentrations. Tracings are representative of 3 separate experiments [44]. 2  2+  2+  +  0  0  2+  2+  0  2+  0  2+  0  0  29 Fig. 9 illustrates the effect of repetitive stimulations of the Mg /Ca -SR with sequential challenges 2+  2+  of either MgCl or CaCl . Both [Mg ] and [Ca ] stimulated transient increases in cytosolic Ca which 2+  2  2  2+  0  2+  0  diminished after a number of repetitive additions of divalent cation (Fig. 9). The diminution of Ci signalling +  after many repetitive stimulations may reflect desensitization of the Mg /Ca -SR to [Mg ] and [Ca ] or 2+  2+  2+  2+  0  0  depletion of intracellular Ca stores as each cation challenge was applied. This phenomenon is distinct from 2+  the rapid desensitization observed in hormone-induced cytosolic Ca transients observed in the thick 2+  ascending limb cell [63] or the sustained elevation seen in hormone-mediated increments in Ca entry in distal 2+  convoluted tubule cells [39,61]. III.4  Mg /Ca -SR activation inhibits hormone-stimulated cAMP synthesis. 2+  2+  Finally, we determined the effect of activation of Mg 7Ca -SR on hormone-stimulated synthesis of 2  2+  cAMP. The MDCT cells possess parathyroid hormone (PTH), calcitonin, glucagon and arginine vasopressin (AVP) receptors which are coupled to adenylate cyclase [39]. PTH, calcitonin, glucagon at 10" M, and 7  AVP, 10" M, increase cellular cAMP levels by about 3-fold. The potent polyvalent cation, neomycin, was 8  used to activate the Mg 7Ca -SR prior to the addition of hormones (Table 2). Pretreatment of the MDCT 2  2+  cells with neomycin inhibited hormone-stimulated cAMP accumulation. Next, we activated the Mg 7Ca -SR 2  2+  with high levels of [Mg ] or [Ca ] (Table 2). Pretreatment of MDCT cells with 10 mM [Mg ] or [Ca} 2+  2+  0  2+  0  2  0  0  inhibited hormone-mediated cAMP generation. IV Discussion:  In this study, we show that the established MDCT cell line possesses a Mg /Ca -SR that responds 2  2+  to [Mg ] and [Ca ] , to transiently release Ca ^ from cytosolic stores. These cells possess components 2+  2+  0  2  0  involved in calcium metabolism such as , hormone receptors, vitamin D receptors, vitamin D metaboliteinduced 24 hydroxylase activity, P-450 metabolites, and the calbindin 28K calcium-binding protein that are important in vitamin D actions [39,64,65]. Accordingly, it is of interest that this cell line also has an  30 Table 2.  Activation of Mg 7Ca -SR inhibits hormone-stimulated cAMP synthesis. 2  Control  2+  PTH  Calcitonin  Glucagon  AVP  56±2*(5)  60±0.1*(4)  105±5*(7)  71±2*(5)  Control  19±l(ll)  Neomycin  22±2(3)  34±1**(3)  33±4**(3)  22±1**(7)  24±3**(4)  23±2(3)  -  -  32±2**(3)  34±3**(3)  24±3(3)  -  -  37±3(3)  27±3**(3)  Ca  2+  Mg  0  2+ 0  n  Table 2 Legend:  Where indicated, neomycin, 50 uM, extracellular Ca ,10 mM, or extracellular Mg ,10 mM, were added 5 min prior to the addition of parathyroid hormone (PTH), 10" M, calcitonin, 10" M, glucagon, 10" M, or arginine vasopressin (AVP), 10" M, and cAMP was measured 5 min later. Values are cAMP accumulation, pmol/mg protein • 5 min. ()" is the number of separate observations. * indicates significance, p<0.01, from control values without hormone and * * indicates significance ofneomycin or Ci or Mg ^ valuesfromthose of the respective hormone treatments [44]. 2+  2+  0  0  7  7  7  8  +  2  Q  extracellular divalent cation-sensing receptor. Using RT-PCR and Southern blotting, we show that the MDCT cells possess transcripts for the Ca -SR. The presence of the Ca -SR protein in MDCT cells was 2+  2+  documented by Western blot analysis using a specific Ca^-SR antiserum. Furthermore, monitoring of cytosolic [Ca ] showed that activation of the Mg /Ca -SR in MDCT cells elicits [Ci \ signals in response 2+  2+  2+  +  to increasing extracellular concentrations of polyvalent cations demonstrating that the receptor is functional. These studies with mouse distal convoluted cells are consonant with reports identifying Ca -SR RNA 2+  31 transcripts in the rat distal convoluted segment [11,13,17]. More recently, Riccardie^ al. showed that the rat Ca -SR is localized to the basolateral membrane of the distal convoluted tubule (15). The presence of a 2+  Mg 7Ca -SR in the distal convoluted tubule may have important ramifications on cellular function within this 2  2+  segment.  32  Chapter 2: Attempts to isolate a specific Mg -sensing Receptor 2+  I. Background The results of Chapter 1, specifically the MDCT cell's ability to sense Mg  2+ 0  in the face of  physiological levels of Ca , as well as the body's ability to manage magnesium independently of calcium 2+  0  [74,76-78], strongly indicate the presence of a extracellular Mg -SR. In order to see if Mg -sensing occurs 2+  2+  in other cell types, we determined the effect of extracellular Mg versus Ca on intracellular signalling in 2+  2+  two different systems. A system, that specifically responds to magnesium, has been demonstrated in the oocyte of the prawn Palaemon serratus [79,80]. The presence of Mg in sea water induces a Ca wave 2+  2+  within the cell similar to that seen with stimulation ofthe Ca -SR [80]. A Ca wave is also observed when 2+  2+  Madin Darby Canine Kidney (MDCK) cells were exposed to 3 mM Mg but not 5 mM Ca (Fig 10). Given +  2+  the results in these two systems it is clear there is a extracellular Mg sensing mechanism that appears to 2+  be more responsive to extracellular Mg than Ca . Accordingly, we attempted to isolate a Mg -sensing 2+  24  2+  receptor from MDCK cells an&Palmaemon serratus oocytes using a genetic screening. Ca -SR's are most 2+  highly conserved, with respect to all known Ca -SR's as well as to metabotropic glutamate receptors and 2+  pheromone receptors, in the transmembrane region [81-83]. Probes and primers used for this homology approach were therefore designedfromthis region. II Methods and Materials: As described in Chapter 1. III Results: III. 1  Intracellular Ca signalling in MDCK, but not HEK cells, in response to [Mg J . 2+  2+  0  MDCK and HEK cells were exposed to extracellular Ca and Mg as in chapter 1 . When we 2+  2+  challenged MDCK cells with 3 mM Mg or 25/uM neomycin, MDCK cells demonstrated a response similar 2+  33 to MDCT cells (Fig. 10). However, 5 mM Ca failed to induce an increase in intracellular Ca . HEK cells, 2+  2+  which do not express the Ca -SR [25], are unresponsive to Ca 2+  2+ 0  or Mg  2+ 0  up to 10 mM.  111.2. Expression of the_Mg response in Xenopus oocytes injected with Palaemon mRNA. 2+  An expression protocol similar tothat used by used by Brown et al.[4] was also attempted using Palaemon RNA. To test for functional expression of a Mg -SR or Ca -SR in Xenopus oocytes the ability 2+  2+  of Mg or Ca to increase CI" currents was assessed. Water (50 nl), or 50 nl of shrimp mRNA (l/-g//il), 2+  2+  was injected into defolliculated Xenopus oocytes. Oocytes were then incubated for 2-3 days, exposed to 15 mM external Mg or Ca , and the chloride current measured as described. Currents of up to 80 nA were 2+  2+  detected in response to Mg (15 mM) in 50 % of mRNA injected oocytes (Fig. 11). In contrast extracellular 2+  Ca (15 mM) did not cause changes in the CI" currents in any of the injected oocytes. Water injected 2+  oocytes were unresponsive to 15mM Mg or Ca (data not shown). 2+  2+  111.3. A Ca -SR cDNA, or related transcript, was not identified by screening a Palaemon serratus 2+  oocyte or MDCK cDNA library .  The Mg -sensing response of thePalaemon serratus oocyte was conveyed to the Xenopus oocyte 2+  with mRNA injection. Based on the hypothesis that an extracellular Mg -SR gene is similar to a Ca -SR 2+  2+  gene, a homology-based screening approach was attempted. A cDNA library from the mouse kidney was obtained and cDNA libraries were made from the MDCT cell line, MDCK cell line, andPalaemon oocytes. These libraries were screened at low stringency using a probe comprising the majority of the transmembrane region of the Ca -SR. This region has the most similarity among Ca -SR's and their related genes [81]. 2+  2+  Other than partial Ca -SR clones from the MDCT, and mouse kidney, no related transcripts were identified. 2+  Multiple attempts to identify a related gene using PCR with primers designed from regions conserved from the Ca -SRto the metabotropic glutamate receptor failed to identify any related gene. The 2+  Ca -SR was not identified in MDCK cells using RT- PCR. 2+  34 IV.  Discussion Preliminary evidence demonstrates that some cells may respond sensitively, to Mg  2+ 0  relative to extracellular Ca . Unfortunately, no mammalian receptors specific for magnesium control have 2+  been discovered. Reports of mammalian Ca -SR homologues have been reported but none have proven to 2+  be specific for cations other than Ca [81]. The putative Mg -SR may be significantly different from the 2+  2+  Ca -SR making homology based screening an ineffective method for its isolation. A homologue has, however, 2+  been identified from the dogfish shark (Sualaus acathias) and reported in abstract form [84], This protein shares 74% amino acid identity to the rat Ca -SR and is 3.3 times more sensitive to Mg that Ca (half 2+  2+  2+  maximal activation of 7.5 mM for Mg ) when expressed in HEK cells. Unfortunately no sequence data is 2+  available. Initial data from Xenopus oocytes, injected with RNA isolated from Palaemon oocytes, was encouraging. Unfortunately, seasonal availability and limited supply of these oocytes made further experimentation impractical. As we failed to identify a specific Mg -SR, and the Ca -SR clearly does respond to low 2+  2+  concentrations of magnesium, we chose to use the MDCT cell for the rest of the experiments concerning characterization of the polyvalent-cation-sensing receptor. This does not preclude the possibility of separate Mg sensing receptor(s) but it allows us to determine a response to a Mg /Ca -sensing system that we 2+  know is located within the MDCT cell line.  2+  2+  35  HEK-293 cells  MDCK cells 1000  M g 2 3 mM +  500  500  I  J  3 minutes  I  5 minutes O  200  TO o  c  Ca 10mM 200 Mg 10 mM J 2+  Ca 5 mM 2+  2+  100 J  100  \  J  10. External polyvalent cations transiently increase cytosolic Ca in MDCK but not HEK cells. Cells were cultured in DMEM/Ham's F-12 (1:1) with 10% fetal calf serum containing 0.6 mM magnesium and 1.5 mM calcium. The cells were loaded with fura-2 for 30 min in a buffer solution containing (in mM): MgCl 0.5, CaCl 1.0, NaCl 145, KC1 4.0, Ka HP0 0.8, KH P0 0.2, glucose 5, and HEPES-Tris 20, pH 7.4. In order to test the effect of [Mg ] , the buffer solution was changed to one without MgCl . This bathing solution was replaced 1 -2 min later with one containing indicated MgCl . To test [Ca ] , the cells were initially bathed with the above solutions containing no CaCl . This was replaced with one containing indicated CaCl . Tracings are representative of 4-10 separate experiments. 2+  2  2  2  4  2  4  2+  0  2  2+  2  0  2  2  36  Extracellular Mg (15 mM), but not Ca , evokes increases in CI" currents in Xenopus oocytes injected with 50 ng prawn mRNA . Tracing is representative of those seen in three separate oocytes. There is no response evoked by water injected oocytes (data not shown). 2+  2+  37  Chapter III. Characterization of Mg Uptake Influenced by Peptide Hormones Acting Through 2+  G-Protein Linked Receptors.  I. Background.  Magnesium reabsorption in the DCT is under the control of a number of hormones and altered by a number of influences. From the experiments of chapter 1, it was discovered that c AMP production, stimulated by hormones known to bind to G-protein linked receptors, was inhibited by high external magnesium [44]. It is interesting, therefore, to define the effect hormones have on this cell line with respect to magnesium uptake. As the dependence of magnesium absorption with magnesium delivery is maintained in the presence of these hormonal factors [85,86], these systems must be inter-related. In this chapter four, peptide hormones which act through G-protein linked receptors to generate cAM, are characterized with respect to magnesium uptake [87]. Glucagon. Glucagon is a potent renal magnesium conserving hormone [88]. Bailly and colleagues reported that the acute infusion of pharmacological concentrations of glucagon in rats with intact parathyroid glands leads to a rapid fall in fractional magnesium excretion from 16±1% to 9±2% [88]. The response to glucagon is even greater in hormone-deprived animals. Bailly et al. showed that fractional magnesium excretion markedly decreased by about 50% (from 71.5±8.0 to 39.6±5.7 nmoles/min) with glucagon administration in rats deficient in endogenous PTH, calcitonin, glucagon and antidiuretic hormone [89]. This was attributed to a doubling of absolute reabsorption within both the loop of Henle (increasefrom6. 5±0.7 to II. 7±0.7 nmoles/min) and the distal tubule (increasefrom0.85±0.1 to 1.75±0.3pmoles/min). Accordingly, glucagon acts within the loop and distal tubule of the rat.  38 Arginine vasopressin. The actions of arginine vasopressin (AVP) within the distal convoluted tubule are poorly understood [90]. AVP has been shown to be an effective magnesium-conserving hormone in anaesthetized and conscious hormone-deprived rats [91-93]. Micropuncture studies of these animals have shown that AVP acts principally within Henle's loop [92]. Elalouf and colleagues failed to discern any change in fractional magnesium absorption in the superficial distal tubule following physiological administration of AVP [92]. In these studies, Elaloufe/ al. reported thatfractionalcalcium absorption significantly increased from 42.0±5.8% to 62.8±7.1%, whereas the change infractionalmagnesium transport with AVP was not significant, although it increased from 45.5±7.8% to 55.3± 15.5%. These changes may have been significant if a greater number of tubules had been sampled. Costanzo and Windhager did not observe any change in calcium absorption in the microperfused rat distal tubule with administration of AVP [94]. These animals had their thyroid and parathyroid removed (TPTXed), but where not hormone-deprived as were those used by Elalouf et al. [92]. In both studies, AVP enhanced sodium absorption in the distal tubule. Parathyroid hormone. A number of hormones stimulate magnesium absorption within the distal tubule. The first to be described was parathyroid hormone (PTH). Infusion of PTH to TPTXed animals increased the reabsorption of magnesium and diminished urinary magnesium excretion [95]. Micropuncture studies showed that part of this hormonal action occurs within the distal tubule [89,96]. An increase in magnesium conservation was observed even in the face of enhanced magnesium delivery to this segment [97100]. The largest changes were observed in TPTXed hamsters where the mean tubular fluid-to-ultrafilterable magnesium (TF/UF ) ratio at the distal sampling site fellfrom0.56±0.08 to 0.33±0.08 following Mg  administration of PTH [101]. This was associated with a fall infractionalmagnesium excretionfromabout 14% to 3%. De Rouffignac et al. and Bailly et al. have shown that PTH and other hormones stimulate magnesium absorption in the rat distal tubule [102]. They used Brattleboro rats with hereditary diabetes insipidus, that lack endogenous ADH, and they infused either glucose or somatostatin to inhibit glucagon  39 secretion. Furthermore, they TPTXed the animals to eliminate circulating PTH and calcitonin. Thus a "hormone-deprived" animal model was created to serve as a basis for evaluating the respective actions of each hormone [102]. Micropuncture studies were then performed to determine the effects of hormone administration; importantly, these studies were all performed with physiological hormone concentrations. Infusion of PTH ,. to hormone-deprived rats leads to diminished magnesium and calcium delivery to early 34  and late distal tubule sampling sites. These studies clearly demonstrate that PTH enhances magnesium absorption within the distal tubule. Calcitonin. Calcitonin infusions have clearly been shown to enhance renal magnesium conservation in the rat [ 103 ]. Poujeol et al. infused calcitonin to TPTXed rats and observed a fall in fractional magnesium excretionfrom4.1±0.4 to 1.0±0.3% which they attributed to an increase in magnesium reabsorption in the loop of Henle [103]. However, subsequent studies with the hormone-deprived rat showed that calcitonin markedly stimulatedfractionalmagnesium absorption in the superficial distal tubule as well as the loop [ 104]. These micropuncture studies indicate that calcitonin enhances magnesium conservation, in part, by actions within the distal tubule[104].  II. Methods and Materials: As previously described in chapter 1. Results  III. 1  Glucagon, arginine vasopressin, PTH, and calcitonin stimulate cAMP accumulation in Mg 2+  depleted MDCT cells.  In the present study, I determined the concentration-dependence of glucagon, AVP, PTH, (Fig. 12) and calcitonin-stimulated cAMP generation in MDCT cells [87]. (PTH and calcitonin induce similar responses as glucagon and AVP in MDCT cells so these data will only be included in a summary figure (Fig. 16 ).  40 These hormones elicited intracellular cAMP accumulation in a concentration-dependent fashion. Second, I determined whether these hormones elicit cAMP accumulation in the Mg^-depleted MDCT cells used here. Glucagon or AVP at submaximal (10" M) and maximal (10" M) concentrations increased cAMP by similar 9  7  amounts in normal and Mg -depleted MDCT cells (Table 3). The concentration-dependence of glucagon 2+  and AVP are far higher than the concentrations normally observed in vivo. Explanations for these discrepancies include absence of normal bathing plasma, diminished circulation of hormone or changes in membrane receptors due to cell culture conditions. Nevertheless, these responses indicate that hormone receptors are present in this cell line which activate intracellular signalling processes. These data confirm the observations of Friedman and Gesek et al. [ 108,109] that receptors for these hormones are present in the MDCT cells which upon stimulation release intracellular cAMP. Intracellular Mg depletion did not alter 2+  hormone-responsive cAMP generation in these cells (Table 3). Finally, glucagon or AVP, PTH , and calcitonin did not induce rapid intracellular Ca transients (data not given) indicating that cytosolic Ca 2+  2+  signalling is not a product of glucagon- or AVP- mediated pathways in MDCT cells [108,109].  2  Mg* uptake in MDCT cells. +  In order to determine Mg uptake, subconfluent MDCT monolayers were cultured in magnesium2+  free medium for 16 h. These cells possessed a significantly lower [Mg ] , 0.22±0.01 mM than cells cultured 2+  f  in normal media, 0.53±0.02 mM. When the Mg -depleted MDCT cells were placed in a bathing solution 2+  containing 1.5 mM MgCl , intracellular Mg concentration increased with time and levelled off at a [Mg ^, 2+  1  2  of 0.52±0.06 mM, (n = 9) which was similar to basal levels observed in normal cells (Fig. 13). The average rate of refill, d([Mg ])/dt, measured as the change in [Mg ] with time, was 164±5 nM/s/n = 6) cells, as 2+  2+  ;  ;  determined over the first 500 s following addition of 1.5 mM MgCl .[87] 2  41  Table 3. Glucagon and AVP stimulate cAMP synthesis in magnesium-depleted MDCT cells cAMP accumulation pmol»mg»protehr • 5min"' 1  Normal Cells Control  22± 2  (6)  Mg -depleted cells 2+  21± 1  (5)  Glucagon, 1(T M  6 0 ± 5*  (4)  6 0 ± 3*  (4)  Glucagon, 1 0 M  9 2 ± 5*  (4)  8 9 ± 3*  (4)  AVP, 3 x 1 0 " M  6 2 ± 4*  (3)  61±3*  (4)  AVP, 3 x  8 9 ± 5*  (4)  81±7*  (4)  9  _ 7  9  10" M 7  Legend to Table 3  Values are mean ± SE; number of experiments is in parentheses. Glucagon and arginine vasopressin (AVP) Ire added 5 min prior to the measurement of cAMP. *P < 0.01, significant vs. control values. There were no significant differences between normal and Mg depleted cells for each of the conditions tested. MDCT, mouse distal convoluted tubule [87]. 2+  III.3  Glucagon stimulates Mg uptake in MDCT cells. 2+  Glucagon, 10" M, added to the refill buffer solution increased the rate of Mg entry into Mg 7  2+  depleted MDCT cells (Fig. 13). Glucagon, nM/s, (n =  5),  10"  7  2+  M, increased the mean Mg entry rate from 1 6 4 ± 5 to 2+  196±11  which represented a stimulation of 2 0 ± 6 % above control values. In all cases where measured,  [Mg ]j returned to basal levels, 0 . 5 2 ± 0 . 0 3 mM. The effect of glucagon on Mg uptake was concentration2+  2+  dependent with maximal rate of stimulation at  10  6  M, 2 7 3 ± 6 nM/s, and half-maximal stimulation at a  concentration about 10" M (Fig. 14). I have previously reported that dihydropyridines inhibit Mg uptake into 7  +  Mg -depleted MDCT cells [61]. To determine whether glucagon-induced Mg entry is mediated through 2+  2+  42 a dihydropyridine-sensitive pathway, I examined the effect of the channel blocker, nifedipine, on the changes in [Mg ]| following placement in the refill buffer solution containing 1.5 mM MgCl . The presence of 10" 2+  5  2  M nifedipine inhibited glucagon-stimulated Mg uptake, 24±2 nM/s, indicating that this pathway is sensitive 2+  to the channel blocker and supporting the notion that glucagon-stimulated uptake is the same as the entry pathway observed in control cells (Table 4).  Table 4 . Hormone-stimulated Mg uptake in MDCT cells is dihydropyridine sensitive 2+  Basal [Mg ] mM 2+  d([Mg ]i)/dt, nM/s 2+  i;  Control  0.22 ± 0.02 (6)  164 ±5 (6)  Control + Nifedipine  0.22 ±0.02 (4)  42 ±21* (4)  Glucagon  0.22 ±0.01 (4)  196±11* (4)  Glucagon + Nifedipine  0.21 ±0.02 (3)  24 ± 2* (3)  AVP  0.22 ± 0.02 (5)  189 ± 6* (6)  AVP + Nifedipine  0.21 ±0.02(3)  75 ± 9* (3)  Table 4 Legend:  Values are mean ± SE; number of experiments is in parentheses. Measurements were performed in presence of 1.5 mM MgCl with and without 10" M Glucagon, or 3 x 10" M AVP according to the methods outlined in legend to Fig.13. Nifedipine, 10" M, was added with the refill solution where indicated. *P < 0.05, significant vs. control values [87]. 7  2  5  7  43  150 -.  0  10'"'  10"''  10"*  [GLUCAGON]  10 "  7  10  M  B  [AVP]  Fig. 12.  M  Glucagon and arginine vasopressin (AVP) stimulate cAMP accumulation in MDCT cells. Glucagon (panel A) or AVP (panel B) were added, at the concentrations indicated, 5 min prior to the measurement of cAMP. Values are means ± SE for 3-9 observations, 2 separate preparations. * indicates significance, p<0.05, compared to control values [87].  44  Fig. 13.Glucagon and arginine vasopressin (AVP) stimulate Mg uptake in Mg -depleted mouse distal convoluted tubule (MDCT) cells. Confluent MDCT cells were cultured in Mg -free media (<0.01 mM) for 16-20 hr. Fluorescence studies were performed in buffer solutions in absence of Mg , and where indicated, MgCl (1.5 mM final concentration) was added to observe changes in intracellular Mg concentration. The buffer solutions contained (in mM): 145 NaCl, 4.0 KC1,0.8 K HP0 ,0.2 KH P0 , 1.0 CaCl , 5.0 glucose, and 10 HEPES/Tris, pH 7.4, with and without 1.5 mM MgCL, Glucagon, 10 M, or AVP, 3 x 10" M, were added to with this buffer solution. Fluorescence was measured at 1 data point/s with 25-point signal averaging, and the tracing was smoothed according to methods previously described [87]. 2+  2+  2+  2+  2  2+  2  2  4  2  7  7  4  45  . 14. Concentration-dependence of glucagon-stimulation of Mg entry in MDCT cells. The rate of Mg influx as determined by d([Mg ]j)/dt was measured with the given glucagon concentrations using techniques performed according to that given in legend to Figure 1. d([Mg ])/dt values were determined over the first 500 s of measurements. Values are means ± SE for 3-6 cells [87]. 2+  2+  2+  2+  ;  46 III.4  A VP stimulates Mg uptake in MDCT cells. 2+  Fig. 13 illustrates the effect of AVP, 3 x 10" M, on Mg uptake in Mg -depleted MDCT cells. The 7  2+  2+  mean uptake rate, d([Mg ])/dt, increased from control levels 164±5 nM/s to 189±6 nM/s/i = 6. Unlike the 2+  ;  actions of glucagon, AVP responses were not immediate with the addition of the hormone to the refill solution. As shown, the response was observed only after 5-10 min after addition of the hormone to the superfusion solution. The reasons for this delay are not apparent at the present time. The following refill studies were uniformly performed 6 min after AVP was added to the MDCT cells. The refill rate, d([Mg ])/dt, was 2+  ;  determined over a standard 500s time interval following the 6 min delay. AVP stimulates Mg entry in a 2+  concentration-dependent manner (Fig. 15). The maximal effect was observed at about 3 x 10~ M which 6  resulted in an increase of d([Mg ])/dt to 188±2 nM/s. This change was significantly less than the response 2+  f  to maximal glucagon concentrations. AVP-stimulated Mg uptake was inhibited by nifedipine indicating that 24  the hormonal response is through activation of channels responsible for Mg entry in MDCT cells (Table 4). 24  III. 5  Calcitonin and PTH have effects on Mg ^ uptake similar to glucagon and AVP. 2  Under the same experimental conditions described in section III.3 and III.4, maximal concentrations of calcitonin [10 " M] and PTH [10 M] stimulate Mg uptake 49±5% and 12±2%, respectively, above 7  7  2+  control levels (Fig. 16). II.6  Glucagon- and A VP-stimulated Mg entry does not require protein synthesis. 2+  Gesek and Friedman have reported that PTH-stimulated calcium uptake in MDCT cells is sensitive to cycloheximide (1 ug/min for 30 min) whereas calcitonin actions are insensitive to protein synthesis inhibitors [41,108]. Accordingly, there may be diverse pathways involved with hormonal regulation of magnesium transport. Pretreatment of MDCT cells with cycloheximide, 1 ug/ml for 30 min, did not alter glucagon or AVP stimulation of Mg entry into MDCT cells (Table 5). It is apparent thatcfe novo protein synthesis is 24  not required for the acute actions of glucagon or AVP on magnesium uptake.  47  Fig. 15.  Concentration-dependence of AVP stimulation of Mg uptake in MDCT cells. The rate of Mg influx as determined by d([Mg ])/dt was measured with the given AVP concentrations using techniques performed according to that given in legend to Figure *. The entry rate, d([Mg ]j)/dt, was determined over 500 s, 5 min after the addition of AVP. Values are means ± SE for 3-6 cells [87]. 2+  2+  2+  f  2+  48  Table 5 . Role of protein synthesis on glucagon or AVP stimulated Mg uptake in MDCT Cells 2+  Control, nM/s  Cyloheximide, nM/s  Control  164 ±5 (6)  163 ±6 (3)  Glucagon  196 ±11 (4)  197 ± 11 (3)  AVP  189 ±6 (6)  192 ±2 (4)  Table 5 Legend  Values are mean ± SE; number of experiments is in parentheses. Mg entry was determined in presence of 10" M Glucagon, or 3 x 10 M AVP, with and without pretreatment with 10~ M cycloheximide. Protein synthesis inhibitor was applied 30 min prior to fluorescence studies. * P < 0.05, significant for cycloheximide vs. control values for each of the respective hormone treatments [87]. 2+  7  7  III.7  8  Exogenous cAMP stimulates Mg uptake in MDCT cells. 2+  Next, I determined if activation of either protein kinase A or protein kinase C may have stimulated Mg entry in Mg -depleted MDCT cells. Friedmane/ al. and Hilal et al. have shown that activation of both 2+  2+  pathways are necessary for PTH-mediated increases in calcium uptake in MDCT cells and distal tubule vesicles, respectively [109,111]. The activation of these kinases with added cAMP orphorbol esters were without effect alone but significantly stimulated calcium entry when administered together [110]. In the present studies, the addition of the long lasting cAMP analog 8-bromo cAMP, 10" M, 6 min prior to the 4  concentration determinations stimulated Mg uptake by 137±6% above control values (Fig. 17). There was 2+  an apparent latent period prior to the effects of 8-bromo cAMP of about 5-10 min, not unlike that observed for the responses of AVP. The addition of PMA, on the other hand, had no apparent effect on Mg  2+  49  Fig 16. Summary of hormone induced stimulation of Mg uptake and cAMP generation in MDCT cells. cAMP and Mg uptake studies were performed as previously described. *P < 0.05, significant vs. control values for Mg uptake. + P < 0.05, significant vs. control values for cAMP generation [87]. 2+  2+  2+  50  entry at any time (Fig. 17). Moreover, cAMP, 10" M, stimulated Mg uptake in the presence of the PMA. 4  2+  I infer from these studies that hormone-stimulated Mg"' uptake may involve intracellular cAMP accumulation. 2 1  III. 8  The effect ofPKA and PKC inhibition on hormone-stimulated Mg * uptake. 2  Finally, I determined the effect of protein kinase A and protein kinase C inhibition on hormonestimulated Mg uptake. Rp-cAMP, a protein kinase A inhibitor was applied 5 min prior to Mg uptake 2+  2+  measurements. Rp-cAMP abolished the effects of glucagon and AVP (Table 6) suggesting that activation of protein kinase A is involved with hormone actions. Ro31-8220, an inhibitor of protein kinase C, was without effect supporting the theory that this signalling pathway is not involved with hormone stimulation of Mg uptake. 2+  Table 6. Glucagon and AVP act through PKA-mediated pathway Control, nM/s  Glucagon, nM/sAVP, nM/s  Control  164 ±5 (6)  196±11* (4)  189 ±6* (6)  Rp-cAMPS  171 ±3 (3)  176 ±5 (3)  177 ±8 (3)  Ro31-8220  167 ± 10(3)  201± 17* (3)  197 ±7* (3)  Table 6 Legend  Values are mean ± SE; number of experiments is in parentheses. Mg entry was determined in presence of 10" M Glucagon, or 3 x 10" M AVP, with and without a 5 min. pretreatment with either the PKA inhibitor, RpcAMPS (10" M) or the PKC inhibitor, Ro31 -8220 (10 M). * P < 0.05, significantly different from control values [87]. 2+  7  7  6  6  51  Fig. 17.  Exogenous cAMP stimulates Mg entry in MDCT cells. Either 8-bromo cAMP, 10 M, orphorbol 12-myristate 14-acetate(PMA), 10" M, were added 6 min prior to determination of d([Mg ]i)/dt with the techniques illustrated in Fig. 1. Values are means ± SE for 3-6 cells and * indicates significance, p<0.01, from control values [87]. 2+  4  7  2+  52  Fig 18.  The phospholipase inhibitor U73122 inhibits glucagon and calcitonin stimulated Mg uptake. U73122 (15 /JM) was added 1 0 min prior to hormone addition. Techniques where as previously describe. Values are means ± SE for 3-6 cells and * indicates significance, p < 0 . 0 1 , from control values [87]. +  53  III.9  Glucagon and calcitonin stimulated Mg ^ uptake is inhibited with phospholipase-C inhibition. 2  It is clear that cAMP production is related to hormonal stimulation and Mg^uptake. However, cAMP +  levels do not always correlate directly with the increase in uptake. Glucagon and calcitonin are both known to alter phosphoinositide metabolism [109] in addition to their effects on cAMP. To determine if phospholipase-C (PLC) was involved in increasing Mg"uptake, the PLC inhibitor, U-73122 (15/JV1), was 24  added 16 min prior to glucagon and calcitonin (10" M) addition and uptake measurements. Glucagon- and 7  calcitonin- stimulated magnesium uptake was inhibited with the phospholipase C inhibitor U-73122. In a separate experiment PTH stimulated Mg uptake was also inhibited by U-73122 (data not shown). 2+  IV  Discussion  Micropuncture studies clearly indicated that glucagon stimulates magnesium transport in the superficial distal tubule [88]. This segment is comprised of the distal convoluted tubule, connecting tubule and initial cortical collecting tubule. Prior to the present studies, information was not available concerning which distal segment was involved with hormone-stimulated distal tubular magnesium reabsorption. Of the segments comprising the distal tubule, evidence from the present studies clearly indicate that the convoluted portion is involved in glucagon-induced magnesium conservation. Indeed, it may be inferred that glucagon enhances active Mg transport in the distal convoluted tubule. Cellular mechanisms of magnesium transport in the 2+  other distal segments, connecting tubule and initial collecting tubule, have not been studied. AVP has been shown to be an effective magnesium-conserving hormone in anaesthetized and conscious hormone-deprived rats [90,91,111], Micropuncture studies of these animals have shown that AVP actions occur principally within Henle's loop [92,111 ]. Using microperfusion studies, Wittner and Di Stefano demonstrated that AVP enhances magnesium absorption in mouse thick ascending limbs through changes in passive transport commensurate with increases in transepithelial voltage [112]. In the micropuncture studies  54  with hormone-deprived rats, Elalouf and colleagues failed to discern any change in fractional magnesium absorption in the superficial distal tubule following physiological administration of AVP [111]. In these studies, Elalouf et al. reported that the fractional calcium absorption increased significantlyfrom42.0±5.8% to 62.8±7.1% whereas AVP increasedfractionalmagnesium transportfrom45.5±7.8% to 55.3±15.5% but this change was not statistically significant [111]. Costanzo and Windhager did not observe any change in calcium absorption in the microperfused rat distal tubule with administration of AVP [93] The animals used in this latter study were TPTXed but not hormone-deprived as were those used by Elaloufet al. [111]. In both studies, AVP enhanced sodium absorption in the distal tubule. In the present studies, AVP increased Mg  2+  entry into an established distal convoluted tubule (MDCT) cell line suggesting that this hormone acts within this segment of the distal tubule and is involved with renal magnesium conservation. Magnesium transport in the distal convoluted tubule is probably transcellular and active in nature because of the high resistance and lumen negative transepithelial voltage of this segment [74]. Accordingly, AVP likely stimulates active Mg  2+  transport in contrast to enhancing passive Mg absorption in the thick ascending limb [112]. 2+  Interestingly, AVP stimulates Mg entry 5-10 min following addition of the hormone. This 2+  pronounced latency period was not observed with glucagon stimulation of Mg uptake. The reasons for these 2+  differences are not known. Friedman and Gesek observed similar differences in action of PTH and calcitonin stimulation of Ca uptake in MDCT cells [41,60]. There was a latency period of 10-15 min prior to PTH2+  stimulation of Ca entry compared to calcitonin actions which occurred immediately upon addition ofthe 2+  hormone [41,60]. Although the basis for this latency period is not known, it may have something to do with translational processes and protein synthesis because Friedman and Gesek showed that incubation of MDCT cells with cycloheximide for 15 min inhibited PTH-stimulated Ca entry but had no effect on calcitonin 2+  actions [41,60]. Furthermore, treatment of the cells with l,25-(OH) D shortened the latency period 2  3  associated with PTH-stimulated increases of [Ca ] and Ca entry but had no effect on Ca entry by itself 2+  2+  (  2+  55 [113]. These workers were unable to explain the origin of the latency period but concluded that it involved transcription because 5,-6-dichloro-l-[3-D-ribofuranosyl benzimidazole blocked the stimulatory response of 1,25-(OH) D without inhibiting PTH actions [41]. Gesek and Friedman speculated that vitamin D may 2  3  increase the number of PTH receptors expressed in the MDCT cells [113]. These investigators were unable to show hormone stimulated calcium entry with either AVP or glucagon [107]. In the present studies, pretreatment of MDCT cells with cycloheximide did not inhibit either AVP- or glucagon- stimulated Mg  2+  uptake nor did it affect the latent period associated with AVP. It is apparent from the above mentioned studies that peptide hormones act through different mechanisms to stimulate divalent cation transport in distal convoluted tubules. Further studies are needed to explain these diverse observations but the hormonal control of calcium and magnesium in the distal convoluted tubule may involve unique intracellular signalling pathways. Recent studies by Gesek and Friedman indicate that PTH-mediated stimulation of calcium transport is through intermediary pathways involving activation of both protein kinase A and protein kinase C as cAMP or phorbol esters had no effect on Ca entry alone but together they enhanced uptake [109]. These studies 2+  are consistent with the observations of Hilale^ al. using isolated membrane vesicles from rabbit distal tubules [111]. Moreover, Lajeunesse et al. have shown that Ca uptake in apical membrane vesicles harvested 45  2+  from rabbits pretreated with PTH is greater than those from control animals [114]. Accordingly, PTH may have additional effects on the apical membrane to cause an increase in Ca uptake. In the present studies, 2+  I show that addition of 8-bromo cAMP stimulates Mg* uptake but PMA does not increase entry rates +  (Fig. 17). I infer that activation of protein kinase A is involved in glucagon and/or AVP actions. However, it is not known how activation of protein kinase A stimulates Mg uptake nor what other pathways may be 2+  involved. On balance, the differences in time-frame of hormone action and the disparity of cAMP synthesis suggests that glucagon and AVP may operate through separate but interactive intracellular signalling pathways. More research is required to sort out these intracellular controlling mechanisms.  56 In summary, glucagon, AVP, calcitonin, and PTH stimulate Mg" uptake into MDCT cells, supporting 9  the results of micropuncture reports that these hormones may act within the convoluted segment ofthe distal tubule [88,112,115,116,117]. The cellular mechanisms by which these hormones stimulate uptake are unclear; however, the involvement of signalling pathways influenced by phospholipase-C and protein kinase-A is clear. The notable difference in hormone responses include the time-frame of action suggesting different intracellular pathways are involved with stimulation of Mg uptake. Further studies are required to determine 24  the intracellular signalling pathway mediated by these hormones.  57 Chapter IV. Inhibition of Hormone Stimulated Mg  2+  Uptake with Activation of the Polyvalent Cation-Sensing Mechanism.  I. Background In chapter 1 I show that activation of the polycation-sensitive mechanism with either extracellular Mg or Ca inhibited PTH-, calcitonin-, glucagon- and AVP-stimulated cAMP synthesis in MDCT cells 2+  2+  [44]. In Chapter 3 I demonstrated the same hormones stimulate Mg uptake. In the present study, I show 2+  that activation of Mg7Ca -sensing in MDCT cells inhibits hormone-stimulated Mg entry in these cells [75]. 2  2+  24  II Methods and Materials: As previously described in chapter 1. III Results III. 1  Activation of the Mg /Ca ~ -sensing mechanism inhibits hormone-stimulated cAMP synthesis. 2+  2  >  Table 7 summarizes the effects of activation of the Mg 7Ca -sensing mechanism on hormone2  2+  stimulated cAMP accumulation in normal MDCT cells. Glucagon, 10 M, and AVP, 10 M, increased 7  8  cellular cAMP accumulation from control values of 19±1 to 105±5 and 71±2 pmol/mg protein*5 min, respectively. Addition of neomycin, 50 uM, 5 min prior to the cAMP determinations abolished hormonestimulated cAMP synthesis (Table 7). Similarly, 10 mM [Mg ] or 10 mM [Ca ] completely abolished 2+  2+  0  0  glucagon- and AVP-dependent cAMP accumulation. Gd , Ni , Ba , and La also inhibited glucagon3+  2+  2+  3+  stimulated cAMP formation indicating that polyvalent cation-sensing in MDCT cells is similar to that observed in the parathyroid gland [4,8,54,75]. Next, I determined the concentration-dependence of extracellular Mg or Ca inhibition on glucagon24  2+  stimulated cAMP synthesis. Fig. 19 summarizes these results. The concentration of extracellular Mg required for half-maximal inhibition was about 1.5 mM and glucagon-stimulated cAMP synthesis was  2+  58  completely inhibited at about 2.5 m M M g C l . These studies were performed with normal Ci concentration, +  2  1.0 m M , in the bathing solution. Extracellular Ci was less potent, as it maximally inhibited hormone-related +  c A M P synthesis at 5.0 m M with a half-maximal inhibition at about 3.0 m M . Accordingly, the M g / C a 2+  2+  sensing mechanism in M D C T cells is responsive within the physiological concentration range of these divalent cations and the potencies of extracellular M g  2 +  and C a  2+  are almost equivalent. It is of interest that these  results were obtained in the presence of normal extracellular concentrations of either 1.0 m M C a , or 0.5 2+  m M M g , in the respective experiments. 2 +  III.2  Activation of Mg /Ca -sensing diminishes hormone-stimulated Mg 2+  2+  2+  uptake into  Mg 2+  depleted MDCT cells. Using our M D C T cell model Mg^-depleted M D C T cells were placed in a bathing solution containing 1.5 m M M g C l . The [Mg ] increased with time and plateaued at 0.52±0.06 mM,(n = 9) which was 2+  2  (  consistent with previous results [61]. The mean rate of refill, d([Mg ];)/dt, measured as the change in 2+  [Mg ] with time, was 164±5 nM/s, (n = 6) experiments, as determined over the first 500 s following addition 2+  ;  of magnesium (Fig. 20).  59 T a b l e 7.  A c t i v a t i o n of M g 7 C a - s e n s i n g inhibits h o r m o n e - s t i m u l a t e d c A M P a c c u m u l a t i o n . 2  2+  Hormone Glucagon  Control (None)  AVP  cAMP accumulation (pmol/mg protein«5min) (11)  105±5 (7)  71±2 (5)  22±2 (3)  22±l (7)  24±3 (4)  24±3 (3)  37±3 (3)  27±3 (3)  23±2 (3)  32±2 (3)  34±3 (3)  22±1 (5)  34±l (5)  34±2 (5)  0  25±1 (5)  41±3 (5)  34±2 (5)  0  24±1 (5)  39±2 (5)  33±l (5)  24±0.3 (5)  32±l (5)  27±l (5)  none  19±1  Neomycin Mg  2+  Ca  2+  Gd  3 +  Ni  0  0  0  2 +  Ba  2+  La „ 3+  a  a  b  b  b  ab  ab  ab  ab  ab  ab  ab  ab  ab  ab  ab  Table 7 Legend  Where indicated, neomycin, 50 uM, or extracellular Mg ,10 mM, Ca ,10 mM, Gd ,0.5 mM, Ni ,0.5 mM, Ba , 0.5 mM or L a , 0.5 mM, were added 5 min prior to the addition of either glucagon, 10 M , or arginine vasopressin, 10~ M , and cAMP was measured 5 min after addition of the respective hormones. indicates significance, p<0.01, of hormone-treated versus values without hormone treatment and indicates significance, p<0.01, of hormone-treated values in the presence of neomycin or Ca or M g versus hormonetreated values in the absence of pretreatment with the polyvalent cations. () is the number of observations [75]. 2+  2+  0  2+  3+  0  2+  0  0  3+  0  7  0  8  a  b  24  24  60  IOO -  A  * <  80  60  40  20  0 J  0  0.1  0.5  1.5  2.5  5.0  1.5  2.5  5.0  [Ca I , mM ;  120  .  B  0  0.1  0.5  [MgToIIlM  Fig. 19. Concentration-dependence of extracellular M g or Ca inhibition of glucagon-stimulated cAMP. MDCT cells were cultured in DMEM/Ham's F-12 (1:1) with 0.2% BSA containing 0.6 m M magnesium and 1.5 m M calcium. At the time of experimentation, the cells were washed with a buffer solution containing (in mM): MgCl 0.5, CaCl 1.0, NaCl 145, KC14.0, K H P 0 0.8, K H P 0 0.2, glucose 5, and HEPES-Tris 20, pH 7.4. Panel A.To test [Ca ] , the cells were initially bathed with the above solutions containing no CaCl . This was replaced with one containing the indicated concentrations. Five minutes following the addition of either M g C l or CaCl , glucagon, 10 M, was added and cAMP measured following a 5 min incubation period. Panel B. In order to test the effect of [Mg ] , the buffer solution was changed to identical to the above but without MgCl . This bathing solution was replaced 10 min later with one containing the indicated M g C l concentrations. Values are means ± SE for 2-3 experiments consisting of 5 individual observations each and * indicates significance, p<0.01, from control values (set to 100%) [75]. 2+  2  2+  2  2  4  2  2+  0  2  7  2  2  2+  0  2  2  4  61 Previously reported data that indicates the Mg uptake is concentration-dependent and selective for 2+  Mg [61]. I have further shown that glucagon and AVP stimulates Mg entry into Mg^-depleted MDCT 2+  2+  cells by 15-20% over basal entry rates [49]. Glucagon and AVP stimulate Mg entry without changes in 2+  transmembrane voltage, -64.7±0.9 mV, (n = 5)[49]. As neomycin stimulates the Ca -SR neomycin was used to activate the Mg 7Ca -SR [4,44]. 2+  2  2+  Neomycin was added about 5 min prior to the addition of 1.5 mM MgCl for measurement of Mg° uptake. +  2  However, the addition of 50 uM neomycin to the extracellular buffer solution had no effect on basal Mg  2+  uptake, (155±5 nM/s,w = 4), into Mg -depleted MDCT cells (Fig. 20). However, pretreatment of cells with 2+  neomycin inhibited glucagon-stimulated Mg entry. The mean Mg uptake rate of glucagon-treated cells 2+  2+  was 196±11 nM/s, (n = 5) and pretreatment with neomycin, 50 uM, diminished uptake to 162±3 nM/s/« = 3) that was not different from control uptake rates (Fig. 21). Accordingly, activation of Mg 7Ca -sensing 2  2+  mechanism with neomycin inhibits glucagon-stimulated Mg entry. 50 uM Neomycin, does not alter the 2+  membrane voltage either without or with glucagon, (-61.3±1.1 mV vs. -62.0±2.3 mV, n=3. Activation ofthe Mg 7Ca -sensing mechanism also inhibits AVP- stimulated Mg uptake (data not shown). Addition of 2  2+  2+  neomycin 5 min prior to the application of AVP, 3 x 10" M, diminished Mg uptake from 189±6 to 163±4 7  2+  nM/s, n = 3. Next, 1 tested whether elevated extracellular Ca may affect Mg entry. I have previously 2+  2+  shown that addition of 10 mM extracellular Ca does not alter basal Mg entry into Mg -depleted MDCT 2+  2+  2+  cells [61]. Extracellular Ca was added 5 min prior to measurement of Mg uptake. As with neomycin 2+  2+  [Ca ] ,5 mM, did not change basal Mg uptake rates but inhibited glucagon-stimulated Mg entry (Fig. 21). 2+  24  24  0  As with extracellular Ca , large concentrations of extracellular Mg inhibited glucagon-stimulated Mg 2+  2+  2+  uptake into MDCT cells (Fig. 21). It should be kept in mind that Mg uptake rate, d([Mg ])/dt, appears to 2+  2+  ;  saturate at about 5 mM [61]. The basal [Mg ]; refill rate was 164 ± 5 nM/s in the presence of buffer 2+  containing no calcium and 5.0 mM MgCl . Glucagon failed to stimulate Mg uptake, 154 ± 6 nM/s, 2+  2  62  . 20. Activation of the Mg /Ca -sensing mechanism diminishes hormone-stimulated Mg uptake into MDCT cells. Intracellular magnesium concentration, [Mg ]) was determined with mag-fura-2. Cells were cultured in media containing no magnesium (magnesium concentration <0.01 mM) for 16 hr. The basal [Mg ] was determined and the cells were subsequently placed in buffer solution containing 1.5 mMMgCl atthe time indicated. The buffer solutions contained (in mM): 145NaCl, 4.0 KC1, 0.8 K HP0 , 0.2 KPLPO,, 1.0 CaCl , 5.0 glucose, and 10 HEPES/Tris, pH 7.4, with and without 1.5 mM MgCl . Where indicated 50 uM neomycin was added 5 min prior to the addition of hormone and 1.5 mM MgCl . Fluorescence was measured at 1 data point/s with 25 signal-point averaging and smoothed according to methods previously reported (10). The Mg uptake, d([[Mg ]j)/dt, was 196 nM/s with glucagon and 165 nM/s in the presence of neomycin. These fluorescence tracings are representative of 9 cells [75]. 2+  2+  24  24  2+  ;  2  2  4  2  2  2  2+  63  220  Glu  Glu.  Fig. 21. Summary of the effects of Mg 7Ca -sensing mechanism activation on hormone-stimulated M g uptake. M g uptake, (d[Mg ]j)/dt, was determined with 1.5 m M extracellular M g in the absence and presence of neomycin, 50 u M , Ca , 5.0 m M , M g , 5.0 m M , as indicated. Neomycin, C a or M g was added 5 min prior to the addition of glucagon, 1 0 M , and M g C l , 1.5 m M . The studies were performed as given in legend to Fig. 2. The Mg " uptake rate was determined over 500 s following addition of glucagon. Values are mean± SE for 3-5 cells. * indicates significance (p<0.05) from control values [75]. 2  2 +  2+  2 +  2+  2 +  2+  2 +  2+  2 +  7  2  24  64 when determined in the presence of 5.0 mM MgCl . These results are consistent with the effects of the 2  polyvalent cations on hormone-mediated cAMP accumulation (Table 7 and Fig. 19). Next, I measured the glucagon-stimulated Mg uptake rate, at concentrations below that which 2+  produces saturation of d([Mg ],)/dt. This was also observed in the presence of glucagon (Fig. 22). However, 2+  uptake rates below this value were increased so that the hormone-stimulated d([Mg^];)/dt was dependent +  on extracellular Mg demonstrating greater fractional transport rates with the lower extracellular magnesium 2+  concentrations used to perform the uptake measurements (Fig. 22, insert). Arthur has recently shown that activation of the calcium-sensing receptor of Madin-Darby canine kidney (MDCK) cells, another distal tubule cell line, with high extracellular calcium, inhibited basal transepithelial calcium transport after 30 min but not after 5 min [96]. I have tested whether the addition of neomycin, 50 uM, for 30 min, might inhibit basal Mg uptake in MDCT cells. The mean uptake rate, 2+  d([Mg ]j)/dt, was 153±10, n = 6 following a 30 min incubation with neomycin (Fig. 23). Accordingly, in 2+  MDCT cells, I have no evidence that activation of Mg 7Ca -sensing inhibits basal Mg uptake. Further 2  2+  2+  studies are necessary to determine if this conclusion is valid for transepithelial Mg transport. 2+  III.3  Activation of Mg /Ca -sensing does not affect cAMP stimulation of Mg ^ uptake. 2+  2+  2  From chapter 3 I have shown that the addition of exogenous 8-bromo cAMP stimulates Mg entry 2+  into MDCT cells [49]. Furthermore, protein kinase A inhibition diminishes hormone-stimulated Mg uptake 2+  [49]. Accordingly, glucagon and AVP act, in part, through a cAMP-dependent pathway.  65  . 22. Glucagon-stimulation of Mg uptake is dependent on the concentration of extracellular Mg used to determine d([Mg ];)/dt. Mg uptake was measured with and without glucagon, 10" M, in the presence of the extracellular MgCl concentrations as indicated. The insert shows the change of hormone-stimulated Mg uptake as a function of extracellular Mg concentration used to perform the refill studies. Values are means ± SE for 3-6 observations and * indicates significance, p<0.05, from control values not treated with glucagon [75]. 2+  2+  2+  2+  7  2  2+  2+  66  Control  Neomycin  cAMP  cAMP + Neomycin  Forskolin Forskolin + Neomycin  .23. Activation ofMg^/Ca * sensing does not alter cAMP-mediated M g uptake. Either forskolin, 1 u M , to generate endogenous cAMP synthesis or 8-bromo cAMP, 10" M , was added 6 min prior to determination of d([Mg ] )/dt with micro fluorescence according to the techniques illustrated in Fig. 3. Neomycin, 50 p M , was added 5 min prior to the addition of forskolin or 8-bromo cAMP. Values are means ± SE for 3-6 cells and * indicates significance, p<0.01, from control values [75]. 2  2 +  4  2+  i  67  In order to determine if activation of the Mg 7Ca -sensing mechanism affects cAMP-mediated processes, 2  2+  I pretreated cells with neomycin to activate the receptor then added 8-bromo cAMP and measured Mg  2+  uptake rate. Fig. 23 summarizes these experiments. 8-bromo cAMP stimulated Mg regardless of the 2+  presence of presence of neomycin. Forskolin stimulates intracellular cAMP production in the presence of activation of Mg /Ca -sensing [73,107]. Accordingly, I performed Mg uptake studies in the presence of 2+  2+  2+  forskolin and neomycin to test whether forskolin alters transport despite activation of Mg 7Ca -sensing. 2  2+  Again, forskolin stimulated Mg"" uptake in the presence of activation of Mg /Ca -sensing by neomycin. 2 1  2+  2+  These studies suggest that activation of the Mg7Ca -sensing mechanism inhibits hormone-stimulated Mg 2  2+  2+  uptake by inhibiting hormone-mediated cAMP generation. III. 4  Activation of Mg /Ca -sensing does not inhibit amiloride-stimulated Mg * uptake. I have 2+  2+  2  shown that amiloride stimulates Mg uptake rates by about 30-40% above basal levels in MDCT cells. 2+  Amiloride hyperpolarizes the plasma membrane by -28±8 mV, thereby creating a more favourable electrical gradient for Mg entry [61]. I pretreated MDCT cells with neomycin, then determined the effect of 2+  amiloride on Mg uptake. Amiloride increased Mg entry, 221±12 nM/s, in the presence of neomycin 2+  2+  indicating that the activation of the Mg 7Ca -sensing mechanism does not alter the cellular 2  2+  actions of this magnesium- conserving diuretic (Fig. 24). The increase in membrane voltage, from basal levels, -65.5±2.0 mV, normally observed with amiloride,-75.3±l .8 mV, was also not altered by neomycin, 74.7±1.2mV, n=5.  IV. Discussion  Activation of a Mg7Ca -sensing mechanism elicits cytosolic Ca signals and diminishes hormone2  2+  2+  mediated intracellular cAMP accumulation (as shown in Chapter 1 & 3) [44]. In the present  68  . 24. Activation of Mg 7Ca sensing does not alter amiloride-stimulated Mg uptake. Mg uptake, d([Mg ] )/dt, was performed in the presence of 1.5 mM MgCl, and with or without 10 uM amiloride. Neomycin, 50 uM, was added where indicated 5 min prior to the addition of amiloride. Values are means ± SE for 3-6 cells and * indicates significance, p<0.01, from control values [75]. 2  2+  i  2+  2+  2+  69 study, I show that activation of this Mg 7Ca -sensing mechanism also inhibits glucagon- and AVP2  2+  stimulated Mg uptake into MDCT cells [75]. As this cell line possesses many of the transport properties 2+  of the intact distal convoluted tubule, I conclude that the Mg 7Ca -sensing mechanism plays an important 2  role in control of magnesium transport in this nephron segment.  2+  70  Chapter V. Mineralocorticoid Hormones Stimulate Hormone Mediated Mg * uptake in MDCT cells. 2  I. Background  Cells of the distal convoluted segment possess mineralocorticoid receptors [ 126-129]. Although there is some dispute concerning their functional purpose within this segment [130, 131], there is evidence that mineralocorticoid hormones enhance both Na conductance and NaCl co-transport in distal convoluted tubule +  cells [ 13 2,13 3 ]. Accordingly, mineralocorticoid may alter Mg entry by acting through changes in other ion 2+  transports [61]. Alternatively, mineralocorticoids may affect hormone-mediated control of Mg transport 2+  within the distal tubule. Doucete/ al. have shown that adrenalectomy reduced glucagon-stimulated adenylate cyclase activity in the thick ascending limb; this reduced cAMP response was prevented with aldosterone administration [134]. Other investigators have shown that vasopressin-sensitive adenylate cyclase is diminished in rat and rabbit collecting tubules harvestedfromadrenalectomized animals [135,136,137,138]. Accordingly, mineralocorticoids could alter hormonal control of transport in the distal tubule through their actions on receptor-mediated signalling pathways. Using the MDCT cell model I sought to determine the effect of the steroid hormones on Mg  2+  transport and the interactions with hormone-stimulated Mg uptake [139]. In the previous studies, it was 2+  shown that aldosterone potentiates glucagon- and AVP-stimulated intracellular cAMP accumulation and Mg" 24  uptake in MDCT cells. The potentiation of hormone actions by mineralocorticoids may have significant effects on renal magnesium balance.  II. Materials and Methods: As previously described in chapter 1.  71 Results  In the previous chapter I have showed that glucagon and AVP stimulate Mg uptake into Mg 2+  2+  depleted MDCT cells in a concentration-dependent fashion [87]. Maximal concentration of glucagon, 10" 6  M, and AVP, 3 x 10" M, enhanced mean Mg uptake rates by 273±6 and 189±6 nM/s, respectively. 7  2+  Hormone-stimulated uptake was inhibited by nifedipine and was not affected by pretreatment with the protein synthesis inhibitor, cycloheximide. It was concluded that hormonal actions were likely through the activation of putative Mg channels that is independent of de novo protein synthesis[87]. 2+  III. 1  Aldosterone potentiates hormone-stimulated Mg * uptake 2  The addition of 10" M aldosterone, 20 min prior to the measurement of Mg entry did not alter the 7  2+  rate of Mg uptake (189±21 nM/s, w = 3) vs control. Incubation of MDCT cells with aldosterone, 10" M, 2+  7  for 16 hr prior to determination of Mg entry also had no effect on basal Mg uptake, (172±8 nM/s/i = 3). 2+  2+  However, pretreatment of MDCT cells with aldosterone potentiated both glucagon- and AVP-stimulated Mg uptake rates (Fig. 25). Hormone-stimulated Mg^-refill was enhanced with aldosterone but aldosterone 2+  treatment had no effect on thefinal[Mg ]j attained following refill; this first concentration was not different 2+  from control values, 0.52 mM. Furthermore, aldosterone potentiated glucagon and AVP at all hormone concentrations above 10" M (Figs. 26 and 27). The glucagon concentration required to produce half-maximal 9  stimulation of Mg uptake in aldosterone-treated cells was about 5 x 10~ M which was not different from 2+  7  control cells. Unlike glucagon, AVP stimulates Mg uptake after a latent period of 5-10 min (Fig.25B). 2+  Pretreatment of cells with aldosterone appeared to shorten the duration of this latent period (Fig.25B). The AVP concentration for half-maximal stimulated Mg uptake is not changed (Fig.27). 2+  Fig. 28 shows that aldosterone potentiated hormone-stimulated Mg uptake was sensitive to 2+  nifedipine, suggesting that Mg entry was being affected. 2+  72  Fig. 25. Aldosterone potentiates glucagon- and AVP- stimulated Mg uptake in Mg -depleted mouse distal convoluted tubule (MDCT) cells. Confluent MDCT cells were cultured in Mg -free media (<0.01 mM) for 16-20 hr. Uptake studies were performed in buffer solutions in the absence of Mg , and, as indicated, MgCl (1.5 mM final concentration) was added to observe changes in intracellular M§ concentration. The buffer solutions contained (in mM): 145 NaCl, 4.0 KC1, 0.8 K HP0 , 0.2 KH P0 , 1.0 CaCl , 5.0 glucose, and 10 HEPES/Tris, pH 7.4, with and without 1.5 mM MgCl . Panel A: glucagon, 10" M, and panel B: AVP, 3 x 10 M, were added to this buffer solution where indicated. Were indicated the MDCT cells were treated with aldosterone, 10" M, for 16 hr prior to analysis. Fluorescence was measured at 1 data point/s with 25-point signal averaging, and the tracing was smoothed as previously described [139]. 24  2+  2+  2+  +  2  2  2  4  2  4  2  7  7  7  73  [GLUCAGON] M  Fig. 26. Aldosterone potentiates glucagon-stimulated Mg entry in MDCT cells. MDCT cells were incubated with and without aldosterone, 10" M, for 16 hr prior to the determination of Mg entry. The rate of Mg"^ uptake, d([Mg ])/dt, was determined over the initial 500 s following addition of the hormone and MgCl . Glucagon was added at the concentrations indicated. Values without aldosterone are from ref 4. Values are means ± SE for 3-6 cells. * indicates significance, p<0.05, of Mg uptake with glucagon vs aldosterone + glucagon [139]. 2+  7  2  2+  i  2  2+  2+  74  [AVP] M  . 27. Aldosterone potentiates AVP-stimulated Mg"" uptake in MDCT cells. Magnesium uptake determinations were performed as given in legend to Fig. 1. AVP was added at the concentrations indicated. The cells were incubated with and without aldosterone, 10" M, for 16-20 hr prior to uptake measurements. Mg uptake rates were determined 6 min following addition of AVP. Tracings are representative of 4-6 cells [139]. 2 1  7  2+  75 111.2  Aldosterone potentiates hormone-stimulated cAMP accumulation in MDCT cells.  Evidence has been given that mineralocorticoids may increase the coupling efficiency of hormone receptors through G-proteins to adenylate cyclase [134]. Accordingly, I determined hormone-stimulated cAMP synthesis in MDCT cells pretreated with aldosterone. Aldosterone did not have any effect on basal cAMP concentrations in control cells (Table 7). However, aldosterone potentiated cAMP accumulation in response to glucagon and AVP. Aldosterone also enhanced cAMP synthesis by parathyroid hormone (PTH) and calcitonin; accordingly, potentiation of hormone-mediated cAMP production by mineralocorticoids may be a general phenomenon (Table 7). These results indicate that mineralocorticoids increases receptormediated cAMP synthesis which may provide the basis for potentiation of Mg uptake in MDCT cells. 2+  Aldosterone potentiates glucagon-stimulated cAMP synthesis in a concentration-dependent manner (Fig. 29). Incubation of cells for 16 hr with various aldosterone concentrations potentiated maximal glucagon responses with a half-maximal aldosterone concentration at about 10 M. 8  111.3  Aldosterone potentiates hormone-stimulated Mg* uptake and cAMP generation through de +  novo protein synthesis. As it has been well established that mineralocorticoids act, in part, through initiating protein production, I tested whether potentiation of hormone action may be through this process. In this study, the MDCT cells were depleted of Mg for 16 hr prior to the addition of aldosterone, with or without 2+  cycloheximide. The cells were incubated for a further 3 hr and uptake measurements were performed in the presence of 1.5 mM MgCl to determine Mg uptake. Mg uptake was 244±14 nM/s (Table 9) in the 2+  2+  2  presence of aldosterone plus glucagon. Addition of cycloheximide with aldosterone, 3 hr prior to uptake analysis resulted in the inhibition of aldosterone potentiation of glucagon-stimulated Mg entry (Table 7 & 2+  8). Cycloheximide did not have any effect on glucagon-stimulated Mg entry in the absence of 2+  mineralocorticoids (Table 8). These observations indicate that aldosterone may potentiate hormone actions through mechanisms dependent on de novo protein synthesis.  76 Next, I determined the effect of cycloheximide on aldosterone potentiation of hormone-stimulated cAMP accumulation. Again, the cells were Mg -depleted for 16 hr then treated with aldosterone, 10" M, 2+  7  with and without cycloheximide. Three hours later hormone-responsive cAMP synthesis was determined by radioimmunoassay measurements. Glucagon, 10" M, stimulated cAMP synthesis from 19±1 to 105±5 7  pmol/mg protein • 5 min (Table 8). Cycloheximide pretreatment did not alter cAMP generation. However, cycloheximide prevented the potentiation of aldosterone on glucagon induced cAMP synthesis (Table 9)  Table 8.  Aldosterone potentiates hormone-stimulation of cAMP synthesis (pmol/mg protein •5 min) in MDCT cells.  Glucagon  Calcitonin  PTH  AVP  Control: 19±1  105±5*  71±2*  56±2*  60±0.1*  (5)  (4)  (5)  (5)  (4)  26±4  251±32**  139±14**  153±26**  286±29**  (4)  (3)  (4)  (3)  (3)  Aldosterone  Table 8 Legend  The hormones, glucagon, 10" M, AVP, 10" M, parathyroid hormone (PTH), 10 M, and calcitonin 10" M, were added 5 min prior to cAMP determinations. Where indicated aldosterone, 10" M, was added to the culture media 16 hr prior to c AMP measurements (measured as pmol/mg protein • 5 min). Values are means ± SE for (n) number of experiments. *indicates significance, p<0.01, vs. control values and ** indicates significance of hormone vs. aldosterone + hormone for each of the respective hormones [139]. 7  8  7  7  7  77 Role of protein synthesis in glucagon-stimulated Mg uptake and cAMP synthesis 2+  Table 9.  in MDCT cells.  d([Mg ]j)/dt  cAMP synthesis  2+  Control  Cycloheximide  Control  Cycloheximide  nM/s  nM/s  Control  164±5  163±6  19±1  26±7  («) Glucagon  (6)  (3)  (5)  (3)  196±11*  197±11*  105±5*  98±6*  (3)  (3)  (4)  (3)  244±14*  195±18*(**)  251±32*  113±14*(£-£-)  (6)  (5)  (3)  (4)  (") Aldosterone +  pmol/mg protein • 5 min  Glucagon (n)  Table 9 Legend  Mg entry, d([Mg ];)/dt, and cellular cAMP accumulation were determined with and without the presence of aldosterone, 10" M for 3 hr and with the addition of glucagon, 10" M, with and without pretreatment with cycloheximide, 1 ug/ml, as indicated. The protein synthesis inhibitor was applied with the aldosterone 3 hr prior to uptake studies or measurement of cAMP accumulation, n is the number of observations and * indicates significance, p<0.05, of hormone vs control values. ("&"&) indicates significance, p<0.05, of cycloheximide vs control values for each of the hormone treatments [139]. 2+  2+  7  7  78  300  I  I  + Nifedipine  +  +  240  * 180  ja  120  a iSf  60  Control  Glucagon  AVP  Aldo.& Glu.  Aldo.& AVP  28. Aldosterone potentiation of hormone-stimulated M g entry is through dihydropyridine-sensitive pathways. MDCT cells were treated with aldosterone, 10" M , for 16 hr prior to determining hormone-responsive M g uptake. Nifedipine, 10 u M , was added with the 1.5 m M MgCl refill solution. Values are means ±3-6 cells. * indicates significance from control values and + indicates significance of aldosterone plus hormone vs the respective glucagon or AVP values [139]. 2+  7  2 +  2  79  ISO 140 130 -  [ALDOSTERONE] M  Fig. 29. Aldosterone potentiates glucagon-stimulated intracellular cAMP accumulation in a concentrationdependent manner. MDCT cells were incubated for 16 hr in magnesium-free buffer solution containing the indicated aldosterone concentrations. Glucagon, 10" M, was added and cAMP was measured following 5 min in the presence of IBMX. Values are mean ± SE for 3-5 observations. * indicates significance, p<0.05, from control values [139]. 7  80  Fig. 30. M g entry, d([Mg ]j)/dt was determined with and without the presence of aldosterone, 10" M for 3 hr, and with or without the addition of glucagon ( 1 0 M) or neomycin (50/iM). Ca and M g were altered as shown. Values are mean± SE for 3-5 observations. * indicates significance, p<0.05, from control values. 2+  2+  7  7  2+  2+  81  III. 4  Stimulation of the M^/Ca^-SR  inhibits Aldosterone stimulated Mg* uptake. Finally I tested +  if stimulation of the Mg 7Ca -SR had any affect on Mg"" uptake. Addition of 50/iM neomycin inhibited Mg"" 2  2+  2 1  2 1  uptake stimulated with both 10"M aldosterone and 10"M glucagon (Fig.30). 7  7  IV. Discussion  In summary, these studies show that aldosterone potentiates glucagon- and AVP-stimulated Mg  2+  uptake into MDCT cells. In additional studies not shown we have further demonstrated that aldosterone potentiates PTH- and calcitonin-stimulated Mg uptake. Aldosterone's action is in part through potentiating 2+  hormone-stimulated cAMP formation. Important to the present study is the observation that activation of the Mg 7Ca sensing receptor inhibits this aldosterone response. 2  2+  82 Chapter VI. 1,25-Dihydroxyvitamin D stimulates Mg uptake into 2+  3  mouse distal convoluted tubule cells  I. Background  Vitamin D metabolites have important effects on mineral metabolism by their actions on epithelial transport. Although it is clear that the hormonally-active vitamin D metabolite, 1 a,25-dihydroxyvitamin D  3  [ 1,25(OH)D], increases calcium and magnesium absorption within the intestine, its actions within the kidney 2  3  are more complex [78,107,142,143]. 1,25(OH),D increases calbindin-D in distal tubule cells including the 3  28k  convoluted and connecting tubule and collecting duct cells [ 144-147]. This calcium-binding protein is thought to be involved in epithelial calcium transport [148-156]. Bindelsef al have shown that 1,25(OH)2D stimulates 3  transepithelial calcium absorption in primary rabbit connecting and cortical collecting tubule cells [157] whereas Friedman and Gesek have reported that it accelerates PTH-dependent calcium transport in immortalized mouse distal convoluted tubules (MDCT) cells [113]. Despite these cellular responses, the effects of l,25(OH) D on overall renal calcium excretion are equivocal [156,158-160]. The actions of 2  3  l,25(OH) D on renal magnesium absorption are also controversial [161-169]. 2  3  Although l,25(OH) D seems to increase calcium absorption within the distal tubule, there is no 2  3  evidence that it has any effect on distal magnesium transport [78,107,142,143]. To date, there are no experiments reporting the direct effects of vitamin D metabolites on cellular magnesium transport. In the present studies, I used our MDCT cell model to investigate the actions of 1,25(OH)D on Mg uptake rates. 2+  2  3  Friedman and Gesek have reported that, in MDCT cells, l,25(OF£) D accelerates PTH-dependent Ca 2  2+  3  uptake, suggesting that vitamin D receptors are present in this cell line [113]. Accordingly, these cells provide a useful model to determine the effects of vitamin D metabolites on magnesium handling in the distal convoluted tubule. The present studies demonstrate that 1,25(OH),D stimulates Mg uptake in MDCT cells, 2+  3  83 independently of PTH, providing evidence that it also affects magnesium absorption in the intact distal tubule[139].  IIMethods: As previously described in Chapter one.  III. Results  III.l  l,25(OH) D stimulates Mg * entry in MDCT cells. MDCT cells were treated with 10" M 2  2  7  3  l,25(OH) D for 16 h prior to experimentation. During this time they were incubated in media without 2  3  magnesium in order to deplete them of intracellular Mg [75]. The cells were then placed in 1.5 mMMgCl 2+  2  and the [Mg ]; monitored by fluorescence. Fig. 31 shows a typical experiment of MDCT cells pretreated 2+  with 1,25(OH)D compared to control cells. The rate of the entry in control cells, measured over the initial 2  3  500 s, was 164±5 nM/s, n=6 (Fig. 32,33). Pretreatment with 1,25(OH)D increased Mg uptake by 78 ±4 2+  2  3  % to 210 ±11 nM/s, n=5 (Fig. 32,33). l,25(OH) D increased Mg uptake in a concentration-dependent 2+  2  3  manner with half-maximal response, 185±3 nM/s, occurring at about 10"M (Fig. 32). 9  Acute treatment of MDCT cells for 20 min with l,25(OH)Dhad no effect on Mg uptake, 178±17 2+  2  3  nM/s, n=3, which suggested that the action of the vitamin Q metabolite required transcription and translation [170]. I tested this by incubating MDCT cells with 50 uM cycloheximide and the 10" M 1,25(OH)D3, for 7  2  16 h prior to uptake studies. l,25(OH) D did not stimulate Mg uptake, (155±18 nM/s, n=4), in 2+  2  3  cycloheximide treated cells supporting my initial hypothesis response (Fig. 33). This data is in accord with the observations of Friedman and Gesek who reported a significant acceleration of PTH-stimulated calcium uptake in these cells at 3 hours and maximal stimulation at 5 hours with l,25(OH) D [113]. 2  3  I have shown that the hormone-induced intracellular second messenger, cAMP, increases Mg entry +  into MDCT cells [75]; accordingly , I measured cAMP concentrations in cells in the presence of  84 1,25(OH)D. There was no difference in cAMP levels in the 1,25(OH)D-treated cells compared to control 2  3  2  3  (Fig. 33). Furthermore, treatment of cells with RpcAMPs, a protein kinase inhibitor, did not diminish 1,25(OH)D -stimulated Mg uptake, 210± 15 nM/s, n=4, indicating that cAMP-mediated pathways are not 2+  2  3  involved with 1,25(OH)D activity (Table 10). These studies indicate that 1,25(OH),D mediates its effects 2  3  3  independently of those involving protein kinase A. III.2  l,25(OH) D and PTH stimulate Mg * uptake by separate pathways. Our studies, as well as 2  2  3  Gesek and Friedman's, have shown that PTH stimulates cAMP formation in MDCT cells [41,65,75 & Fig. 16, chapter 3]. Evidence has also been provided that PTH enhances magnesium reabsorption in the distal tubule of the rat, hamster, dog, and MDCT cells (PTH, 10" M, increased Mg uptake from control levels of 7  2+  164±5 to 209±21 nM/s) [41,85,88,93,94]. The PTH induced uptake and cAMP generation are similar to the glucagon and calcitonin uptake described in chapter 3 (Fig. 18) in that they are inhibited by the protein kinase A inhibitor Rp-cAMPS and phospholipase C inhibitor U73122 (data not shown). In order to compare the effects of the steroid action that does not involve protein kinase A or protein kinase C pathways, I used PTH as a prototypical hormone that requires cAMP, in part, to elicit its responses. In order to determine if 1,25(OH)D and PTH act through common mechanisms, I used maximal concentrations of 1,25(OH)D3,10" 2  7  3  2  M, and PTH, 10" M, to test if they had additive responses. 7  l,25(OH) D + PTH increased cAMP formation to a similar extent as PTH alone (Fig. 34). 2  3  However, Mg uptake with 1,25(OH) D ,251±16 nM/s, was greater than either with l,25(OH)D , 210±11 2+  2  3  3  nM/s, or PTH, 209±9 nM/s alone. Accordingly, the steroid metabolite, 1,25(OH)D, and the peptide hormone, 3  PTH, seem to alter Mg entry rates by different intracellular pathways. Or, alternately, a single pathway 2+  can't be maximally stimulated by either l,25(OH) D or PTH. 2  3  85  0.6  0.5  1^5(OH) D 2  3  0.4  ^  0.3  0.2 1.5 m M M g  0.1  .31.1,25(OH) D stimulates Mg uptake in Mg -depleted mouse distal convoluted tubule (MDCT) cells. MDCT cells were cultured in Mg -free media (<0.0T mM) for 16 hr. Uptake studies were performed in buffer solutions in absence of external magnesium, and where indicated, MgCl (1.5 mM final concentration) was added to observe changes in intracellular Mg concentration, [Mg ] . The buffer solutions contained (in mM): 145 NaCl, 4.0 KC1, 0.8 K HP0 , 0.2 KH P0 , 1.0 CaCl , 5.0 glucose, and 10 HEPES/Tris, pH 7.4, with and without 1.5 mM MgCl . Where indicated, l,25(OFf) D , 10" M, was added to this buffer solution from a stock solutions. Fluorescence was measured at 1 data point/s with 25-point signal averaging, and the tracing was smoothed according to methods previously described . 2+  2  2+  3  2+  2  +  2+  f  2  4  2  2  7  2  3  4  2  86  250 -,  [1^5(OH) D ] M 2  3  .32. Concentration-dependence of l,25(OH)D-stimulation of Mg entry in MDCT cells. MDCT cells were treated with the given l,25(OH) D concentrations for 16 h prior to fluorescence determinations. The rate of Mg influx as determined by d([Mg ])/dt was measured with the given 1,25(OH)D concentrations using techniques performed according to that given in legend to Figure 34. d([Mg ]j)/dt values were determined over the first 500 s of fluorescence measurements. Values are means ± SE for 3-6 cells and * indicates significance, p<0.05, from control values. 2+  2  3  2  2+  3  2+  ;  2  3 2+  87  300  100  260  80  a  * 220  60  40  Qa  140  g IT) C  I I. sr. E o E S  20  100  Control  1^5(OH) D 2  3  Cyclo.+1^5(OH) D 2  3  33. Summary of l,25(OH) D -stimulated M g uptake in M D C T cells. The M D C T cells were treated with l,25(OH) D , 10" M , for 16 h prior to the determination of M g uptake or c A M P measurements. Mg * uptake was determined with microfluorescence according to methods given in legend to Fig. 34. In those cells indicated, cycloheximide, l/^g/ml, was added with the 1,25(OH)2D 16 h prior to M g uptake and c A M P determinations. Values are mean ±SE and * indicates significance from control values. 2 +  2  3  7  2  2 +  3  2  3  2 +  88  300  100  *• 260  80  s  * 60  220  £a E  +  + S  S i  180  40  140  20  a  <  100  Control  PTH  1^5(OH) D 2  3  l,25(OH) D 2  3  PTH  34. PTH stimulates cAMP formation and M g entry into MDCT cells. PTH, 10" M , and 1,25(OH) D , 10" M, were added where indicated. cAMP was measured by radioimmunoassay and d([Mg ] )/dt by fluorescence. Values are means ± SE for 4-5 preparations. * indicates significance, p<0.01, of M g uptake rates and + indicates significance of cAMP concentrations following PTH or l,25(OH) D + PTH compared to the control values. • indicates significance, p<0.01, of M g2+ uptake o f 1,25(OH) D versus 1,25(OH) D + PTH. 2 +  7  2  7  1+  i  2 +  2  3  2  3  2  3  3  89  Table 10. l,25(OH) D does not stimulate Mg uptake through a protein kinase A pathway. 2+  2  3  Control  l,25(OH) D  nM/s  nM/s  Control  164±5  210±11*  (") RpcAMPS  (6)  (4)  171±3  196±5*  (n)  (3)  (3)  2  3  Table 10 Legend  Mg entry was determined in the presence ofl,25(OH) D , 10" M, with and without a 5 min pretreatment with the protein kinase A inhibitor RpcAMPS, 10" M. The values are means ± SE with (n) observations and * indicates significance, p<0.05, from control values. 2+  7  2 6  III.3  Stimulation of the Mg /Ca -SR 2+  2+  3  does not inhibit l,25(OH)JD  stimulated Mg * uptake. 2  }  Stimulation of the Mg 7Ca -SR with addition of 50 pM neomycin did not change Mg uptake into 2  2+  2+  1,25(OH)D-treated cells (Fig. 35). This is in contrast to the actions of polyvalent cation-sensing on hormones 2  3  acting through G-protein linked receptors whereby both cAMP generation and Mg uptake are inhibited. 2+  Clearly, 1,25(OFf)D alters Mg entry into MDCT cells by intracellular pathways that are distinctive to those 2+  2  3  of the peptide hormones, PTH, glucagon, calcitonin, and AVP.  90  VI  Cont.  Neo.  VitD  VitJ) + Neo.  3  3  PTH  PTH + Neo.  Fig. 35. Summary of the effects of activation of Mg 7Ca -sensing on hormone-stimulated M g uptake. M g uptake, (d[Mg ] )/dt, was performed on Mg -depleted cells treated with/without 1,25(OH) D (note: Vit.D above= l,25(OH) D ), as indicated. M g uptake was determined with 1.5 m M extracellular M g in the absence and presence of neomycin, 50 uM. Neomycin was added 5 min prior to the addition of PTH, 1 0 M, and MgCl , 1.5 m M . The M g uptake rate was determined over 500 s following addition of PTH. Values are mean ± SE for 3-5 cells. * indicates significance (p<0.05) from control values. 2  2+  2+  2+  2+  2+  i  2  2 +  2  3  2 +  7  2 +  2  3  91 Next, I determined the effects of chronic (16 h) elevation of extracellular Ca , 5.0 mM, and neomycin, 50 2+  ,uM, on l,25(OH)2D3-stimulated Mg uptake. Ca or neomycin were added with l,25(OH) D 16 h prior 2+  2+  2  3  to uptake studies. In support of our earlier findings, high extracellular Ca for 16 h did not alter basal Mg 2+  2+  uptake, 165±7 nM/s. The effect of 16 h high Ca and neomycin on l,25(OH) D -stimulated Mg uptake 2+  2+  2  3  was no different from acute treatment (as seen in Fig. 35).  IV. Discussion  The present studies show that activation of the Mg 7Ca -sensing mechanism inhibits hormone 2  2+  stimulated Mg uptake into MDCT cells. 1,25(OH)D stimulated uptake, in contrast, was not inhibited with 2+  2  3  stimulation of the Mg 7Ca -sensing mechanism. These results further support my hypotheses that the 2  2+  Mg /Ca -SR has an important role in controlling magnesium absorption by acting in at least two nephron 2+  2+  segments; the thick ascending limb and the distal convoluted tubule.  92 GENERAL CONCLUSIONS  1:  MDCT cells have proven to be a good model for studying distal convoluted tubule Mg transport 2+  (Table 11). 2:  The Ca -SR is present in MDCT cells and there is evidence these cells have a mechanism which 2+  also responds selectively to extracellular Mg . 2+  3:  Stimulation of the Mg 7Ca -SR inhibits hormone stimulated Mg uptake (Fig.36). 2  2+  2+  GENERAL DISCUSSION  Identification of extracellular polyvalent cation-sensing receptor in MDCT cells. In this study,  I show that the established MDCT cell line possesses aCa -SR that responds to [Mg ] and [Ca ] , to 2+  2+  2+  0  0  transiently release Ca jfromcytosolic stores. This immortalized cell line is representative of the intact distal 2+  convoluted tubule as it possesses many of the properties characteristic of this segment including chlorothiazide-sensitive NaCl cotransport, amiloride-inhibitable Na conductance, and parathyroid hormone +  and calcitonin-stimulated calcium and magnesium transport [39,41,49,61 ]. Accordingly, it is of interest that this cell line also has an extracellular divalent cation-sensing receptor. Using RT-PCR and Southern blotting, I show that the MDCT cells possess transcripts for the Ca -SR. The presence of the Ca -SR protein in 2+  2+  MDCT cells was documented by Western blot analysis using a specific Ca -sensing receptor antiserum. 2+  Furthermore, determination of cytosolic Ca with fluorescence showed that Ca -SRin MDCT cells elicit 2+  2+  93 intracellular signals in response to increasing extracellular concentrations of polyvalent cations demonstrating that the receptor is functional. These studies with mouse distal convoluted cells are consonant with reports of Ca -SR transcripts in the rat distal convoluted segment [11,13,17]. More recently, Riccardi et al. showed 2+  that the rat Ca -SR is localized to the basolateral membrane of the distal convoluted tubule [11]. The 2+  presence of a Ca -SR in the distal convoluted tubule may have important ramifications on cellular function 2+  within this segment. The importance of the Ca -SR in the control of [Mg ] and [Ca ] homeostasis is indicated by the 2+  2+  2+  0  0  fact that individuals with familial hypocalciuric hypercalcemia (FHH) manifest hypermagnesemia and hypercalcemia which is due, in part, to excessive renal reabsorption [26,66,67,68]. Additionally, mice that are heterozygous or homozygous with inactivating mutations of the Ca -SR show significant increases in [Mg""],, 2+  2 1  and [Ca ] relative to normal mice [26]. Artie et al performed clearance studies on hypoparathyroid patients 2+  0  and concluded that inappropriate renal calcium conservation is due to an inherent abnormality ofthe kidney [68]. Conversely, they suggested that inappropriate magnesium reabsorption might be due predominantly to elevated circulating PTH levels. Our functional studies addressed this issue (see below discussion). In all studies to date, [Ca ] has been found to be a more potent stimulator of Ca -SR-induced 2+  2+  0  intracellular signalling than [Mg ] . The threshold value for [Ca ] has been reported to be of the order of 2+  2+  0  0  1 -5 mM for renal cells whereas a similar cytosolic Ca response requires the presence of 5-20 mM [Mg ] 2+  2+  0  [39]. These relative potencies of [Ca ] and [Mg ] recapitulates their actions in bovine parathyroid cells 2+  2+  0  0  and mXenopus oocytes injected with cRNA of cloned Ca -sensing receptor [4,56]. Thus, it was of interest 2+  that the polyvalent cation-sensitive mechanism of MDCT cells was apparently as sensitive to [Mg ] as it 2+  0  was to [Ca ] . This is particularly noteworthy as the [Mg ] studies were performed in the presence of 2+  2+  0  0  normal or elevated [Ca ] . The functional consequences of changes in the amino acid sequence ofthe Ca 2+  2+  0  SR has been investigated by expressing a variety of mutated receptors in HEK 293 cells [32]. Some ofthe  94 mutations diminish Ca -SR signalling, others enhance the sensitivity to external [Ca ] , and still others are 2+  2+  0  completely nonfunctional with no intracellular signalling as determined by changes in [Ca ] [28,29,33]. From 2+  i  these transfection studies of mutated Ca -SRs, Bai et al. suggested that discrete but interrelated cation 2+  binding sites may exist in this receptor. Ca binding to the Ca -sensing receptor per se has yet to be 2+  2+  examined [32]. These discrete sites remain to be identified but it is apparent from these studies that changes in the extracellular domain of the Ca -SR may alter sensitivity to the various ligands [32]. The present 2+  studies with the endogenous polyvalent cation-sensitive mechanism of the MDCT cell show that the intracellular signalling is responsive to both [Mg ] and [Ca ] and that there appears to be little interaction 2+  2+  0  0  between these cations. Accordingly, the Ca -sensing receptor may function as a [Mg ] receptor (Mg 2+  2+  2+  0  sensing receptor) even in the presence of relatively high concentrations of [Ca ] . The sites involved in 2+  0  binding [Mg ] and [Ca ] and the cooperative association in intracellular signalling remain to be determined. 2+  2+  0  0  Alternatively, the results of my studies may indicate the presence of separate receptors for [Ca ] and 2+  0  [Mg ] in MDCT cells. 2+  0  Evidence for a Mg *-sensing receptor. The present studies suggest that a receptor for extracellular 2  Mg may exist in the MDCT cell line. There are two reports of the Mg -SR in the literature. First, 2+  2+  Goudeau and Goudeau have reported that the Mg^-SRis present in the shrimp oocyte [79,80]. This receptor is involved in developmental activation of the oocytes. In an effort to identify this receptor, I obtained shrimp oocytes and isolated poly (A ) RNA to inject into Xenopus oocytes for heterologous expression. The receptor +  was successfully expressed in Xenopus oocytes . Next, we attempted to clone the receptor by homology screening libraries prepareredfromthe RNA of the oocytes. This was not successful. We concluded that the shrimp Mg -SR is different from the Ca -SR. 2+  2+  Cellular mechanisms of the Ca -SR. Activation of the polyvalent cation-sensing mechanism in 2+  MDCT cells with neomycin inhibited PTH-, calcitonin-, glucagon- and AVP-stimulated cAMP release (Table  95 2). It has long been known that hypermagnesemia and hypercalcemia inhibit hormone-mediated cAMP accumulation in the proximal tubule, loop of Henle, and the collecting duct. Hypermagnesemia and hypercalcemia inhibit the PTH-mediated increase in cAMP in the proximal tubule and cortical thick ascending limb [69,70]. The elevation of [Ca ] also mitigates vasopressin-stimulated increases in cAMP production 2+  0  in the medullary thick ascending limb of Henle's loop [70,71 ] and PTH, calcitonin, vasopressin and glucagonstimulated cAMP accumulation in the cortical thick ascending limb [70]. Jones et al. have shown that [Ca ] 2+  0  reduces the hydrosmotic response of cortical collecting ducts to AVP and cAMP [72]. Chen et al. have shown that polyvalent cations inhibit agonist-stimulated cAMP accumulation in bovine parathyroid cells [56]. This inhibition was totally prevented following preincubation of the cells with pertussis toxin which is known to ADP-ribosylate and uncouple the guanine nucleotide regulatory (G)-protein, G , from the cell surface ;  receptors which are coupled to inhibition of adenylate cyclase [54]. Accordingly, the parathyroid gland Casr appears to be linked to adenylate cyclase via a G protein [54]. This also appears to be the case for MDCT (  cells. Glucagon-and A VP-stimulate magnesium and calcium transport in the rat distal tubule and magnesium entry into MDCT cells [43,87]. We postulate that activation of the polyvalent cation-sensing mechanism with high levels of [Ca ] or [Mgf ] may inhibit glucagon-and AVP-stimulated magnesium and calcium transport. 2+  +  0  0  Further studies are needed to establish the functional roles of the polyvalent cation-sensing receptor(s) within the distal tubule. In summary, a Ca -SR is present in an immortalized mouse distal convoluted tubule (MDCT) cell 2+  line. This cell line has been extensively used to characterize magnesium and calcium transport in the distal convoluted segment of the nephron. The use of this cell line should allow insights into expression, control, and function of the Ca -sensing receptor in distal convoluted tubule cells. This is particularly important as in 2+  vivo micropuncture and microperfusion approaches do not allow for study of cellular mechanisms of transport. In vitro microperfusions have not been performed in this portion of the tubule because of the  96 difficulty in isolating intact segments. Cellular mechanisms of peptide hormone actions and stimulation of Mg * uptake. In order to 2  determine the cellular actions of extracellular Ca -SR on Mg transport and the influences on hormonal 2+  2+  controls, we characterised Mg uptake in MDCT cells. In the present study, we show that PTH, calcitonin, 2+  glucagon, and AVP stimulate Mg entry into MDCT cells in a concentration-dependent manner. 2+  Furthermore, these hormones appear to act through cAMP-, phospholipase C- and protein kinase Cdependent pathways. Although the details of these signalling pathways are not known this data provides some explanation in interpreting the cellular actions of the Ca -SR in MDCT cells. By inference this information 2+  provides some detail as to what is occurring in the distal convoluted tubule. The cellular mechanisms underlying the hormonal actions on distal magnesium absorption are largely unknown at the present time. Morel et al. have shown that PTH, calcitonin, and glucagon, stimulate receptormediated cAMP release in the DCT [ 173,174,106]. They also reported that AVP receptors may be present in the DCT but there were marked species differences in adenylate cyclase responsiveness [107]. There was little AVP-stimulated cAMP production in the DCT of the rabbit and human, intermediate response in the mouse, and greatest in the rat [ 106,174]. We have shown that PTH, calcitonin, glucagon, and AVP stimulates Mg uptake into MDCT cells [49, unpublished observations]. Friedman and Gesek reported that these 2+  hormones also stimulate cellular cAMP accumulation [108,109]. We determined whether cAMP may influence Mg entry into MDCT cells. The addition of the c AMP analogue, 8-bromo-cAMP, increased Mg" 2+  +  uptake whereas inhibition of protein kinase A with RpcAMPS prevented hormone-stimulated uptake [49]. Accordingly, receptor-mediated cAMP release and activation of protein kinase A plays a role in hormonestimulated Mg uptake in MDCT cells. However, it is apparent that other signaling pathways are present for 2+  hormone-mediated Mg uptake in MDCT cells. Hormone-stimulated Mg^uptake rates do not correlate with 2+  +  the measured intracellular cAMP levels in MDCT cells [49]. Furthermore, phospholipase C inhibition, with  97 U-73122, but not protein kinase C inhibition with Ro31 -8220, abolished PTH- and calcitonin-stimulated Mg  +  uptake. This was true for all of the hormones tested, PTH, calcitonin, glucagon, AVP [49, unpublished observations]. These hormones do not elicit receptor -mediated intracellular Ca transients suggesting that 2+  Ca signalling is not involved with the responses [39,108]. We have demonstrated, however, there is a 2+  requirement for some portion of the PLC pathway above the level of PKC. There may in fact be non-specific activity of the U-73122 or involvement of alternative phospholipases contributing to these observations. As the hormones used are not associated with Ca? transients the possibility arises that DAG, and its downstream +  metabolites, may be involved. Binding of the released G^ subunit, to an undetermined phospholipase, may also play a crucial role in this pathway. A variety of PLC isoforms have been identified in the distal nephron which lends some support to this theory [175]. It is well known that multiple receptors can converge on a single G protein and in many cases a single receptor can activate more than one G protein and thereby modulate multiple intracellular signals [ 176]. It is evident that c AMP-protein kinase A and phospholipase C pathways are necessary for hormone-stimulated Mg entry into MDCT cells. The details of how peptide hormones act 2+  on Mg transport in MDCT cells and intact distal tubules are unknown. However, these studies provide a 2+  background for understanding the actions of the Ca -SR. 2+  The effect of the Ca -SR on hormone-stimulated Mg * uptake. In the present study, we show 2+  2  that activation of this Mg 7Ca -sensing mechanism inhibits hormone-stimulated Mg uptake into MDCT 2  2+  2+  cells. The evidence provided here is that activation of Ca -SR by extracellular polyvalent cations diminish 2+  hormone-stimulated Mg uptake, in part, by inhibiting hormone-mediated c AMP formation. The effect of this 2+  receptor on phospholipase C and protein kinase C pathways is not known. As this cell line possesses many of the transport properties of the intact distal convoluted tubule, we conclude that the Mg 7Ca -sensing 2  2+  mechanism plays an important role in control of magnesium transport in this nephron segment. These conclusions are consonant with the results of earlier micropuncture studies. Le Grimellec and  98 colleagues used magnesium- or calcium-loaded rats having intact parathyroid glands. They performed freeflow micropuncture experiments to determine the effects of hypermagnesemia and hypercalcemia on distal tubular absorption [177,178]. They showed that magnesium delivery from the early distal tubule sampling site to the final urine relative to inulin delivery was markedly increased in hypermagnesemic and hypercalcemic rats. They interpreted this data to indicate "a drastic inhibition (by an unknown mechanism) of reabsorption taking place in the terminal segments of the nephron or a proportionately more important contribution of the deeply located nephrons to magnesium excretion than expectedfromtheir number" [177,178]. In support of these early micropuncture experiments, in vivo microperfusion studies indicated that the rate of magnesium absorption within the distal tubule was altered by extracellular magnesium and calcium [ 100]. Distal tubules were perfusedfroma proximal site and tubule fluid sampledfromearly and late sites of superficial distal tubules. Magnesium absorption within the superficial distal tubule of TPTXed rats was highly dependent on delivery of magnesium to this segment [98,99]. Thefractionof delivered magnesium absorbed in distal tubules amounted to 34±10% in rats with normal plasma magnesium and calcium concentrations, 0.78±0.04 mM and 2.24±0.04 mM, respectively [100]. However,fractionalmagnesium absorption decreased significantly to 6±3% when the animals were made hypermagnesemic (plasma magnesium 3.58±0.20 mM) and to 14±7% in hypercalcemic rats (plasma calcium 4.24±0.36 mM) [78]. The animals used in these studies were TPTX but were intact with respect to other circulating hormones. The cellular basis for diminished fractional absorption in hypermagnesemia and hypercalcemia remained unexplained until a Ca -SR was 2+  identified in the kidney and located to the distal tubule [11]. The Ca -SR is localized along the length of the nephron from the proximal tubule to the collecting 2+  system with particular abundance in the basolateral membrane of the thick ascending limb and the apical membrane of the inner medullary collecting duct [11,17]. The functions of renal polyvalent cation-sensing receptor(s) are not fully understood. Hebert has recently summarized the salient features of increases in  99 extracellular Ca and Mg " on loop and collecting duct function [123]. Hypercalcemia and hypermagnesemia 2+  24  inhibit NaCl, calcium, and magnesium absorption in the thick ascending limb [97] and water permeability in the medullary collecting duct [10]. Hebert and colleagues speculate that inhibition of salt reabsorption in the thick ascending limb and water transport in the medullary collecting duct together with diminished calcium absorption in the thick ascending limb would wash out the calcium cations minimizing the possibility of urinary stone formation [123]. The functional roles of iheCasr within the proximal tubule and segments of the distal tubule have not been determined. The present studies show that activation of the Mg /Ca -sensing 2+  2+  mechanism inhibits hormone-stimulated Mg uptake into MDCT cells. These results suggest that this 2+  receptor might play an important role in controlling magnesium absorption acting in at least two nephron segments; the thick ascending limb and the distal convoluted tubule. The mechanisms by which the Mg 7Ca -sensing mechanism inhibits magnesium transport in the 2  2+  thick ascending limb and distal tubule are becoming clearer. In the cortical thick ascending limb, magnesium and calcium transport is passive, dependent on the transepithelial voltage and the permeability of the paracellular pathway [86]. Wang et al. have reported that neomycin inhibits apical K channels and possibly +  apical Na, K, CI cotransport through signalling pathways involving cytochrome P-450 metabolites (7). Accordingly, activation of the Ca -SR would lead to a decrease in transepithelial voltage and diminished 2+  passive calcium and magnesium transport in the thick ascending limb [78]. Unlike the thick ascending limb, magnesium transport within the distal tubule is active and transcellular in nature so that the receptor must affect active magnesium absorption. My results indicate that activation of the Mg7Ca -sensing mechanism 2  2+  inhibits hormone-stimulated Mg " uptake and hormone-mediated accumulation of cAMP in MDCT cells. As 24  cAMP enhances Mg entry into MDCT cells, I speculate that activation of the Mg 7Ca -sensing 2+  2  2+  mechanism may act, in part, through diminished hormone responsive cAMP release. We were unable to show any effects of neomycin activation of Mg /Ca -sensing on membrane voltage either with glucagon 2+  2+  100  or amiloride suggesting that these effects were independent of voltage. We infer from these studies that the Mg 7Ca -sensing mechanism in the distal convoluted tubule plays an important role in renal magnesium 2  2+  conservation in addition to its affects within the loop of Henle. It has long been known that hypermagnesemia and hypercalcemia inhibit hormone-mediated cAMP accumulation in the proximal tubule, loop of Henle, and the collecting duct. Hypermagnesemia and hypercalcemia inhibits the PTH-mediated increase in cAMP in the proximal tubule and cortical thick ascending limb [13,14]. The elevation of extracellular Ca also mitigates vasopressin-stimulated increases 2+  in cAMP production in the medullary thick ascending limb of Henle's loop [123] and PTH-, calcitonin-, vasopressin- and glucagon-stimulated cAMP accumulation in the cortical thick ascending limb [123]. Finally, Sands et al. have shown that AVP-elicited water permeability in rat kidney terminal inner medullary collecting ducts is inhibited with elevated plasma calcium [10]. Accordingly, hypermagnesemia and hypercalcemia, probably through activation of the Mg 7Ca -sensing mechanism, have significant effects on hormone2  2+  mediated cAMP generation along the length of the nephron. These actions are also apparent on hormonestimulated cAMP accumulation in MDCT cells. We have previously shown that cAMP, in part, mediates hormone-stimulated Mg uptake [87]. Accordingly, it is likely that functional responses within the distal 2+  convoluted tubule that are mediated by cAMP are also modulated by hypermagnesemia and hypercalciuria through Mg /Ca sensing. 2+  2+  Examples of Ca -SR controls on Mg * transport: steroid hormones. Mineralocorticoid receptors 2+  2  are present in DCT cells which are thought to be involved in expression of NaCl cotransport, Na  +  conductance, and sodium pump activity [ 132,133,134]. The effects of aldosterone on distal tubule magnesium absorption have not been studied with micropuncture techniques. Clearance studies have shown that chronic aldosterone administration results in renal magnesium-wasting but this has been explained by extracellular volume expansion leading to diminished NaCl and magnesium reabsorption within the loop [86]. We have  101 studied the effects of aldosterone on Mg entry into MDCT cells [139]. Incubation of aldosterone, for 16 2+  hr prior to determination of Mg uptake failed to have any effect on basal magnesium transport. However, 2+  pretreatment of MDCT cells with aldosterone potentiated hormone-stimulated Mg uptake. This was 2+  associated with potentiation of hormone-mediated cAMP release in aldosterone-treated MDCT cells. Cycloheximide, an inhibitor of protein synthesis, abolished the potentiation of aldosterone on glucagon- and A VP-stimulated cAMP release and Mg uptake [139]. Accordingly, aldosterone may enhance hormone2+  stimulated Mg entry by increasing cAMP and its responses. This notion is supported by the observations 2+  of others [136]. Rajerison et al. demonstrated that adrenalectomy reduced AVP-stimulated adenylate cyclase activity in membrane fractions prepared from rat kidney medulla [137]. Doucet et al. have shown that glucagon- and AVP-responsive c AMP generation is diminished in thick ascending limb and collecting tubule segments harvested from adrenalectomized rats compared to animals treated with physiological doses of aldosterone [134,135]. These investigators postulate that aldosterone induces a protein(s) which stimulates hormone-sensitive adenylate cyclase activity. Studies with kidney membrane fractions and isolated segments in the absence of aldosterone demonstrated an impairment of coupling between hormone receptors and adenylate cyclase catalytic units was responsible for diminished cAMP generation [134]. Steroid hormones have significant effects on expression and posttranslational targeting of heterotrimeric G-proteins so that associated channels are covalently modified [ 131,140]. The mechanism(s) through which steroids control Gxproteins (synthesis and/or degradation vs. activity of each unit) associated with Mg uptake in MDCT cells 2+  s  is not known. The role of mineralocorticoids in the physiological maintenance of renal magnesium handling also requires further research. Vitamin D metabolites have important effects on mineral metabolism by 1,25(OH)Dactions on 3  2  3  epithelial transport [142,143]. The distal tubule, including the convoluted segment, also possesses 1,25(OH)2D3 receptors [144,145,146,147]. We have demonstrated that l,25(OH)2D3 increases Mg^ entry rates in MDCT +  102 cells. The response is concentration-dependent, involves transcriptional processes involving de novo protein synthesis and does not appear to be related to cAMP-mediated stimulation of Mg^ uptake. These studies +  suggest that vitamin D 3 may modulate magnesium transport in the distal tubule. Important to the present study is that activation of the extracellular Ca^ -SR does not inhibit 1,25(OH)2D3-stimulated Mg^ transport. +  +  Accordingly, there are some hormonal controls of Mg^ transport that are not modulated by extracellular +  cation-sensing, i.e. hypermagnesemia and hypercalcemia. Summary. In summary, a Mg 7Ca -sensing mechanism is present in MDCT cells which upon 2  2+  activation inhibits hormone-mediated cAMP accumulation and peptide hormone-stimulated Mg uptake in 2+  Mg -depleted cells. It is not known if these responses or others are present in normal distal tubule cells. 2+  Also, the pathways by which the Mg 7Ca -sensing mechanism alter these activities are yet to be fully 2  2+  elucidated. However, the functional responses observed in the present studies are in keeping with earlier microperfusion studies demonstrating that hypermagnesemia and hypercalcemia diminish magnesium absorption within the distal tubule [98,100]. These studies show that the Mg 7Ca -sensing mechanism is 2  2+  important in the regulation of renal magnesium transport at both the level of the distal convoluted tubule as well as the loop of Henle. It is envisioned that either hypermagnesemia or hypercalcemia could inhibit divalent cation absorption in the loop and hormone-mediated absorption in the distal convoluted tubule. The latter response would be appropriate to mitigate excessive magnesium and perhaps calcium reabsorption in the face of increased delivery to this segment.  103  Table 11. Controls of Magnesium Reabsorption in Intact Distal Tubule Compared to MDCT Cells  Peptide hormones:  Parathyroid Hormone Calcitonin Glucagon Arginine Vasopressin PGE Aldosterone l,25(OH) D  Prostaglandins: Mineralocorticoids: Vitamin D: Magnesium restriction Metabolic acidosis Metabolic alkalosis Hypermagnesemia Hypercalemia Phosphate-depletion Potassium-depletion Diuretics: Furosemide Amiloride Chlorothiazide 2  2  3  Intact DT Increase (*) Increase (*) Increase (*) Increase (*) ? ?  ?  Increase (*) Decrease (*) Increase (*) Decrease (*) Decrease (*) Decrease (*) ?  No effect (*) ?  Increase (*)  MDCT Increase (*) Increase (*) Increase (*) Increase (*) Increase (*) Increase (*) Increase (*) Increase (*) Decrease (*) Increase (*) Decrease (*) Decrease (*) Decrease (*) Decrease (*) No effect (*) Increase (*) Increase (*)  Data derived from intact distal tubule was obtained from micropuncture and microperfusion studies. ? indicates not known; u.p. unpublished results.  104  Model of Magnesium Transport in DCT Apical  Cell  0.2 m M Mg * 2  Mg  2 +  Basolateral  J  0.6 m M Mg *  0.5 m M Mg -  2  2 +  Na  +  dihydropyridines  Na  \  thiazides  Na  PKA  VcAMP^.  +  Peptide hormone (PTH, calcitonin, glucagon, AVP)  amiloride  Ca -10 mV  Figure 36.  -70 mV  /'Mg ' 2  OmV  Schematic representation of the Mg 7Ca -SR in the distal convoluted tubule and its inhibitory effect on hormone stimulated Mg uptake. 2  2+  2+  105  FUTURE RESEARCH  The focus of future and current research with this cell line includes identification of a specific M g 2+  SR. As I could not isolate a M g -SR by homology (with a Ca -SR) through homology screening of libraries 2+  2+  it would suggest that the receptor may be quite different. Accordingly, future, experimentation should use an expression based approach. It will be important to use tissue with higher abundance of the M g -SR and low 2+  abundance of the Ca -SR as determined by physiological means. Mg Finally, determining the genetic controls 2+  on expression of the Ca -SR, and how this may also be influenced by hormone and external cationic 2+  stimulation, should be established.  106 Bibliography:  1  Watson, S., Arkinstall, S.(1994) Superfamily of seven transmembrane proteins. In "The G-protein linked receptor facts book" (S. Watson, S. Arkinstall eds.), pp.2-6. Academic Press, Harcourt Brace and Company, San Diego, CA.  2  Hall, JM., Caulfield, MP., Watson SP., Guard, S.: Receptor subtypes or species homologues: relevance to drug discovery: Trends in Pharmacological Sciences. 14(10):376-83, 1993.  3  Jackson, TR.: Structure and function of G protein coupled receptors: Pharmacology & Therapeutics. 50(3):425-42, 1991  4  Brown, EM., Gamba, G., Riccardi, D., Lombardi, M., Butters, R., Kifor, O., Sun, A., Hediger, MA., Lytton, J., Hebert, SC.: Cloning and characterization of an extracellular Ca -sensing receptor from bovine parathyroid. Nature 366:575-580, 1993 2+  5  Brown, EM., Vassilev, PM., and Hebert, SC.: Calcium ions as extracellular messengers. Cell 83: 676-682, 1995  6  Kifor, O. Diaz, R., Butters, R., Brown, E.: The Ca sensing receptor (CaR) activates Phospholipases C, A2, and D in bovine parathyroid and CaR-transfected, human embryotic kidney (HEK293) cells. J. of Bone Research 12(5):715-725, 1997  7  Wang, W-H., La, M., and Hebert, SC.: Cytochrome P-450 metabolites mediate extracellular Ca -induced inhibition of apical K channels in the TAL. Am. J. Physiol.(271 Cell Physiol). 40: C103-C111, 1996.  2+  2+  +  8  Mathias, RS., and Brown EM. :Divalent cations modulate PTH-dependent 3', 5'-cyclic adenosine monophosphate production in renal proximal tubular cells. Endocrinology 128: 3005-3012, 1991.  9  Chou, Y-HW., Brown, EM., Levi, T., Crowe, G., Atkinson, AB., Amqvist, HJ., Toss, G. :The gene responsible for familial hypocalciuric hypercalcemia maps to chromosone 3q in four unrelated families. Nature Genetics 1:295-299.1993  107 10  Sands, JM., Naruse, M., Baum, M., Hebert, SC., Brown, EM., and Harris, HW. :Apical extracellular calcium/polyvalent cation-sensing receptor regulates vasopressin-elicited water permeability in rat kidney inner medullary collecting duct. J. Clin. Invest. 99: 1399-1405, 1997.  11  Riccardi D., Lee W-S., Lee K, Segre G.V., Brown EM., Hebert, SC. localization of the extracellular Ca -sensing receptor and PTH/PTHrP receptor in rat kidney. Am. J. Physiol. 27LF951-F956, 1996 2+  12  Paulais M., Baudouin-Legros M., Teulon J.: Functional evidence for a Ca7polyvalent cation sensor in the mouse thick ascending limb. Am. J. Physiol. 271 :F1052-F1060, 1996  13  Riccardi, D., Park, J., Lee, W-S., Gamba, G., Brown, EM., Hebert, SC.: Cloning and functional expression of a rat kidney extracellular calcium/polyvalent cation-sensing receptor. Proc. Natl. Acad. Sci. U.S.A. 92:131-135, 1995  14  Aida, K., Koishi, S., Tawata, M., Onaya, T. : Molecular cloning of a putative Ca -sensing receptor cDNA from human kidney. Biochem. Biophys. Res. Comm. 214:524-529, 1995  15  Riccardi, D., Hall, AE., Chattopadhyay, N., Xu, NJZ., Brown, EM. and Hebert, SC.: Localization of Ca /polyvalent cation-sensing receptor protein in rat kidney. Am. J. Physiol. 244 (Renal Physiol. 43): F611-F622, 1998.  2  2+  2+  16  Cattopadhyay, N., Baum, M., Bai, M., Riccardi, D., Hebert, H., Harris, W., Brown, E. : Ontogeny of the extracellular calcium-sensing receptor in rat kidney. Am. J. Physiol. 244 (Renal Fluid Electrolyte Physiol. 40): F736-F743, 1996.  17  Yang, TX., Hassan, S., Huang, YNG., Smart, AM., Briggs, JP., and Schnerman, JB. : Expression of PTHrP, PTH/PTHrP receptor and Ca -sensing receptor RNAs along the rat nephron. Am. J. Physiol. 41 (Renal Physiol.): F751-F758, 1997. 2+  18  Ruat, M., Snowman, AM., Hester, LD., Snyder, SH.: Cloned and expressed rat Ca -sensing receptor: differential cooperative responses to calcium and magnesium. J. Biol. Chem. 271:59725975,1996  19  Bai, M., Trivedi, S., Lane, C, Yang, Y., Quinn, S., Brown, E. :Protein kinase C phosphorylation at position 888 in the Ca sensing receptor (CaR) inhibits coupling to Ca store release. J. of Biol. Chem. 273(33): 21267-21275. 1998  2+  2+  2+  108 20  Takaichi, K., Uchida, I., and Kurokawa, K.: High Ca inhibited AVP-dependent cAMP production in thick ascending limb of Henle. Am. J. Physiol. 250 (Renal Fluid Electrolyte Physiol. 79):h-F776, 1986.  21  Celeste, M., Ferreira, De J., Bailly, C: Extracellular Ca decreases chloride reabsorption in rat CTAL by inhibiting the cAMP pathway. Am. J. Physiol., 275 (Renal Physiology 40): F198-F203, 1998  22  Desfleurs, E., Wittner, A., Simeone, S., Pajaud, S., Moine, G., Rajerison, R., Di Stefano, A.: Calcium-Sensing Receptor : Regulation of electrolyte transport in the thick ascending limb. Kidney and Blood Pressure Research 822, 1-12. 1998  23  Chabardes, D., Firsov, D., Aarab, 1., Clabecq, A., Bellanger, A-C, Siaume-Perez, Elalouf, J-M.: Localization of mRNAs encoding Ca -inhibitable adenylyl cyclases along the renal tubule. J. of Biol. Chem., 271(32) 19264-19271. 1996  2+  2+  2+  24  Stow JL., Almeida, JB. distribution and role of heterotrimeric G-proteins in the secretory pathway of polarized epithelial cells. J. of Cell Science 17,33-39. 1993.  25  Pearce S., Bai, M. Quinn, S., Kifor, O., Brown, E., Thakker, R. Functional characterization of calcium-sensing receptor mutations expressed in human embryotic kidney cells. J. Clin. Invest. 98(8): 1860-1866, 1996.  26  Ho, C, Conner, DA., Pollack, MR., Ladd, DJ., Kifor, O., Warren, HB., Brown, EM., Seidman, JG., Seidman, CE. : A mouse model of human familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism. Nature Genetics 11:389-394,1995  27  Aida, K. Koishi, S., Inoue, M. Nakazato, M., Tawata, M., Onaya, T. : Familial hypocalciuric hypercalcemia associated with mutation in the human Ca -sensing receptor gene. J. of Clin. Endocrinology and Metabolism. 80(9): 2594-2598,1995 2+  28  Bai, M., Janicic, N., Trivedi, S., Quinn, S.J., Cole, DEC, Brown, EM., Hendy, GN. :Markedly reduced activity of mutant calcium-sensing receptor with an inserted alu element from a kindred with familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism. J. Clin. Invest. 99:1917-1925, 1997  109 29  Heath, III., Odelberg, S., Jackson, CF., Teh, BT., Hayward, N., Larsson, C, Buist, NRM., Krapcho, KJ., Hung, BC, Capuano, I., Garrett, JE., Leppert, MF. : Clustered inactivating mutations and benign polymorphisms of the calcium receptor gene in familial benign hypocalciuric hypercalcemia suggest receptor functional domains. J. Clin. Endocrinol. Metab. 81:1312-1317, 1996  30  Chou,Y., Pollak, M., Brandi, G., Toss, H., Amqvist A., Atkinson, S. Papapoilos, S., Marx, S.E., Brown, E., Seidman, J., Seidman, C. : Mutations in the human Ca -sensing receptor gene that cause familial hypocalciuric hypercalcemia. Am. J. of Human Genetics. 56:1075-1079. 1995 2+  31  Pearce, S.H. Bai, M., Quinn, S.J., Kifor, O. Brown, EM. Thakker, RV. functional characterization of calcium-sensing receptor mutations expressed in human embryonic kidney cells. J Clin.Invest. 98(8): 1860-6, 1996.  32  Bai, M., Quinn, S., Trivedi, S., Kifor, O., Pearce, SHS., Pollak, MR., Krapcho, K., Hebert, SC., Brown, EM. Expression and characterization of inactivating and activating mutations in the human Ca -sensing receptor. J. Biol. Chem. 271:19537-19545, 1996 2+  33  Pollak, MR., Brown, EM., Estep, HL., Mclaine, PN., Kifor, O., Park, J., Hebert, SC., Seidman, CE., Seidman, JG. : Autosomal dominant hypocalcemia caused by a Ca -sensing receptor gene mutation. Nature Genetics 8:303-307, 1994 2+  34  Brown, E.: Mutations in the calcium-sensing receptor and their clinical implications. Hormone Research 48: 199-208. 1997  35  Pearce, S.H., Brown, EM. :Calcium-sensing receptor mutations: insights into a structurally and functionally novel receptor. J. of Clin. Endocrinology & Metabolism. 81(4): 1309-11, 1996  36  Quamme, GA.: Magnesium homeostasis and renal magnesium handling. Mineral & Electrolyte Metabolism. 19(4-5):218-25, 1993.  37  Costanza, L. and Windhager, E.: Renal regulation of calcium: In The Kidney: Physiology and Pathophysiology edited by D.W. Seldin and G. Giebisch. New York: Raven, p.2375-2393, 1992  38  Kriz, W., and Bankir, L. : A standard nomenclature for structures of the kidney. Am. J. Physiol. 254 (Renal Fluid Electrolyte Physiol.23): F1-F8, 1988  39  Friedman, PA., Gesek, FA.: Cellular calcium transport in renal epithelia: measurement, mechanisms, and regulation. Physiological Reviews 75:429-471, 1995  110 40  Friedman, PA., Gesek, FA. : Sodium entry mechanisms in distal convoluted tubule cells. Am. J. Physiol. 268:F89-F98, 1995  41  Gesek, FA., and Friedman, PA. On the mechanism of parathyroid hormone stimulation of calcium uptake by mouse distal convoluted tubule cells. J. Clin. Invest. 90: 749-758, 1992.  42  Pizzonia, JH., Geseck, FA., Kennedy, SM., Coutermarsh BA., Bacskai, BJ., Friedman, PA..: Immunomagnetic separation, primary culture and characterization of cortical thick ascending limb plus distal convoluted tubule cells from mouse kidney. In Vitro Cell. & Dev. Biol. 27A: 409-416, 1991.  43  Friedman, P.A. and Gesek, FA.: Mechanism of calcium transport stimulated by chlorothiazide in mouse distal convoluted tubule cells. J. Clin. Invest. 90: 429-438, 1992.  44  Bapty, BW., Dai, L-J., Ritchie, G., Jirik, F., Canaff, L., Hendy, GN., Quamme, GA.: Extracellular Mg - and Ca -sensing in mouse distal convoluted tubule cells. Kidney Int., 53: 583-592, 1998. 2+  45  2+  Fan, G., Goldsmith, P.K., Collins, R., Dunn, C.K., Krapcho, K.J., Rogers, K.K., Spiegel, AM.: N-linked glycosylation of the human Ca receptor is essential for its expression at the cell surface. Endocrinology 138:1916-1922, 1997 2+  46  Grynkiewicz, G., Poenie, M., Tsien, R.Y.: A new generation of Ca indicators with greatly improved fluorescence properties. J. of Biol. Chem. 260(6):3440-50, 1985.  47  Malgaroli, A., Milani, D., Meldolesi, J., Pozzan, T.: Fura-2 measurement of cytosolic free Ca in monolayers and suspensions of various types of animal cell. J. of Cell Biology. 105(5):2145-55, 1987  48  Dai, L.J., Quamme, GA.: Cyclic nucleotides alter intracellular free Mg in renal epithelial cells. Am. J. Physiol., 262,.F1100, 1992.  49  Dai, L-J., Bapty, BW., Ritchie, G., and Quamme. GA.: Glucagon and arginine vasopressin stimulates Mg uptake in mouse distal convoluted tubule cells. Am. J. Physiol. 274 (Renal Physiol. 43): F328-F335, 1998.  2+  2+  2+  2+  Ill 50  Emanuel, RL., Adler, GK., Kifor, O., Quinn, SJ., Fuller, F., Krapcho, K., Brown, EM. : Calciumsensing receptor expression and regulation by extracellular calcium in the AtT-20 pituitary cell line. Molecular Endocrinology 10:555-565,1996  51  Garrett, JE., Tamir, H., Kifor, O., Simin, RT., Rodgers, KV., Mithal, A., Gagel, RF., Brown, EM. : Calcitonin-secretory cells of the thyroid express an extracellular calcium receptor gene. Endocrinology 136:5202-5211, 1995  52  Freichel, M., Zink-Lorenz, A., Holloschi, A., Hafher, M., Flockerzi, V., Raue, F. : Expression of a calcium-sensing receptor in a human medullary thyroid carcinoma cell line and its contribution to calcitonin secretion. Endocrinology 137:3842-3848, 1996  53  Quarles, LD., Ffartle, JE., Siddhanti, SR., Guo, R., Hinson, TK. : A distinct cation-sensing mechanism in MC3T3-E1 osteoblasts functionally related to the calcium receptor. J. Bone Miner. Res. 12:393-402, 1997  54  Brown, EM., Fuleihan, GEH., Chen, C, Kifor, O.R. : A comparison of the effects of divalent and trivalent cations on parathyroid hormone release, 3', 5'-cyclic-adenosine monophosphate accumulation, and the levels of inositol phosphates in bovine parathyroid cells. Endocrinology 127: 1064-1071, 1990.  55  Brown, EM., Enyedi D., Leboff M., Rotberg J., Preston J., Chen C. : High extracellular Ca and Mg stimulate accumulation of inositol phosphates in bovine parathyroid cells. FEBS Lett. 218:113-118, 1987 2+  2+  56  Chen, C.J.,Anast, CS., Posillico, JT., and Brown, EM.: Effects of extracellular calcium and magnesium on cytosolic calcium concentration in fura-2-loaded bovine parathyroid cells. J. Bone Mineral Res. 2: 319-327, 1987.  57  Hawkins, D., Enyed, IP., Brown, EM.: The effects of high extracellular Ca and Mg concentrations on the levels of inositol 1,3,4,5 tetrakisphosphate in bovine parathyroid cells. Endocrinology 124:838-844, 1989  58  Nemeth, EF., Scarpa, A: Rapid mobilization of cellular Ca in bovine parathyroid cells by external divalent cations. Evidence for a cell surface calcium receptor. J. Biol. Chem. 262: 5188-5196, 1987  2+  2+  2+  112 59  Shoback, DM., Thatcher, JG., Brown, EM. : Interaction of extracellular calcium and magnesium in the regulation of cytosolic calcium and PTH release in dispersed bovine parathyroid cells. Mol. Cellular Endocrinol. 38:179-181, 1984  60  Gesek, F.A., and Friedman, PA.: Calcitonin stimulates calcium transport in distal convoluted tubule cells. Am. J. Physiol. 264 (Renal Fluid Electrolyte Physiol.33): F744-F751, 1993.  61  Dai, L-J., Raymond, L., Friedman, PA., Quamme, GA.: Cellular mechanisms of amiloride stimulation of Mg uptake in mouse distal convoluted tubule cells. Am. J. Physiol. 272 (Renal Physiol. 41): F249-F256, 1997. 2+  62  Ruat, M., Snowman, AM., Hester, LD., Snyder, SH. : Cloned and expressed rat Ca -sensing receptor: differential cooperative responses to calcium and magnesium. J. Biol. Chem. 271:59725975,1996  63  Dai, L-J., Quamme, GA.: Hormone-mediated Ca transients in isolated renal cortical thick ascending limb cells. Pfliigers Archiv. 427:1-8, 1994  64  Clemens, TL., Mcglade, SA., Garrett, KP., Craviso, GL., Hendy, GN. : Extracellular calcium modulates vitamin D-dependent calbindin-D28K gene expression in chick kidney cells. Endocrinology 124:1582-1584., 1989  65  Sneddon, WB., Gesek, FA., Friedman, PA. : l,25-(OH) vitamin D up-regulates the expression of the parathyroid hormone receptor in distal convoluted tubule cells. J. Am. Soc. Nephrol. 4:729, 1993 (Abstract)  66  Marx, SJ., Spiegel, A.M., Brown, EM., Koehler, JO., Gardner, DG., Brennan, MF., Aurbach, GD.: Divalent cation metabolism: familial hypocalciuric hypercalcemia versus typical primary hyperparathyroidism. Am. J. Med. 65:235-242, 1978  67  Marx, SJ., Attie, MF., Levine, MA., Spiegel, A.M., Downs, RW Jr., Lasker, RD. : The hypocalciuric or benign variant of familial hypercalcemia: clinical and biochemical features in fifteen kindreds. Medicine (Baltimore) 60:235-242, 1981  68  Attie, MF., Gill, Jr J., Stock, JL., Spiegel, AM., Downs, AM. Jr., Levine, MA., Marx, SJ. : Urinary calcium excretion in familial hypocalciuric hypercalcemia. Persistence of relative hypocalciuria after induction of hypoparathyroidism. J. Clin. Invest. 72:667-676, 1983  2+  2+  2  3  113 69  Slatopolsky, E., Mercado, A., Morrison, A., Yates, J., Klahr, S. : Inhibiting effects of hypermagnesiuria on the renal action of parathyroid hormone. J. Clin. Invest. 58: 1273-1279., 1976  70  Takaichi, K., Kurokawa, K. : High Ca -inhibits peptide hormone-dependent cAMP production specifically in thick ascending limbs of Henle. Miner. Electrolyte Metab. 12:342-346, 1986  71  Takaichi, K., Kurokawa, K. : Inhibitory guanosine triphosphate-binding protein-mediated regulation of vasopressin action in isolated single medullary tubules of mouse kidney. J. Clin. Invest. 82:1437-1444, 1988  72  Jones, SM., Frindt, TG., Windhager, EE. : Effect of peritubular [Ca] or ionomycin on hydrosmotic response of CCD to ADH or cAMP. Am. J. Physiol. 254:F240-F253, 1988  73  Chen, C.J., Barnett, J.V., D.A. Congo, and Brown, EM.: Divalent cations suppress 3',5'adenosine monophosphate accumulation by suppressing a pertussis toxin-sensitive guanine nucleotide-binding protein in cultured bovine parathyroid cells. Endocrinology 124: 233-239, 1989.  74  Quamme GA. : Magnesium: Cellular and renal exchanges. In: The Kidney: Physiology and Pathophysiology edited by D.W. Seldin and G. Giebisch. New York: Raven, p.2339-2356, 1992  75  Bapty, BW., Dai, L-J., Ritchie, G., Canaff, L., Hendy, GN., Quamme, GA.: Activation of Mg /Ca -sensing inhibits hormone-stimulated Mg uptake in mouse distal convoluted tubule cells. Am. J. Physiol. (Renal Physiology. 44): F353-360),1998.  2+  2+  2+  2+  76  Shafik, L, Quamme, G.: Early adaptation of renal magnesium reabsorption in response to magnesium restriction. Am. J. Physiol. 257:F974-F977, 1989.  77  Carney, SL., Wong, NLM., Quamme, GA., Dirks JH. : Effect of magnesium deficiency on renal magnesium and calcium transport in the rat. J. Clin.lnvest.65: 180-188, 1980.  78  Quamme, GA. Renal magnesium handling: New insights in understanding old problems. Kidney Int. 52: 1180-1195, 1997.  79  Goudeau, M.,and Goudeau, H. External Mg triggers oscillations and subsequent sustained level of intracellular free Ca , correlated with changes in membrane conductance in the oocyte of the prawn Palaemon serratus. Developmental Biology 177: 178-189, 1996. 2+  2+  114  80  Goudeau, M.,and Goudeau,H.: Depletion of intracellular Ca stores, mediated by Mg stimulated IP3 liberation or thapsigargin, induces a capacitative Ca influx in prawn oocytes. Developmental Biology, 193,225-238. 1998 2+  2+  2+  81  Hinson, TK., Damodaran, J., Chen, J., Zhang, X., Qumsiyeh, MB., Seldin, MF., Quarles, LD.: Identification of putative transmembrane receptor sequences homologous to the calcium sensing G-protein coupled receptor. Genomics 45, 279-289, 1997.  82  Naito,T., Saito,Y.,Yamamoto,J., Nozaki, Y., Tomura, K. Hazama, M., Nakaanishi, S., Brenner, S.: Putative pheromone receptors related to the Ca -sensing receptor in Fugu. Proc.Nat. Acad. Sc. USA 95,5178-5181, 1998 2+  83  Ryba, NJP., Tirindelli, R.: A new multigene family of putative pheromone receptors. Neuron 19, 371-379, 1997.  84  Nearing, J.,Bai, M., Albanese,T., Abe, T., Hebert, SC., Brown, EM., and Harris, HW.: Cloning and expression of a homologue of the calcium (Ca )/polyvalent cation sensing receptor (Car) protein that acts as a magnesium (Mg ) sensor in the dogfish shark (Squalus acanthias). Am. Soc. Nephrol., Programs and Abstracts, 30 AGM, AO 194. 1997 2+  2+  th  85  Massry, SG., and Coburn, JW. : The hormonal and non-hormonal control of renal excretion of calcium and magnesium. Nephron 10: 66-112, 1973.  86  Quamme, GA., and Dirks, JH.: Magnesium metabolism. In: Clinical Disorders of Fluid and Electrolyte Metabolism, edited by R.G. Narins, McGraw-Hill Inc., 1994, p.373-398.  87  Dai, L-J., Bapty, B., Ritchie, G., Friedman, PA., Quamme, GA.: Glucagon and arginine vasopressin stimulate Mg uptake in mouse distal convoluted tubule cells. Am. J. Physiol 274(2 pt2):F328-35,1998 2+  88  Bailly, C , and Amiel, C : Effect of glucagon on magnesium renal reabsorption in the rat. Pflugers Arch. 392: 360-365, 1982.  89  Bailly, C , Roinel, N., and Amiel, C : Stimulation by glucagon and PTH of Ca and Mg reabsorption in the superficial distal tubule of the rat kidney. Pflugers Arch. 403: 28-34, 1985.  90  Koeppen, BM., and Stanton, BA.: Sodium chloride transport: distal nephron. In: The Kidney: Physiology and Pathophysiology. Raven Press Ltd. NY. 2nd Edition. Edited by Seldin, D.W., and G. Giebisch. 1992, pp 2003-2039.  115 91  Bouby, N., Trinh-Trang-Tan, M-M., Bankir, L.: Stimulation of tubular reabsorption of magnesium and calcium by antidiuretic hormone in conscious rats: study in Brattleboro rats with hereditary hypothalamic diabetes insipidus. Pflugers Arch. 402: 458-464, 1984.  92  Elalouf, JM., Roinel, N., de Rouffignac, C: Effects of antidiuretic hormone on electrolyte reabsorption and secretion in distal tubules of rat kidney. Pflugers Arch. 401: 167-173,1984.  93  DeRouffignac, C, Corman, B., N. Roinel:. Stimulation by antidiuretic hormone of electrolyte tubular reabsorption in rat kidney. Am. J. Physiol. 244 (Renal Fluid Electrolyte Physiol. 13): F156-F164, 1983.  94  Costanzo, LS., and Windhager, EE.: Effect of PTH, ADH and cyclic AMP on distal tubule Ca and Na reabsorption. Am. J. Physiol. 239 (Renal Fluid Electrolyte Physiol. 8): F478-F485, 1980.  95  Massry, SG., Coburn, JW., Kleeman, CR.: Renal handling of magnesium in the dog. Am. J. Physiol. 216: 1460-1467, 1969.  96  Arthur, JM.: The calcium-sensing receptor (CaR) inhibits calcium transport in a model of renal distal tubule. J. Am. Soc. Nephrol. 8: 557A, 1997 (Abstract).  97  Quamme, GA., Dirks, JH.: Effect of intraluminal and contraluminal magnesium on magnesium and calcium transfer in the rat nephron. Am. J. Physiol. 238 (Renal Fluid Electrolyte Physiol. 7): F187-F198, 1980.  98  Quamme, GA., Effect of furosemide on calcium and magnesium transport in the rat nephron. Am. J. Physiol. 241 (Renal Fluid Electrolyte Physiol. 10): F340-F347, 1981.  99  Quamme, GA., Carney, SL., Wong, NLM., Dirks, JH.: Effect of parathyroid hormone on renal calcium and magnesium reabsorption in magnesium deficient rats. Pflugers Arch. 386: 59-65, 1980.  100  Quamme, GA., and J.H. Dirks. Effect of intraluminal and contraluminal magnesium on magnesium and calcium transfer in the rat nephron. Am. J. Physiol. 238 (Renal Fluid Electrolyte Physiol. 7): F187-F198, 1980.  101  Harris, CA., Burnatowska,MA., Seely, JF., Sutton,RAL., Quamme, GA., Dirks, JH.: Effects of parathyroid hormone on electrolyte transport in the hamster nephron. Am. J. Physiol. 246 (Renal Fluid Electrolyte Physiol. 15): F745-F756, 1984.  116  102  deRouffignac, C. , Elalouf, JM., Roinel, N., Bailly, C, and Amiel, C: Similarity of the effects of antidiuretic hormone, parathyroid hormone, calcitonin and glucagon on rat kidney. In: Nephrology, edited by R.R. Robinson: Springer, 1984, p340-357.  103  Poujeol, P., Touvay, C, Roinel, N. and deRouffignac, C: Stimulation of renal magnesium reabsorption by calcitonin in the rat. Am. J. Physiol. 239 (Renal Fluid Electrolyte Physiol. 8): F524-F532, 1980.  104  Elalouf, JM., Roinel, N., De Rouffignac, C: Stimulation by human calcitonin of electrolyte transport in distal tubules of rat kidney. Pflugers Arch. 399: 111-118, 1983  105  Bailly, C, Imbert-Teboul, M., Chabardes, D., Hus-Citharel,A., Montegut, M., Clique, A., Morel, F.: The distal nephron of rat kidney: a target site for glucagon. Proc. Natl. Acad. Sci. USA 77: 3422-3424, 1980.  106  Morel, F.: Sites of hormone action in the mammalian nephron. Am. J. Physiol. 240 (Renal Fluid Electrolyte Physiol.9): F159-F164, 1981.  107  Morel, F., Imbert-Teboul, M., Chabardes.D.: Receptors to vasopressin and other hormones in the mammalian kidney. Kidney Int. 31: 512-520, 1987.  108  Friedman, PA. and Gesek, FA.: Calcium transport in renal epithelial cells. Am. J. Physiol. 264 2(Renal Fluid Electrolyte Physiol. 38): F181-F198, 1993.  109  Friedman, PA., Coutermarsh, BA., Kennedy, SM., Gesek, FA.: Parathyroid hormone stimulation of calcium transport is mediated by dual signalling mechanisms involving protein kinase A and renal protein kinase C. Endocrinology 137: 13-20, 1996.  110  Hilal, G., Claveau, D., Zuo,Q.,Brunette, MG.: Interaction of second messengers on Ca uptake by the renal distal luminal membranes. J. Am. Soc. Nephrol. 6: 950, 1995.(Abstract)  111  Ahloulay, M., Dechaux, M., Laborde, K., Bankir, L.: Influence of glucagon on GFR and on urea and electrolyte excretion: direct and indirect effects. Am. J. Physiol. 269 (Renal Fluid Electrolyte Physiol. 38): F225-F235, 1995.  112  Wittner, M., Di Stefano, A.: Effects of antidiuretic hormone, parathyroid hormone and glucagon on the cortical and medullary thick ascending limb of Henle's loop of the mouse nephron. Pflugers Arch.415: 707-712, 1990.  2+  117  113  Friedman, PA., Gesek, FA.: Vitamin D accelerates PTH-dependent calcium transport in distal convoluted tubule cells. Am. J. Physiol. 265 (Renal Fluid Electrolyte Physiol. 34): F300-F308,1993.  114  Lajeunesse, D., Bouhtiauy, I., Brunette, M.G.: Parathyroid hormone and hydrochlorothiazide increase calcium transport by the luminal membrane of rabbit distal nephron segments through different pathways. Endocrinology 134: 35-41, 1994.  115  Bailly, C, Roinel, N., Amiel, M.: PTH-like glucagon stimulation of Ca and Mg reabsorption in Henle's loop of the rat. Am. J. Physiol. 246 (Renal Fluid Electrolyte Physiol. 15): F205-F212,1984.  116  Di Stefano, A., Wittner, M., Nischke, R., Braitsch, R., Greger, R.,. Bailly, C, Amiel, C, Elalouf, JM., Roinel, N , de Rouffignac, C: Effect of glucagon on Na , CI, K , Mg and Ca transport in cortical and medullary thick ascending limbs of mouse kidney. Pflugers Arch. 414: 640-646,1989.  3  +  +  2+  2+  117  deRouffignac, C: Multihormonal regulation of nephron epithelia: achieved through combinational mode? Am. J. Physiol. 269 (Regulatory Integrative Comp. Physiol. 38): R739-R748, 1995.  118  Le Grimellec C, Roinel, N., Morel, F.: Simultaneous Mg, Ca, P, K, Na and CI analysis in rat tubular fluid. II. During acute Mg plasma loading. Pflugers Arch. 340: 197-210, 1973.  119  Le Grimellec C, Roinel, N., Morel, F.: Simultaneous Mg, Ca, P, K, Na and CI analysis in rat tubular fluid. III. During acute Ca plasma loading. Pflugers Arch. 346: 171-189, 1974.  120  Quamme, GA.: Effect of hypercalcemia on renal tubular handling of calcium and magnesium. Can. J. Physiol. Pharmacol. 60: 1275-1280, 1980.  121  Quamme GA, Dirks JH. : Effect of intraluminal and contraluminal magnesium and calcium transport in the rat nephron. Am J Physiol 238:F187-F198, 1980.  122  Quamme, GA.: Effect of furosemide on calcium and magnesium transport in the rat nephron. Am. J. Physiol. 241 (Renal Fluid Electrolyte Physiol. 10): F340-F347, 1981.  123  Hebert SC.: Extracellular calcium-sensing receptor: implications for calcium and magnesium handling in the kidney. Kidney Int. 50:2129-2139, 1996  118  124  Wong, NLM., Dirks, JH., Quamme, GA.: Tubular maximum reabsorption capacity for magnesium in the dog. Am. J. Physiol. 244 (Renal Fluid Electrolyte Physiol. 13): F78-F83, 1983.  125  Di Stefano, A., Roinel, N., de Rouffignac, C., Wittner, M.: Transepithelial Ca and Mg transport in the cortical thick ascending limb of Henle's loop of the mouse is a voltage-dependent process. Renal Physiol. Biochem. 16: 157-166, 1993.  126  Doucet, A, Katz , Al.: Mineralocorticoid receptors along the nephron: [H] aldosterone binding in rabbit tubules. Am. J. Physiol. 241 (Renal Fluid Electrolyte Physiol. 10): F605-F611, 1981.  127  Farman, N., Bonvalet, JP.: Aldosterone binding in isolated tubules. III. Autoradiography along the rat nephron. Am. J. Physiol. 245 (Renal Electrolyte Physiol. 15): F606-F614, 1982.  128  Farman, N., Vandewalle, A., Bonvalet, JP.: Aldosterone binding in isolated tubules. II. An autoradiography study of concentration dependency in the rabbit nephron. Am. J. Physiol. 245 (Renal Fluid Electrolyte Physiol. 12): F69-F77, 1982.  129  Krozowski, ZS., Rundle, SE., Wallace, C., Castell, MJ., Shen, JH., Dowling, J., Funder, JW., Smith, Al.: Immunolocalization of renal mineralocorticoid receptor with an antiserum against a peptide deduced from the complementary deoxyribonucleic acid sequence. Endocrinology 125 :192-198, 1981.  130  Koeppen, BM., Stanton, BA.: Sodium chloride transport: distal nephron. In: The Kidney: Physiology and Pathophysiology, edited by D.W. Seldin and G. Giebisch. New York: Raven, 1992, p.2003-2040.  131  Rossier, BC., Palmer, LG.: Mechanisms of aldosterone action on sodium and potassium transport. In: The Kidney: Physiology and Pathophysiology, edited by D.W. Seldin, and G. Giebisch. New York, Raven, 1992, p. 1373-1410.  132  Chen, Z., Vaughn, DA., Blakeley,P., Fanestil, DD.: Adrenocortical steroids increase renal thiazide diuretic receptor density and response. J. Am. Soc. Nephrol. 5: 1361-1368, 1994.  133  Velazquez, H., Bartiss, A., Bernstein, P., Ellison, DH.: Adrenal steroids stimulate thiazidesensitive NaCl transport by rat distal tubules. Am. J. Physiol. 270 (Renal Fluid Electrolyte Physiol. 39): F211-F219, 1996.  2+  2+  3  119 134  Doucet, A., Barlet-Bas, C, Siaume-Perez, S., Khadouri, C, Marsy.S.: Gluco-and mineralocorticoids control adenylate cyclase in specific nephron segments. Am. J. Physiol. 258 (Renal Fluid Electrolyte Physiol. 27): F812-F820, 1990.  135  El Mernissi, G., Barlet-Bas, C. Khadouri, C., Cheval, L., Marsy, S., Doucet, A.: Short-term effect of aldosterone on vasopressin-sensitive adenylate cyclase in rat collecting tubule. Am. J. Physiol. 264 (Renal Fluid Electrolyte Physiol. 33): F821-F826, 1993.  136  Jackson, BA.,Braun-Werness,JL., Kusano, E., Dousa, TP.: Concentrating defect in the adrenalectomized rat. Abnormal vasopressin-sensitive cyclic adenosine monophosphate metabolism in the papillary collecting duct. J. Clin. Invest. 72: 997-1004, 1983.  137  Rajerison, RM., Marchetti, J.,Roy, C, Bockaert, J., Jard, S.: The vasopressin-sensitive adenylate cyclase of the rat kidney. Effect of adrenalectomy and corticosteroids on hormonal receptorenzyme coupling. J. Biol. Chem. 249: 6390-6400, 1974.  138  Schwartz, M.J., Kokko, JP.: Urinary concentrating defect of adrenal insufficiency. Permissive role of adrenal steroids on the hydroosmotic response across the rabbit cortical collecting tubule. J. Clin. Invest. 66: 234-242, 1980.  139  Dai, L-J., Ritchie, G. Bapty, B., Quamme, G.: Aldosterone potentiates hormone-stimulated Mg uptake in distal convoluted tubule cells. Am. J. Physiol. 274 (2 Pt 2): F336-341,1998.  140  Schafer, JA., Hawk, CT.: Regulation of Na channels in the cortical collecting duct by AVP and mineralocorticoid. Kidney Int. 41: 255-268, 1992.  141  Dai, L-J., Friedman, PA., Quamme, GA.: Cellular mechanisms of chlorothiazide and potassium depletion on Mg uptake in mouse distal convoluted tubule cells. Kidney Int., 51: 1008-1017, 1997.  2+  +  2+  142  Van OS, CH. : Transcellular calcium transport in intestinal and renal epithelial cells. Biochim Biophys Acta 906:195-222, 1987  143  Kumar R. : Vitamin D metabolism and mechanism of calcium transport. J Am Soc Nephrol 1:30-42, 1990  144  Liu, LT., Ng, M., Iacopino, AM., Dunn, ST., Hughes, MR., Bourdeau, JE. : Vitamin D receptor gene expression in mammalian kidney. J Am Soc Nephrol 5:1251-1258, 1994  120  145  Roth, J., Brown, D., Norman, AW., Orci, L.: Localization of the vitamin D-dependent calciumbinding protein in mammalian kidney. Am J Physiol 243:F243-F252, 1982  146  Stumpf, WE., Sar, M., Narbaitz, R., Reid, FA., De Luca, HF., Tanaka, Y. : Cellular and subcellular localization of l,25(OH)-vitamin D in rat kidney, comparison with localization of parathyroid hormone and oestradiol. Proc Natl Acad Sci USA 77:1149-1153, 1980 2  3  147  Manillier, C, Farman, N., Bonjour, JP., Bonvalet, JP. : l,25(OH) D binding along the rat nephron: Autoradiographic study in isolated tubular segments. Am J Physiol 248:F296-F307, 1985  148  Wasserman, RH., Fullmer, CS.: Vitamin D and intestinal calcium transport: facts, speculations and hypotheses. J Nutr 125: SI971-SI979, 1995  149  Borke, JL., Caride, A., Verma, AK., Penniston, JT., Kumar, R.: Plasma membrane calcium pump and 28-kDa calcium binding protein in cells of rat kidney distal tubules. Am J Physiol 257:F842-F849, 1989  150  Feher, JJ., Fullmer, CS., Wasserman, RH.: Role of facilitated diffusion of calcium by calbindin in intestinal calcium absorption. Am J Physiol 262:C517-C526, 1992  151  Bouhtiauy, I., Lajeunesse, D., Brunette, MG.: Effect of vitamin D depletion on calcium transport by the luminal and basolateral membranes of the proximal and distal nephrons. Endocrinology 132:115-120, 1993  152  Bouhtiauy, L., Lajeunesse, D., Christakos, S., Brunette, MG. : Vitamin-D-dependent calcium binding proteins increase calcium reabsorption by different mechanisms. I. Effect of CaBP-28K. Kidney Int. 45:461-468, 1994  153  Bouhtiauy, L., Lajeunesse, D., Christakos, S., Brunette, MG. : Two vitamin-D3-dependent calcium binding proteins increase calcium reabsorption by different mechanisms. I. Effect of CaBP-9K. Kidney Int. 45:469-474, 1994  154  Koster, HPG., Hartog, A., Van Os, CH., Bindels, RJM.: Calbindin-D28k facilitates cytosolic calcium transport without interfering with calcium signalling. Cell Calcium 18:187-196, 1995  2  3  3  121 155  Timmermans, JAFL, Bindels, RJM., Van Os, CH. : Stimulation of plasma membrane Ca pump by calbindin-D28k and calmodulin is additive in EGTA-ffee solutions. J Nutr 125:S1981-SI986, 1995  156  Costanzo, LS., Sheehe, PR., Weiner, IM.: Renal actions of vitamin D in D-deficient rats. Am J Physiol 226:864-870, 1974  157  Bindels, RJM., Hartog, A., Timmermans, J., Van Os, CH.: Active Ca transport in primary cultures of rabbit kidney CCD: stimulation by 1,25-dihydroxy vitamin D3 and PTH. Am J Physiol 261:F799-F807, 1991  158  Puschett, JB., Beck, WS., Jelonek, A., Fernandez, PC: Study of the renal tubular interactions of thyrocalcitonin, cyclic adenosine 3'5-monophosphate, 25-hydroxycholecalciferol and calcium ion. J Clin Invest 53:756-767, 1974  159  Huji, K., Bonjour, J-P., Fleisch, H. : Renal handling of calcium: influence of parathyroid hormone and 1,25-dihydroxyvitamin D3. Am J Physiol 236:F349-F356, 1979  160  Puschett, J.B.: Micropuncture study of the acute renal tubular transport effects of 25hydroxyvitamin D3 in the dog. Miner Electrolyte Metab 4:178-188, 1980  161  Burnatowska, MA., Harris, CA., Sutton, RAL., Seely, JF. : Effects of vitamin D on renal handling of calcium, magnesium, and phosphate in the hamster. Kidney Int. 27:864-870, 1985  162  Hanna, S. : Influence of large doses of vitamin D on magnesium metabolism in rats. Metab Clin Exp 10:735-743, 1961  163  Hanne, S., Alcock, N., Lazarus, B., Mallan, B. : Changes in gut and urinary ciliates following low calcium and magnesium diet and administration of vitamin D. J Lab Clin Med 61:220-229, 1963  164  Miller, ER., Ullrey, HG., Zutan,. CL., Hoefer, JA., Luecke, RW. : Mineral balance studies with the baby pig: Effects of dietary vitamin D level on Ca, P, and Mg balance. J Nutr 85:255-259, 1965  2+  2+  3  165  Richardson, JA., Welt, LG. : Hypomagnesemia of vitamin D administration. Proc Soc Exp Med 118:512-514, 1965  122 166  Lifshitz, F., Harrison, HC, Harrison, HE, : Effects of vitamin D on magnesium metabolism in rats. Endocrinology 81:849-853, 1967  167  Anast, CS., Gardner, DW. : Magnesium metabolism. Disorders of Mineral Metabolism 3:424506,1981  168  Delvin, EE., Salle, BL., Glorieux, FH., Adeleine, P., David, S.: Vitamin D supplementation during pregnancy: effect on neonatal calcium homeostasis. J Pediatrics 109: 328-334, 1986  169  Mortensen, L., Hyldstrap, L., Charles, P.: Effect of vitamin D treatment in hypoparathyroid patients: a study on calcium, phosphate and magnesium homeostasis. European J Endocrinol 136:25-30, 1997  170  Norman, AW., Nemere, I., Zhou, L-X., Bishop, JE., Lowe, KE., Maiyar, AC, Collins, E., Taoka, T., Sergeev, I., Farach-Carson, MC. : l,25(OH)-vitamin D , a steroid hormone that produces biologic effects via both genomic and nongenomic pathways. J Steroid Biochem Mol Biol 41:231240,1992. 2  171  3  Andersson, M., Malmendal, A., Linse, S., Ivarsson, I., Forsen, S., Svensson, A.Structural basis for the negative allostery between Ca - and Mg -binding in the intracellular Ca -receptor calbindin D9K. Protein Sci 6:1139-1147, 1997. 2+  2+  2+  172  Dai, L.-J., Bapty, B., Ritchie, G., Quamme, GA.: PGE stimulates Mg uptake in mouse distal convoluted tubule cells. Am. J. Physiol. 275: 833-839, 1998  173  Bailly, C, Imbert-Teboul, M., Chabardes, M., Hus-Citharel, A., Montegut, M., Clique, A., Morel, F.: The distal nephron of rat kidney: a target site for glucagon. Proc. Natl. Acad. Sci. USA 77: 3422-3424, 1980.  174  Chabardes, D., Imbert-Teboul, M., Gagnon-Burnette, M., Morel, F.: Different hormonal target sites along the mouse and rabbit nephrons: Biochemical Nephrology, edited by W.G. Guder, and U. Schmidt, Bern: Huber, 1978, p.447-454.  175  Lea, JP., Ertoy, D., Hollis, JL., Marrero, MB., Sands, JM.: Immunolocalization of phospholipase C isoforms in rat kidney. Kidney Int. 54(5): 1484-90, 1998.  176  Hamm, HE.: The many faces of G protein signaling. J. Biol. Chem. 273: 669-672, 1998.  177  Le Grimellec, C, Roinel, N., Morel, F.: Simultaneous Mg, Ca, P, K, Na and CI analysis in rat tubular fluid. IV. During acute Mg plasma loading. Pflugers Arch. 340: 197-210, 1973.  178  Le Grimellec, C, Roinel, N., Morel, F.: Simultaneous Mg, Ca, P, K, Na and CI analysis in rat tubular fluid. III. During acute Ca plasma loading. Pflugers Arch. 346: 171-189, 1974.  2+  2  

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0089210/manifest

Comment

Related Items