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Novel magnesium influx pathways in isolated renal thick ascending limb cells Dai, Long-jun 1992

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NOVEL MAGNESIUM INFLUX PATHWAYS IN ISOLATED RENAL THICK ASCENDING LIMB CELLS by LONG-JUN DAI B.M.Sc.,Weifang Medical School,China, 1978 M.Sc.,Capital Institute of Medicine,China,1986  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Medicine)  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA October 1991 © Long-jun Dai, 19 1  In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission.  (Signature)  Department of The University of British Columbia Vancouver, Canada  Date  DE-6 (2/88)  ^C^f  ii ABSTRACT This study was performed to identify the specific magnesium influx pathways in single cortical thick ascending limb (cTAL) cells of the loop of Henle. The segment of thick ascending limb of the Henle's loop plays an important role in renal magnesium homeostasis. Upto 50-60% of the filtered magnesium is reabsorbed in this part and various hormones control the balance of magnesium in the same level. Primary cell cultures were prepared from porcine kidneys using a double antibody technique. Intracellular free magnesium concentration ([Me] i) was determined using magnesium fluorescent dye, mag-fura-2 and the measurement of magnesium refill rates (d[Me] jdt) was used as a means to characterize the magnesium pathway in the plasma membrane. Isolated cTAL cells were pretreated with Me-free media for 16-24 hours and d[Me] ;/dt was tested in magnesium-rich buffer solution. Basal [Me] ; was 0.52±0.02 mM (mean ± S.E.) in normal cells and 0.27±0.03 mM in magnesium-depleted cells. Cells cultured (16 h) in high magnesium media (5 mM) maintained basal [Me] ; , 0.48± 0.02 mM, in the normal range. The decrease of [Mg"J i in Mg-depleted cells was associated with a significant increase in net magnesium transport, (control, 0.19±0.03 and low Mg , 0.35 ±0.01 nmol.mg 1 protein.minl as assessed by 28Mg uptake. [Me], returned to normal basal levels, 0.53±0.07 mM, with a refill rate of 210±25 nM.s-i . Me entry was not changed by 5.0 mM Ca" or 2 mM Sr", Cd", Co", nor Ba" but was inhibited by MezLa":-.1Gd"k:Ni"^::Zek:Be" at 2 mM. Intracellular Ca" and 'Ca uptake was not altered by magnesium depletion or Me refill, indicating that the entry is relatively specific to Me. Me uptake was inhibited by nifedipine (117±20 nM.s 4 ), verapamil (165±34 nM.s -1 ), and diltiazem (194± 19 nM.s -1 ) but enhanced by the  111 dihydropyridine analogue, Bay K 8644 (366±71 nM.s 4 ). These antagonists and agonists were reversible with removal and [Me] subsequently returned to normal basal levels. Mg 2 + ;  entry rate was concentration and voltage dependent and maximally stimulated after 4 h in Me-free media. Cellular magnesium depletion results in increase in a Me refill rate which is dependent, in part, on de novo protein synthesis. These data provide evidence for novel Me entry pathways in cTAL cells which are specific for Me and highly regulated. These entry pathways may be magnesium channels and involved with renal Me homeostasis.  iv  Table of Contents Page ABSTRACT^  ii  List of Figures^  vii  List of Tables ^  viii  Acknowledgement^  ix  INTRODUCTION^  1  I. Chemical properties of magnesium ^  1  II. Function of Mg' in metabolic processes ^  2  II.1. Role in the evolutionary process ^  2  11.2. Role in metabolic functions ^  3  III.Magnesium homeostasis ^  5  III.1. Distribution in the body ^  5  111.2. Absorption and elimination ^  6  IV. Renal handling of magnesium ^  8  W.1. Glomerular filtration ^  9  IV.2. Proximal tubule^  9  W.3. Loop of Henle ^  11  IV.4. Distal tubule and collecting duct ^  13  V. Significance and purpose ^  13  MATERIALS AND METHODS^  15  I. Methods^  15  I.1. Isolation of cortical thick ascending limb (cTAL) cells ^15 I.1.1. Culture dishes coated with anti-goat IgG ^ 15 1.1.2. Dispersion of inner cortex tissue ^  15  1.1.3. Isolation of cTAL cells^  16  1.2. Measurement of intracellular free Me+ and Ca 2 +^ 17 1.2.1. Cell loading with fluorescent probes^  19  1.2.2. De-esterification^  19  1.2.3. Determination of [Me] and [Cal  19  ;  ;^  1.3. 'Mg, "Ca, 121), and 32P uptake measurement^  22  1.4. Cellular cAMP assay^  22  1.5. Statistics^  22  II. Materials^ RESULTS^ I. Characteristics of primary cTAL cells ^  23 24 24  I.1. Morphological appearance ^  24  1.2. Hormonal responses ^  24  1.3. Functional characteristics ^  24  II. Basal Me+ levels ^  26  II.1. [Me] in normal cTAL cells^  26  11.2. [Mgl in Mg-depleted cTAL cells^  26  III.Refill ---- a model for Me+ pathway study^  30  ;  ;  vi N. The study of magnesium influx pathway^  36  IV.1. The relationship between [Me] and Mg" refill rate ^ 36 ;  W.2. The effect of external Me+ on Mg' refill rate ^ 36 IV.3. The effect of membrane potential on Mg" refill rate ^39 N.4. The effect of protein synthesis inhibitors on Mg" refill rate ^41 IV.5. Specificity of Mg" influx pathway ^  43  IV.5.1. The effect of inorganic cations on Mg" influx ^ 43 IV.5.2. The effect of organic Ca" channel blockers on Mg" entry^ DISCUSSION^  46 51  I. The role of intracellular free magnesium in regulation of magnesium transport across the plasma membrane^  52  I.1. The membrane potential determines the driving force for magnesium movement ^  53  1.2. Protein synthesis is involved in the regulation of magnesium entry pathways ^ 57 II. The specificity of magnesium influx pathway^  58  II.1. The effect of inorganic cations on magnesium influx ^59 11.2. The effect of organic Ca" channel blockers on magnesium influx ^60 III. Summary^ REFERENCES^  62 63  VII  List of Figures Figure^  Page  1. The metabolism of magnesium^ 2. Summary of the tubular handling of magnesium^  7 10  3. Excitation spectra of mag-fura-2 as a function of free Me ^18 4. Measurement of intracellular Me concentration with mag-fura-2^ 21 5. Different responses of primary cTAL cells to PTH and glucagon ^25 6. [Me] in normal and Mg-depleted cTAL cells ^  27  7. Distribution of [Me] in single confluent cTAL cells^  28  8. Effect of Me-free medium incubation on [Mgl  29  ;  ;  ;^  9. Intracellular free Me concentration in normal and Mg-depleted cTAL cells ^31 10. Dose-dependent Me refill rate in Mg-depleted and normal cTAL cells ^33 11. [Cal in normal and Mg-depleted cTAL cells ^ ;  34  12. Stimulation of net 'Mg uptake in Mg-depleted cTAL cells ^35 13. The relationship between [Mgl and Me refill rate^  37  14. Membrane potential-dependence of Me refill in Mg-depleted cTAL cells ^38 15. Effect of protein synthesis inhibitors on the adaptive increase in Me influx ^ 16. Specificity of Me influx pathway ^  42 45  17. Effect of dihydropyridine antagonists and agonists on Me influx pathway ^47  viii  List of Tables Table 1. Physiologic roles for magnesium^  Page 4  2. Factors which alter renal magnesium reabsorption ^  12  3. Effect of voltage on Mg' refill rate in Mg-depleted cTAL cells ^  40  4. Inhibition of Me+ influx with inorganic cations ^  44  5. Effect of organic Ca 2 + channel blockers and activators on Me+ influx pathway^ 6. Effect of Na' channel antagonists on Me+ influx pathway^  48 50  ix  Acknowledgement  I would like to thank Dr. Gary A. Quamme for his guidance and support throughout this work. I am grateful to Don Huysmans, Gordon Ritchie, and Kim Britton for their technical assistance.  1  INTRODUCTION  Magnesium is the eighth most abundant element in nature forming an estimated 2.1% of the earth's crust. In the human body it is the second most abundant intracellular cation (second only to potassium), and it is the fourth most abundant cation (1,2,3). The abundance and special properties of magnesium account for its key role in many metabolic functions in the body. All cells contain the two major divalent cations, Ca" and Mg 2 +. However, our knowledge of the biochemical and physiological roles of these cations differs greatly. Calcium has been and continues to be extensively studied, primarily because of its role in regulation of stimulus-secretion coupling, in initiation of muscle contraction and as a messenger for numerous hormones and transmitters. In contrast, very little attention has been focused on the physiological role of Me+ within cells. Recently, more attention has been paid to magnesium. Intracellular Mg 2 + plays a major biological role as a general stabilizer of membrane structures, the ubiquitous chelator of intracellular ATP, an important but largely passive participation in phosphate group transfer reactions, and a necessary factor for hundreds of enzymes, and probably as a regulatory cation for intracellular metabolism and hormonal responses.  I. Chemical properties of magnesium Magnesium has an atomic weight of 24.312. Its atomic number is 12; its valence, 2. The nucleus of the magnesium atom contains 12 neutrons and 12 protons. The configuration of the orbital electrons is as follows: K shell, 2; L shell, 8; and M shell, 2. The tendency to attain the stable electronic configuration of the inert gases with eight  2  electrons in the outer shell causes magnesium to give up two electrons, thus forming a Mg' ion with two positive electrical charges. The size of a cation is one factor that determines its solubility and ligand-binding characteristics (4). The binding energies of complex ions are determined, in part, by the ionic radius; the smaller the ionic radius, the greater the binding energy and the greater the tendency to form complex compounds. Among the biologically important cations, Mg" possesses the smallest ionic radius. It has an ionic radius two-thirds that of Ca" and Na' and half of IC+ (Mg', 0.78 A; Ca", 1.06 A; Nat, 0.98 A; and Kt, 1.33 A). Mg' ions form complexes to a special degree, because of its small ion volume and divalence, complexing not only with other ions but also with many molecules of a dipolar nature. Ligands for Mg' contain highly electronegative electron donors; Mg' is most stably complexed by phosphate and carboxylate anions or by the lone pair of electrons of nitrogen. Owing to its greater polarizing ability, Mg' has a larger hydration energy than Ca2 +, and magnesium salts of long-chain organic acids are more soluble than the analogous calcium salts (4). The charge and electronic configuration of Mg2 + and Ca" are similar but the smaller ionic radius of Mg' results in stronger bonds with unidentate ligands and may cause steric stain with some multidentate ligands. The different polarizing abilities result in different charge distributions in the metal complexes. The result is a large biological difference between the two cations.  II. Function of Mg' in metabolic processes Role in the evolutionary process  3  Aikawa (4) postulated that 3.5 billion years ago the Mg' ion combined with porphyrin rings, owing to the chelating properties of Me, to form chlorophyll. Formation of the chlorophyll molecule resulted in photosynthesis, and therefore made an oxygen-rich environment possible. Magnesium in the chlorophyll molecule is essential for the process of capturing photons from the sun and converting them to adenosine triphosphate (ATP). The Mg-porphyrin complex, excited by the sun, is capable of undergoing reversible photochemical oxidation or reduction, ie, accepting or donating electrons between partner molecules. This process leads to the formation of ATP and oxygen; both are necessary for the next higher step in the evolutionary process, oxidative phosphorylation. The first primitive cell, in all probability, was not as complicated as the present-day cells. If this very ancient cell functioned in a manner similar to a present cell, a minimum of about 100 different protein molecules would have been required in order to maintain protein synthesis and anaerobic energy production. Incorporated on this ancient cell was a series of metal ions. The main metal ion catalyst at that time, perhaps the only one, must have been the Mg' ion, since almost all of the reactions involved in fundamental cell reactions, such as protein biosynthesis and anaerobic energy production, require Me+ ions. Also, Mg' ions are known to catalyze several prebiotic condensation reactions. The further assumption that Me ions were important early in bio-evolution arises from its presence in seawater and sea sediment, an environment similar to that from which our cell system has assumed evolved.  11.2. Role in metabolic functions Me+ plays a critical and necessary role in intracellular metabolism. It has been  4  identified as a cofactor in over 300 enzymatic reactions involving energy metabolism and protein and nucleic acid synthesis (Table 1). Me+ participates in such reactions through  Table 1. Physiologic roles for magnesium* A. Enzyme substrates (ATPMg, GTPMg) 1. Kinases (hexokinase, creatine kinase, protein kinase) 2. ATPase or GTPase (Na, K ATPase, Ca ATPase) 3. Cyclases (adenylate cyclase, guanylate cyclase)  B. Direct enzyme activation  1. Phosphofructokinase 2. Creatine kinase 3. 5-Phosphoribosyl-pyrophosphate synthetase 4. Adenylate cyclase 5. Na, K, ATPase  C. Influence membrane properties 1. Nerve conduction 2. Ca channel activity 3. Ion transporters * From Rude (5).  its ability to form a chelate, ie, an organometallic coordination complex. A variety of modes of this action have been identified: 1, alteration of the structural conformation of substrates, eg, allowing more ready cleavage of the terminal phosphate of ATP and its attachment to other molecules; 2, coordination of a substrate to the active enzyme site; 3, binding directly to an enzyme to stabilize it in its active conformation; and 4, binding catalytic subunits together to make an active enzyme. All enzymatic reactions that involve ATP show an absolute requirement for Me. Me+ coordinates with the ATP molecule to form the true substrate and, in addition may stabilize the terminal phosphate bond of  5 ATP to facilitate transfer of phosphate to other molecules. It also serves to neutralize the negative charge density on the ATP molecule and facilitate its binding to the enzyme participating in biochemical reactions. There is a growing body of information that suggests magnesium may have a function as a regulator of certain cell processes. All enzyme reactions that involve ATP show an absolute requirement for magnesium (4). These reactions encompass a very wide spectrum of synthetic processes. It has been postulated that Mg 2 + acts as a long-term regulatory element that controls the "set point" for intracellular metabolism and hormonal response. In this sense, intracellular Mg 2 + might be regarded as a static rather than a dynamic regulator of cell function (6-9). This is supported by in vitro studies that show a primary disturbance in energy metabolism within the mitochondria during magnesium deficiency, resulting in restricted protein synthesis (10,11). Furthermore, magnesium is important in governing key rate-limiting steps in the cell cycle, particularly at the onset of DNA synthesis and at mitosis (12). Thus, the concentration of intracellular Mg 2 + may affect the regulation of protein synthesis and the cell cycle, which supports the hypothesis that magnesium has a central role in the regulation of metabolism and growth (6).  III. Magnesium homeostasis III.1. Distribution in the body The average adult human body contains approximately 24 gm (1 mole) of magnesium. Magnesium is distributed unevenly, with the greatest concentration in tissues having the highest metabolic activity, such as the brain, heart, liver and kidney. Approximately 60% of body magnesium is in the bone; a third of this is exchangeable and is believed to act  6  as a reservoir for maintenance of normal serum magnesium levels in times of increased utilization or decreased intake. An additional 35% of magnesium is found in the muscle, particularly in the skeletal and cardiac structures. Approximately 1% of total magnesium is in the extracellular fluid compartment; one third of this is nonspecifically bound to plasma protein, leaving the remainder as the diffusible or ionized component (6). Intracellular magnesium is compartmentalized. Most of the magnesium expressed as percent of total magnesium is found in microsomes, followed by mitochondria and nucleus (13). In mitochondria, magnesium is further compartmentalized. From the total mitochondrial magnesium, 4% is localized in the outer membrane, 50% is localized in the intermembranous space, 5% is localized in the inner membrane, and 41% is localized in the matrix (14). About 15% of total intracellular magnesium is found in the cytosol, of which 90% is thought to be complexed to ATP and other Me-binding ligands and the remainder, 10%, is ionized (2,13).  111.2. Absorption and elimination The homeostatic mechanisms for maintaining the serum magnesium concentration within limits are poorly understood. The major factors that appear to regulate magnesium balance are absorption from the gastrointestinal tract and excretion by the kidney, the latter being the more important for controlling serum magnesium concentration. A small amount of magnesium also enters and leaves the bone, in skeletal formation and resorption (Fig.1). The average American diet contains 200 to 350 mg/day of magnesium (15). Approximately 30 to 50% (100 to 150 mg/day) of this is absorbed, depending on intake: the more  7  Fig.1. The metabolism of magnesium. Approximately 30% of ingested magnesium is absorbed; the kidney filters an average of 2.5 g, excreting 100 mg/day to maintain balance. A small amount of magnesium is used by the bone for synthesis and bone resorption. [Abstracted from Rude (6)]  8  magnesium ingested, the less efficient the absorption. A benefit of the chloride salt of magnesium is that it does not require preliminary gastric conversion in the stomach. Magnesium is absorbed throughout the intestine; the predominate site is the distal small intestine. There are three mechanisms by which magnesium crosses the intestine: diffusion, solvent drag, and active transport. Studies in both humans and experimental animals indicate that passive diffusion through the paracellular pathway accounts for the majority of magnesium absorbed (16). The exact transport mechanisms involved in intestinal magnesium absorption are unclear. Vitamin D and its metabolite 1,25-dihydroxy-vitamin  D have been shown to enhance magnesium absorption in some studies, but to a lesser extent than for calcium (17,18). A more sensitive degree of magnesium regulation occurs at the renal level via a filtration-reabsorption process (2,19). This will be discussed in section IV.  IV. Renal handling of magnesium Although no single homeostatic control has been demonstrated for magnesium, the cellular availability of this cation is closely regulated by the kidney. Present evidence suggests that the renal handling of magnesium is normally a filtration-reabsorption process (2,20). Segmental magnesium transport has been described with the aid of micropuncture techniques in a large number of animal species. In all mammals studied to date, magnesium is handled in different ways along the nephron segments. Fig.2 summarizes the segmental handling of filtered magnesium. Up to 80% of the total plasma magnesium (1.5-2.0 mM) is filtered through the glomerular membrane. Of the filtered magnesium (1.2-1.6 mM), 20-25% is reabsorbed by the proximal nephron whereas 60-70% of filtered  9  sodium and calcium are reabsorbed in this segment. Accordingly, the delivery of magnesium to the thick ascending limb of the Henle is relatively much greater than that of sodium and calcium. It is now evident from micropuncture studies that proportionally greater amounts of magnesium (50-60%) are reabsorbed in the loop compared with sodium (20-25%) or calcium (30-35%). Because the terminal nephron segments, including the distal convoluted tubule and collecting tubule, reabsorb only a small portion of the filtered magnesium (5%), the loop of Henle plays a major role in the determination of magnesium reabsorption and it is these segments where major regulatory factors act to maintain magnesium balance (21).  W.1. Glomerular filtration The ultrafilterable portion of plasma magnesium is approximately 70-80%. Of the filterable magnesium, only 70-80% is thought to be in the ionic form (Mg 2 +), the remainder being largely complexed to anions, particularly phosphate, citrate, and oxalate. The ultrafilterability of plasma magnesium is not affected by magnesium deficiency, magnesium excess, or elevation of plasma calcium concentration (22). This suggests that hormonal changes with these conditions have no effect of the glomerular filtration of magnesium.  IV.2. Proximal tubule The proximal convoluted tubule reabsorbs about 25% of the magnesium filtered at the glomerulus (Fig.2). Luminal magnesium concentration rises along the length of the proximal convoluted tubule, and in the late proximal convoluted tubule the magnesium  10  Fig.2. Summary of the tubular handling of magnesium. Inset: schematic illustration of the cellular transport of magnesium within the thick ascending limb of the loop of Henle. [Abstracted from Quamme (23)]  11 concentration in tubule fluid may exceed by as much as 1.5-2.0-fold the plasma ultrafilterable concentration. This observation suggests that the proximal tubule epithelium possesses low permeability for magnesium relative to sodium or calcium (24). Under various circumstances in which it has been indirectly examined, the tubular fluid magnesium concentration closely follows water reabsorption by proximal tubule (25,26). This can be explained by a widening of the intercellular pathways with increased permeability, or, alternatively, a diminution in the active cellular transport of magnesium. However, unidirectional magnesium fluxes and permeabilities have not been studied in vitro in proximal convoluted tubules and an exact definition of active and passive transport mechanisms remains to be determined.  IV.3. Loop of Henle The early micropuncture studies indicated that the loop of Henle was the major site of magnesium reabsorption (27,28). Some 50-60% of the filtered magnesium was reabsorbed between the last accessible portion of the proximal tubule on the surface of the kidney and the early distal tubule. More recent micropuncture studies have shown that the major site of the marked magnesium reabsorption must be located in the thick ascending limb of Henle's loop (29). Fig.2 demonstrates passive movement of magnesium through the paracellular pathway dependent on transepithelial voltage, and active movement through the cell dependent on ATP-dependent mechanisms. The mechanism by which magnesium moves across the luminal and basolateral membranes is also speculated (Fig.2). Magnesium may cross the luminal membrane in a mediated manner, down its electrical gradient which is established  12  Table 2. Factors which alter renal magnesium reabsorption Decrease reabsorption Extracellular volume expansion Hypermagnesemia Hypercalcemia Loop diuretics - furosemide, ethacrynic acid Osmotic diuretics - urea, mannitol Phosphate depletion Acute metabolic acidosis Carbohydrate, protein, alcohol ingestion Increase reabsorption Extracellular volume depletion Magnesium depletion Hypocalcemia cAMP-mediated hormones Parathyroid hormone Glucagon Calcitonin Antidiuretic hormone * Abstracted from Quamme and Dirks (20).  by the active Na-K-ATPase system on the basolateral membrane. The transfer of magnesium across the basolateral membrane is against its electrical gradient and requires active transport. Table 2 lists some of the factors which affect renal magnesium homeostasis, many of which occur in the loop of Henle. Even though there are many questions regarding the specificity of the effects observed on magnesium reabsorption in the segments that compose the loop of Henle, a clear picture has emerged indicating that the loop of Henle is the major modulator of renal magnesium homeostasis (30). Most of the interactions that account for changes in overall urinary magnesium excretion appear  13  to operate at the level of the loop of Henle. IV.4. Distal tubule and collecting duct Under normal conditions the distal convoluted tubule has the ability to reabsorb a small but still significant portion of delivered magnesium accounting for some 2-5% of the filtered magnesium. The transport of magnesium within the distal convoluted tubule is load-dependent. Experimentally, as the delivery of magnesium is elevated either by high magnesium concentration in the perfusate or by inhibition of proximal reabsorption by furosemide, the absolute reabsorption of magnesium increases (21,24). Distal magnesium reabsorption is normally close to capacity, whereas the rate of calcium and sodium transport is ordinarily under capacity. Most of the studies reported to date suggest that the collecting ducts play a very limited role in renal magnesium transport and may account for the reabsorption of less than 1-3% of the filtered magnesium (Fig.2).  V. Significance and purpose Various studies suggest that the availability of magnesium for human consumption can influence human health (4). Magnesium deficiency has been linked to several diseases, including atherosclerosis, myocardial infarction, congestive heart failure, hypertension, cancer, renal stones, premenstrual syndrome and so on (6,31,32). Clinical and laboratory studies have shown that the administration of MgCl 2 or ATP-MgC1 2 has proven beneficial in recovery from various diseases, such as haemorrhagic and endotoxin shock, postischemic hepatic failure, traumatic brain injury, myocardial infarction, congestive heart failure, and acute renal failure (31-39). In order to know the mechanisms by which magnesium protects cells and/or the body against various diseases, it is necessary to investigate  14  magnesium transport processes and their regulations. Although we have considerable understanding of transepithelial magnesium movement and some of the factors that control transport, little is known about the intracellular regulation of magnesium. The purpose of the present study is to characterize the controls of intracellular Me+ concentration in isolated cortical thick ascending limb (cTAL) cells. As previously mentioned, magnesium is essential to living body and the thick ascending limb of Henle's loop is the major site of magnesium reabsorption as well as the major modulator of renal magnesium homeostasis. It is of physiological and pathophysiological importance to study the mechanisms by which the kidney deals with the dynamic magnesium balance at the level of the thick ascending limb.  15  MATERIALS AND METHODS  I. Methods I.1. Isolation of cortical thick ascending limb (cTAL) cells Double antibody immunoadsorption was used for isolation of cTAL cells. This method is based on the unique distribution of Tamm-Horsfall protein along surface membranes of the cTAL of virtually all common laboratory animals and humans (40,41). We isolated cTAL cells from pig inner cortex using the method described by Allen and colleagues (42) with minor modifications. I.1.1. Culture dishes coated with anti-goat IgG. All procedures were performed under sterile conditions. 5 ml of phosphate-buffered saline (PBS; composition in mM: 137 NaCI, 2.7 KCI, 8.1 Na 2HPO 4, pH 7.4) containing 80 pig of affinity-purified rabbit anti-goat IgG was added to each of four culture dishes (Corning, 80 mm), and the dishes were incubated overnight at 4°C. Immediately before the antibody-coated dishes were to be used for immunoadsorption, the antibody solution was aspirated and the dishes were washed four times with 3 ml of 1% bovine serum albumin in PBS. Finally, the dishes were tilted near upright for a few moments and excess liquid was removed by aspiration. 1.1.2. Dispersion of inner cortex tissue. Young pigs (30-50 days old) were sacrificed with a lethal dose of pentobarbital sodium administered through cardiac puncture. The kidneys were removed and placed in the ice cold buffer solution, and then sliced. Tissue from the innermost third stripe of the cortex was dissected and washed three times with ice cold HEPES-buffered Krebs solution (HBK; composition in mM: 5 KCI, 145 NaCl, 1 Na2HPO4, 5 glucose, 1 CaC1 2, 0.5 MgC12, and 10 HEPES; pH 7.4). Approximately 40 ml  16  of 0.1% collagenase in HBK with 1% bovine serum albumin (BSA) was added to the cortical tissue (about 12 gm from two kidneys). The tissue was incubated at 37°C for about 5 min in a shaker. Cell dispersion was evidenced by the appearance of numerous large tubule fragments. The digestion was stopped by adding appropriate amounts of ice cold HBK. Cell suspension was collected through a tea strainer and centrifuged at 700 rpm for 2 min. 1.1.3. Isolation of cTAL cells. The cell pellet was washed three times with HBK, then, resuspended in 10 ml of DMEM medium containing 100 Al of sterile goat anti-human uromucoid serum (50 mg protein/m1). The incubation was continued for 30 min on ice with occasional swirling. The cells were then collected by centrifugation, washed twice with PBS, and resuspended in 4 ml of PBS. 1 ml of cell suspension was applied to each of four antibody-coated dishes in four successive equal aliquots. Each aliquot was allowed to stand on the plates for 5 min; nonadherent cells were removed with one wash with 5 ml of PBS. The dishes were washed six times with 5 ml of PBS following the fourth incubation. To dislodge freshly isolated cTAL cells from the dishes, 5 ml of PBS was added to each dish and the dish was tapped sharply on the side several times with a scalpel handle. The suspended cells were then pipetted into a sterile tube and collected by centrifugation. The cell pellet was resuspended in 3 ml of DMEM containing 10% fetal calf serum, 5 mM L-glutamine, 50 units/ml penicillin, and 50 µg/ml streptomycin. The cTAL cell suspension was plated on glass coverslips or plastic multiwell dishes. The cTAL cells grew on the appropriate support in 95-5%, air-CO 2 . About one week after seeding, the cTAL cells were used for the experiment.  17  1.2. Measurement of intracellular free Me and Ca" The fluorescent Me and Ca" indicators, mag-fura-2 and fura-2, are widely used for the direct measurement of intacellular free Me concentration ([Mg 21 ] ; ) and intracellular free ce+ concentration ([Cal). Mag-fura-2 has similar properties as fura-2. When magfura-2 is loaded into the cell, the dye has only two molecular forms, free and Me-bound. Both mag-fura-2 and its Mg" complex fluoresce strongly, but their excitation peaks differ in wavelength. Mag-fura-2 shifts its excitation peak from about 385 to 340 nm upon binding Me. The extent of the shift between the two wavelengths depends on the amount of intracellular Mg' concentration. Fig.3 displays the changes of intensity in the two wavelengths under different Me concentrations. Measurements at two wavelengths suffice in principle to indicate the ratio of bound to free dye and hence the [Me] ; or [Ca2 +],(43-45). Since the magnesium indicator mag-fura-2 is constructed using the same aminophenol structure utilized in the calcium indicators fura-2 and indo-1, the molecule shares the same desirable insensitivity to pH fluctuations near the physiological concentration (pK = 5.0) (43). The binding ratio of mag-fura-2 to magnesium is 1:1, with the dissociation constant (KO of 1.5 mM. The IC for magnesium is slightly above the level of ionized magnesium believed to characterize most cell types. Although the affinity of mag-fura-2 for Ca" (IC = 53 AM) is somewhat greater than the affinity for Me, it is two to three orders of magnitude above the physiological ionized calcium concentration of most cells, so that interference arising from cytosolic calcium elevation is only likely to become significant under very extreme perturbations. For example, a basal [Cal ; level of 200 nM would correspond to 0.37% of mag-fura-2 complexed to Ca'. Increasing [Cal i to 1µM would  18  MAGNESIUM  0 mM Mg  Rmax = 0.607 (340/385, 50 Mg) Rmin = 0.132 (340/385, 0 Mg) Ch2 f/b = 3.75 (385, 0/50) Kd = 1.5 mM Mg (385, 50%)  0.2 0.5 1  5 10 25 50  0 300^320  1 ^  340  LI  1^1^1^ ^ ^ ^ 1 ^ I^i^I 420 380 400 360  Exitation wavelength nm  Fig.3. Excitation spectra of mag-fura-2 as a function of free Me. Fluorescence emission is collected at 500 nM. Solutions contain 5 mM Ka, 137 mM NaCI, 1 mM EGTA, and 14 mM HEPES/Tris, pH 7.4, 23°C.  19  correspond to <2% as a Ca-mag-fura-2 complex. Contrast this with 27% of the magfura-2 in a Me+ complex that occurs with a basal [Mel of 0.4 mM. Thus, even with 1 AM [Cal,, the concentration of the Ca-mag-fura-2 complex is <10% of the Mg-magfura-2 complex. Furthermore, such situations can be independently assessed by the parallel use of cytosolic calcium indicators. 1.2.1. Cell loading with fluorescent probes. Isolated cTAL cells were loaded with 10 AM fura-2/AM or 5 AM mag-fura-2/AM according to previously described techniques (46). The fluorescent dye, dissolved in dimethylsulfoxide (DMSO), was added directly to the medium with the aid of Pluronic F-127 (0.05%) and incubated for 30 min at 23°C. The final concentration of DMSO in the incubation medium did not exceed 0.2%. 1.2.2. De-esterification. Mag-fura-2/AM is hydrophobic and thus it passes easily into cells across the plasma membrane. Once inside cells, cytosolic esterases cleave the acetoxymethyl groups from the mag-fura-2 molecule rendering a compound which is highly charged and which cannot cross cellular membranes and thus is theoretically trapped in the cytosol. Since the incomplete de-esterification of the dye in cytoplasm will interfere with the [Mel measurement, cells loaded with mag-fura-2 must be de-esterified completely (43). Loaded cells were washed 2X with a buffered salt solution (in mM): 145 NaCI, 4 KC1, 1 CaCl 2, 1 KH213 04f 18 glucose, and 20 HEPES/Tris (pH 7.4) with or without Mg' depending on the different purposes of the experiments. The cells were incubated a further 20 min to ensure complete de-esterification and finally washed once with fresh buffer solution.  1.2.3. Determination of [Mel and [Cal. Cover glasses with cells loaded with either mag-fura-2 or fura-2, were mounted in a plexiglass chamber containing 250 tcl buffer and  20  placed on the mechanical stage of a Nikon inverted microscope. The fluorescence signal was monitored at 505 nm with excitation wavelengths alternating between 340 and 385 nm for mag-fura-2 and 335 and 385 nm for fura-2 using a Deltascan spectrofluorometer (Photon Technology International Inc., So. Brunswick). Averaged light intensities over excitation periods at each of the two wavelengths were used by the computer to calculate 340/385 or 335/385 ratios after background substraction. Data were stored in sequential file. At the end, [Me] ; or [Cal, was calculated as described by Grynkyewicz et al (47) and Malgaroli et al (48) based on the equation: [Me] ; = IC(R-R.A.-R)S,2/S„ equation 1. where IC D is the association constant, R. is the fluorescence ratio at the excitation wavelengths 340/385 nm for uncomplexed mag-fura-2 (zero magnesium) and 335/385 nm for uncomplexed fura-2 (zero calcium); R. is the ratio of fluorescence at the wavelengths 340/385 nm for mag-fura-2 saturated with Mg" and 335/385 nm for fura-2 saturated with Ca"; Sf, and S„ are the fluorescence intensities at 385 nm for mag-fura-2 with zero Mg" and excess Mg" and for fura-2 with zero Ca" and excess Ca", respectively. R is the ratio of fluorescence at wavelength ratios 340/385 or 335/385 of the sample to be measured. Using ratios, dye content and instrumental sensitivity are free to change between one ratio and another since they cancel out in each ratio. Of course, stability is required within each individual ratio measurement; also R, R ini. and R. should all be measured on the same instrumentation so that any wavelength biases influence all of them equally (45). Representative tracing of fluorescent intensity ratios are smoothed according to the method of Savitsky and Golay (49). Figure 4 displays representative determination of the [Mel measurement.  21  0 mM m g 2+ 30uM Dig. 100mM EDTA  100mM Mg 2+  30uM Dig . 0 mM EDTA  385 nm  Rmax^r 0.4  -  Rmin 0.3  -  340 / 385  0.2 0^200  400^600^800  TIME seconds  Fig.4. Measurement of intracellular Me concentration with mag-fura-2. Upper panel: dual wavelength traces of fluorescences from 385 and 340 nm excitation of a single cTAL cell loaded with 5 AM mag-fura-2 in DMEM medium for 30 min at 23 °C. ' Then cell medium is substituted for deesterification solution for 20 min at 23 °C. After recording the intensity in both wavelengths, cell solution is replaced with minimum solution and maximum solution respectively. Both minimum solution and maximum solution contain 145 mM NaCI and 20 mM HEPES/Tris. The concentrations of digitonin, EDTA, and Mg' are indicated in the figure. Lower panel: ratio of 340 to 385 nm fluorescence after subtraction of their respective autofluorescence.  22  1.3. 'Mg, "Ca, 'Rh and 3211 uptake measurement Mg, "Ca, "Rh and nP uptakes were performed by methods similar to those previously  28  described (46). Incubations were routinely performed with transport solution containing (in mM): 137 NaC1, 5.4 KC1, 1.0 Na 2HPO 4, 0.1 28Mg, "Ca, 'Rh or 3213 , 14 HEPES/Tris, pH 7.4 for 5 min. Incubations were terminated by rapid aspiration of the transport solution and addition of ice-cold stop solution containing (in mM): 145 NaCl, and 20 HEPES/Tris, pH 7.4. The cells were solubilized in 0.5% Triton X-100 for 1 hour. 28Mg, "Rh or "Ca uptake by the cells was measured by liquid scintillation on 150 Al of extract solution. 'Mg, "Rb and "Ca uptake was normalized according to total protein content determined by using the Lowry method (50).  1.4. Cellular cAMP assay Hormone-induced cAMP formation was determined after 10 min of incubation with agonist or vehicle and 10' M isobutylmethyl xanthine (IBMX) in cTAL cells cultured for 4-6 days on plastic supports. cAMP concentrations were assayed by radioimmunoassay with kit no. 6021 (Amersham). Protein was determined by the Lowry method after solubilization of cells with 1% SDS.  I.S. Statistics All results are expressed as mean ± S.E. where indicated. The changes in [Mel with time were determined by linear regression analysis of the tracing over the time interval of interest. Significance was determined by one-way analysis of variance. A probability of p < 0.05 was taken to be statistically significant.  23  II. Materials Dulbecco's modified Eagles' medium (DMEM) containing D-glucose (5.0 gm/L), Lglutamine (5 mM) and 10% FCS was from GIBCO. Goat anti-human uromucoid (TammHorsfall glycoprotein) serum was purchased from Organon Teknika. Affinity-purified rabbit anti-goat IgG, parathyroid hormone, arginine vasopressin, calcitonin, glucagon, and atrial natriuretic peptide were from Sigma. '16, 'Ca and cAMPI'll were obtained from Amersham and 'Mg from Martin-Maretta, Oakridge National Laboratories. Mag-fura2, fura-2, and pluronic F-127 were purchased from Molecular Probes. All other chemicals were from Sigma or Fisher Scientific Company.  24  RESULTS I. Characteristics of primary cultured cTAL cells I.1. Morphological appearance. The isolated cTAL cells when grown to confluence had a morphological appearance of epithelial cells. They had cuboidal structure when grown on filters and developed small domes, five to six cells in diameter, when cultured on solid supports for an extended period of time. The morphological appearance is consistent with the observations of Allen et al.(42). 1.2. Hormonal responses. The primary cultured cTAL cells were responsive to various hormones known to release cAMP in the thick ascending limb (51). Basal cAMP concentration was 25.9 ± 1.2 pmol.mg -1 .10 min -1 . Parathyroid hormone (PTH, M), antidiuretic hormone (10-8 M) and calcitonin (10' M) stimulated cAMP release by 1.5, 3.2, and 1.8 fold, respectively, in cells cultured for 5-6 days on plastic supports. Glucagon (7 x M) failed to stimulate cAMP formation in concert with the observations of Allen et al. (42). However, when cAMP production was measured on the cell suspension prepared from the renal tissue, glucagon stimulated cAMP release significantly (control level, 185.52 fmol/mg.protein; after glucagon, 290.65 fmol/mg.protein, n = 6, p < 0.05). Interestingly, calcium signals were observed in cultured cTAL cells stimulated by both parathyroid hormone and glucagon, but only phase I was induced by glucagon (Fig.5). The presence of receptors for parathyroid hormone, calcitonin, antidiuretic hormone and glucagon supported the notion that these cells originated from the segment of the loop of Henle. 1.3. Functional characteristics. The cTAL cells demonstrated Na/K/Cl cotransport as indicated by bumetanide sensitive 86Rb uptake, amounting to 40% of control uptake which was 0.47 ± 13 nmol.mg 1 protein.mint (n = 3). These cells also possessed sodium-  25 1000 -  A PIN  800 -  600 -  400 -  200 -  0 200^  1000 -  400^  600  B  800 GLUCAGON 800 -  1  y  400 -  200 -,  -.......*,  '',-.._.-----",..,•-..-,-  0  ,  600  200  0  Time sec.  Fig.5. Different responses of primary cTAL cells to PTH and glucagon. Intracellular Ca" concentration of single primary cultured cTAL cells were determined with fura-2 as described in the methods. (A) The effect of parathyroid hormone (PTH, 10 4 M) on [Cal in 'a single cTAL cell. Basal [Ca") level was 86.3 ± 5.5 and peak value was 520.0 ± 63.8 nM (n = 4). Intracellular free calcium was maintained in a level above the basal level, 208.8 ± 48.3 nM, afer addition of PTH. (B) The effect of glucagon (2.8 x 10' M) on [Cal in a single cTAL cell. The basal level is 73.8 ± 7.5 nM and peak value is 690.0 ± 256.8 nM (n = 4). Only one phase could be seen in the calcium transient induced by glucagon. ;  ;  ;  26  dependent phosphate transport function demonstrated by 3213 uptake. 321) uptake in the presence of Na+ was significantly greater than that in the absence of Na+, 73.7 ± 2.0 and 38.52 ± 3.5 pmol.mg 1 protein.min -1 respectively (n = 6, p < 0.01). The above characteristics indicated that the cultured cells retained many of the functions typical of thick ascending limb cells.  II. Basal Mg2 + levels II. 1. [Me] in normal cTAL cells. The basal Me level in normal cTAL cell is ;  relatively stable during the period of observation as illustrated in Fig.6. The mean basal concentration of intracellular Me was 0.52 ± 0.02 mM, n=30, and following a normal distribution (Fig.7). The mean [Me] for cTAL cells is significantly greater than for ;  MDCK cells, 0.47 ± 0.01 mM (46) or isolated cardiomyocytes, 0.46 ± 0.01 mM (52). 11.2. [Me] in Me-depleted cTAL cells. Me-free medium was used to induce ;  magnesium depletion in cTAL cells. The magnesium concentration was less than 0.01 mM as determined by atomic absorption. [Me] was decreased by 50% of normal levels ;  in the cTAL cells incubated in Me-free medium for 16 hours (Fig.6), from 0.52 ± 0.02 to 0.26 ± 0.01 mM. No further [Me] decline was found following long culture periods ;  of Mg2 +-free medium incubation (Fig.8). Intracellular Me concentrations in depleted cTAL cells were also normally distributed around the mean value (Fig.7). The effect of high magnesium medium incubation on basal [Me] was also tested in this study. Me;  rich medium incubation (5.0 mM Me, 16 hours) did not change the basal [Me], levels, 0.48 ± 0.02 mM (n = 5). The range of [Mg 2 +] is quite wide in both normal and Me-depleted cTAL cells ;  27  Normal cell  Mg-depleted ceN  !III-  11111^I^I^I^I-^I^I^I^I^I^I  400^800^1200^1600^2000  TIME seconds Fig.6. [Me] in normal and Mg-depleted cTAL cells. Intracellular free Me concentrations were measured as indicated in Fig.4. Magnesiun concentrations were 0.6 mM in normal medium and less than 0.01 mM in Me-free medium as determined by atomic absorption. The calcium concentration in both media was 1.0 mM. ;  28  Distribution of Intracellular Mg 2+in cTAL Cells 10  A  0.1^0.15^0.2^0.25^0.3  0.35  0.4  0.45  Basal [Mg2-91 mM  Fig.7. Distribution of [Me] in single confluent cTAL cells. [Me] was determined as given in Fig.4. A. Distribution of [Mel in normal cTAL cells. The cells were incubated in DMEM medium with 0.6 mM Me. The mean value was 0.52 ± 0.02 mM, n = 30; B. Distribution of [Mel in Medepleted cells. The cTAL cells were pretreated with Me-free DMEM medium for 16-20 hrs. Me concentration in this medium was less than 0.01 mM measured by atomic absorption. The mean value was 0.26 ± 0.01 mM, n = 47. The basal [Me], level in Me-depleted cells was significantly lower than that in normal cells (p < 0.01). ;  ;  29  0.8 —  0.6 —  0.2 —  1  ,  1  0^10^20^30  40^50  TIME hrs Fig.8. Effect of Me-free medium incubation on [Me] . Confluent cTAL cells were pretreated with Me-free DMEM medium for various times. [Mel was determined as given in the methods. Each time point was done in 5 cTAL cells. The value was presented as mean ± S.E. ;  30  (Fig.7). The values of coefficient of variation are 21.1% in normal cells and 26.4% in Me-depleted cells. Presumably, the biological distributions of [Me] ; are somehow narrower than those we obtained in this study. The disagreement may be due to experimental errors in the measurement of R.„., and R,„ in some cells.  III. Refill of Me- ---- a model for Me pathway study Fig.9 shows the kinetic changes of [Me], in three cTAL cells under different experimental conditions. The basal [Me] ; was 0.58 mM in normal cTAL cell (Fig.9, trace 1), and 0.3 and 0.26 mM in Mg 2 +-depleted cTAL cells (Fig.9, trace 2 and 3). The elevation of external Mg 2 + to 5.0 mM in normal cells results in no change in [Me] ; over the time of study. In Mg 2+-depleted cells, [Me]; increased after addition of 5.0 mM MgC12 to the bathing medium (Fig.9, trace 2). The increase in [Me] ; was linear with time, and abruptly stopped when the [Mg 2 +1 was near normal level, 0.52 mM (from a to b in Fig.9, trace 2). If Me-depleted cells were kept in Me-free solution, the [Me] ; maintained its low level during the period of observation (Fig.9, trace 3). The slope of increase in [Me]; after addition of Me is called the magnesium refill rate (d([Me],)/dt), given in units of nM.s 4 and calculated via following regression equation: c([Mg2+]i)/dt =  (21‘40(ET) E M1T z isp4:^(zmy^  equation 2.  The refill rate in cell 2 (from a to b in Fig.9, trace 2) is 220.5 nM.s'. The mean value of magnesium refill rate is 210.5 ± 24.9 nM.s -1 (n = 6). It is necessary to mention that this is a concentration change with time not a flux rate or conductance. If we calculate this value in terms of flux it would be about 1.31 x 10 6 ions.cell-'.s.l.  31  1  0.8 -  A 1 1 40 4  0.2 -  0 0 5.0 mM Mg2+ 0  0 0  Pm-  0 mM Mg 2 +  i^ i^ 1^ 1^ i^  1000^  2000^  3000  TIME seconds  Fig.9. Intracellular free Me concentration in normal and Me-depleted cTAL cells. Confluent cTAL cells were cultured in either normal (0.6 mM Me) or Me-free medium (<0.01 mM) for 16-20 hrs. Fluorescence studies were performed in buffer solutions in absence of Me, and, as indicated, MgC12 (5.0 mM final concentration) was added to observe changes in intracellular free Mg 2 + concentration. Fluorescence was measured at 1 data point/s with 25-signal averaging, and tracing was smoothed according to methods described by Savitzky and Golay (49).  1  32  The Me refill rate observed in magnesium-depleted cTAL cells was dose-dependent and saturable. The maximal refill rate occurred at 5.0 mM of external magnesium concentration (Fig.10). When high Me (5.0 and 50 mM) buffer solution was administered to normal cTAL cells, the Me-refill phenomenon was also observed but it was much less than that in magnesium-depleted cTAL cells (Fig.10). Interestingly, the basal [Mel level was not changed by prolonged incubation in high magnesium medium (see 11.2). In order to exclude the effect of calcium on magnesium refill intracellular Ca 2 + concentrations were determined under the same condition. Basal [Cal ; levels were not changed by either Me depletion or the addition of high concentration of Me (Fig.11). To demonstrate the source of Me during the refill process, net 'Mg uptake was measured in Me-depleted cTAL cells. Fig.12 illustrates the effect of cellular magnesiumdepletion on net 'Mg uptake into cells. Net 'Mg uptake was significantly greater in isolated cells grown in magnesium-deficient medium compared to those cells cultured in normal medium containing 0.6 mM magnesium. Under the same experimental condition Me depletion had no effect on "Ca uptake (0.81±0.07 vs 0.90±0.07 nmol.mg protein' '.min'', n = 8, p > 0.05). These observations confirmed the notion that these epithelial cells can intrinsically adapt their transport rates according to the availability of magnesium. This model provides us at least two points: first, magnesium depletion can enhance Me refill rate; second, most of Me, if not all, comes from extracellular sources during magnesium refill. That is to say, extracellular Me enters the intracellular space through an influx pathway in the plasma membrane. Theoretically, the refill rate is determined by whether the pathways are open or closed and their density in the membrane.  33  300 —  2 a  Mg-depleted cTAL cells  200  171 -  w CC  100 Normal cTAL cells  IL w CC  0  ^ ^  10  20  ^ ^ ^ 30 40 50  [Mg 21 0 mM Fig.10. Dose-dependent Me-refill rate in Mg-depleted and normal cTAL cells. Mg-depleted cTAL cells' were obtained by magnesium-free medium incubation for 16-24 hrs. Me-refill rate, d(Nel)/dt, was assessed at the external magnesium concentrationa indicated. Values are means ± S.E.  34  200180160-  ^5 . 0 mM Mg 2+  140-  C  120 100 -  0  6040-  Mg-depleted cell  200^ 0^400^800^1200^1600^2000  TIME seconds Fig.11. [cal in normal and magnesium-depleted cTAL cells. Intracellular Ca2 + concentrations were determined as indicated in methods. ;  35  400 7 *— E Oa  300 —  7 E 75 200 -  E  0  100 — cn  CONTROL  LOW Mg MEDIA  Fig.12. Stimulation of net 'Mg uptake in magnesium-depleted cTAL cells. cTAL cells were grown to confluence (7-14 days) in DMEM containing 0.6 mM magnesium. Monolayers were then cultured in normal (0.6 mM) or low magnesium (<0.01 mM) niedia for 16 hrs prior to study. Magnesium uptake was determined with 0.1 mM 'Mg over 5 min. Incubations were terminated by rapid aspiration of the transport solution and addition of icecold stop solution. Cells were solubilized in 0.5% Triton X-100 and 28Mg was determined on the extraction solution. * indicates statistical significance (p < 0.05) from control.  36  IV. The study of magnesium influx pathway IV.1. The relationship between [Me] ; and Me refill rate As indicated in Fig.8, [Me] ; was reduced with the duration of Me-free medium incubation. Fig.13 illustrates the relationship between [Mg 2 +] ; and the Me refill rate. During 24 hrs' incubation, [Mg"] ; reduced by 50%, from 0.52 ± 0.02 to 0.26 -±0.01 mM. No further reduction was observed up to 48 hrs Me-free medium incubation. Compared with the changes of [Me] ; , significant increase in Me refill rate was only found in the first 24 hrs Me-free medium incubation. During the second 24 hrs of magnesium depletion, both [Me] ; and the Me refill rate maintained a relatively stable level. This observation indicates that the Me refill rate is determined by intracellular free Mg' concentration. It is necessary to perform further studies to demonstrate the possible role of [Me] ; in the process of Me refill rate.  IV.2. The effect of external Me on Me refill rate Fig.14 shows the effect of external Me on Me refill rate in Me-depleted cTAL cells. No significant Me refill rate was observed when external Mg' concentration was 0.005 mM or less. Me refill rate was linearly increased with the elevation of external Me concentration, from 0.05 mM to 5.0 mM, but stabilized between 5.0 mM and 50 mM of external Me concentrations (Fig.14). This may be the basis for beneficial effect of the administration of MgC1 2 or ATP-MgC1 2 in various magnesium deficiency-associated diseases, as cells may take up magnesium via these pathways in the plasma membrane when activated.  37  1  0 .8 -  to 200  mn  -  m 100 Co  N.1  • 10^20  ^  0 30  40^50  TIME hrs Fig.13. The relationship between [Me], and Me-refill rate. [Me], was measured as indicated in the methods and Me-refill rate was calculated according to equation 3. Each point was calculated from data from at least 4 cells. Data are presented as mean ± S.E.. [Me], and Me-refill rate in 4, 8, 24 and 48 hrs were significantly lower and higher than the control level (P < 0.05) respectively. There was no difference between 24 and 48 hrs points for both values (P > 0.05).  CD  38  300 —  -65 mV  0 mV  0^10^20^30^40^50  [Mg 2+]0 mM  Fig.14. Membrane potential-dependence of Me+ refill in Me-depleted cTAL cells. Cells were cultured in magnesium-free media for 16-24 hrs. The refill rate of [Me], d([Me],)/dt, was assessed at the external magnesium concentrations indicated. Refill was determined in normal Medepleted cells with an apparent transmembrane voltage of -65 mV with respect to outside, and in cells treated with 100 p,M ouabain and 2µM gramicidin D to abolish the potential (see text for methods). Values are means ± S.E.  39  At external Mg' concentrations above 0.05 mM, Mg' refill rate in the Me-depleted cells with normal membrane potential was significantly higher than that in the Me - depleted cells without membrane potential (Fig.14). This result suggests that membrane potential plays a major part in the Me+ refill process.  IV.3. The effect of membrane potential on Me+ refill rate As indicated in Fig.14, membrane potential plays a role in Mg' refill process. In order to confirm the significance of membrane potential in this process, we determined the rate of refill of cells in which the transmembrane voltage was abolished. First, cTAL cells were treated with 100 /AM ouabain and 2 AM gramicidin D, with and without a transmembrane magnesium chemical gradient. Gramicidin D, a sodium ionophore, was used to ensure complete equilibrium across the plasma membrane. Second, a bathing solution composed of high K+, low Nal- was used which would be expected to depolarize the transmembrane electrical gradient. In the Me-depleted cells with normal membrane potential the concentration dependence of the Me+ flux can be described by Michaelis-Menten kinetics (53,54). The J. (maximal d([Me] )/dt) is in the order of 210 ± 18 nM.s -1 and the Km (apparent ;  affinity), the magnesium concentration which results in half-maximal refill rate, is in the order of 0.27 ± 0.12 mM. The d([Me] )/dt was significantly diminished when the trans;  membrane potential was abolished (Table 3). There was virtually no uptake of Mg" when there was no transmembrane magnesium concentration gradient. Uptake was not appreciable until the outside-to-inside gradient was 50-to 0.25 mM. Abolishing the voltage decreased the J. to about 103 ± 1 nM.s -1 and increased the apparent Km value to 6.2  40  Table 3. Effect of voltage on Mg 2 + refill rate in Mg 2 +-depleted cTAL cells  Refill with 0.25 mM Refill with 5.0 mM [Mel d([Me],)/dt (n) [Mel d([Me] ;)/dt (n) Control^0.24±0.01 97±34 Ouabain+ 0.24±0.02 Gramicidin  (2) 0.27±0.07 210±56  5±13 (5) 0.25±0.04  Depola-^0.28±0.05 -2±15 (5) rization  0.27±0.08  Refill with 50 mM [Mel d([Mg 2 +] ; )/dt (n)  (6)  0.25±0.01 208±20  (4)  46±16  (5)  0.26±0.07 92±19  (5)  18±14  (5)  0.26±0.06  (5)  7±12  In order to abolish the transmembrane electrical potential, cTAL cell monolayers were treated with: ouabain 100 I.L.M and gramicidin 2 AM or depolarization solution containing HC1,118mM; NaCI, 25 mM; CaC1 2 ,1 mM; KH,PO 4f 1 mM; glucose, 18 mM and HEPES-TRIS, 14 mM; pH 7.4.  41  ± 3.1 mM (53). Me influx was completely inhibited in the presence of high IC+, low Na+ solutions. These studies support the notion that Me entry into the cell is altered by changes in the transmembrane voltage. IV.4. The effect of protein synthesis inhibitors on Me refill rate The adaptation in transport rates or channel activity may require de novo synthesis of protein. Accordingly, as illustrated in Fig.15, actinomycin D, an inhibitor of transcription (55), cycloheximide, an inhibitor of translation (56), and 3'-deoxyadenosine (cordycepin), an inhibitor of polyadenylation involved in the processing of heterogeneous nuclear RNA (57-60) were used to determine the role of protein synthesis in the adaption of d([Me] )/dt to magnesium depletion. The inhibitors were added to the culture media 20 ;  min prior to placing the cells in Me-free media and during the 4 hr adaptive phase. Basal Me levels and refill rates were determined after 4 hr in Me-free solutions in the presence of the inhibitor. Actinomycin D treatment inhibited the adaptive response by 86%, but had no effect on basal [Me] , and refill in cells cultured in media containing normal amounts of ;  magnesium (data not shown). In order to determine whether actinomycin D directly altered transport, cTAL cells were first adapted for 16 hr in Me-free media and then exposed to actinomycin D for 4.5 hr. Actinomycin D did not significantly alter Me refill in cells adapted prior to treatment with the protein synthesis inhibitors. Cycloheximide inhibited the adaptive response, as measured by Me refill rate, by 60% and cordycepin by 55%. Again, no effect was observed on basal [Me] in cells cultured in normal ;  medium. These results suggest that protein synthesis is partially involved with the adaptation of transport sites in cTAL cells following placement in Me-free medium.  42  300  4 hrs 141141V likb.  16 hrs  0)  200 1.11 -  I3C -J U1.1-1 QL C4^100  -  -  0  CONTROL COR . ACT . CYC .  Fig.15. Effect of protein synthesis inhibitors on the adaptive increase in Mg' influx. Effect of protein synthesis inhibitors on adaptation of cTAL cells to low magnesium media. With the inhibitors present during adaptation, the cTAL monolayers were treated with actinomycin D (5 µg/ml), cyclohexamide (50 /LM), or cordycepin (100 /AM) for 20 minutes prior to and following placement of the cells into magnesium-free medium for 4 hr. In the studies in which the inhibitor was present after adaptation, cTAL cells were adapted for 16 hr prim' to treatment with the respective protein synthesis inhibitors, the inhibitors were present for 4.5 hr before daMelydt was determined. Basal [Mel and the refill rate was monitored by methods given in the text. *indicates significance (p < 0.05) from respective control values.  43  IV.5. Specificity of Me influx pathway IV.5.1. The effect of inorganic cations on Me influx A number of inorganic cations were used to determine the specificity of the Me influx pathway. First, the effect of 5 mM external Ca' was tested by adding it with the 5.0 mM MgC12 refill solution. Calcium had no effect on the Me refill rate (Table 4) and Me depletion had no effect on 'Ca uptake (0.81±0.07 vs 0.90±0.07 nmol.mg protein-1 .Mill 1, n = 8, p > 0.05) suggesting that the putative Me pathway is distinct from Ca' entry. This observation is in concert with the in vivo micropuncture studies (19) which clearly show that there is no inhibition of magnesium reabsorption with elevation of luminal or apical calcium in the loop of Henle. There were no changes in [Cal ; during these manipulations over the duration of study (Fig.11). Fig.16 demonstrates the effect of I2 on the Me influx pathway. La', 5.0 mM, completely inhibited the change in [Me], when added concurrently with MgC1 2. Removal of the La' resulted in an immediate increase in [Me] ; in the presence of external magnesium; the rate of change, 278 ± 35 nM. s -1, was similar to control cells and abruptly stopped at or near normal cellular [Me] ; . Similar studies were performed, but with fura2 to measure [Cel t ; [Cal, was normal in Me-depleted cells and  Le  had no effect on  calcium levels over the time period of study. These studies indicate that the entry of Me is due to the presence of a mediated pathway rather than simple diffusion of Me across the plasma membrane into the cell. Table 4 lists the results of a number of other cations on the Me refill rate. Strontium, cadmium, cobalt and barium were without effect on Me entry. Manganese, nickel, zinc, gadolinium and beryllium inhibited Me entry. The potency sequence  44  Table 4. Inhibition of Mg' Influx with Inorganic Cations Cation Concentration Increment in [Mel Inhibition Increment in [Mg 2 1, (presence of cation)^(following removal of cations) mM nM.sec-1 % of control nM.sec' (n)  Control  257 ± 37  100 ± 14  (14)  Mn 2 +  2.0  40 ± 69*  15 ± 27*  189 ± 71  (4)  I...a 3 +  2.0  81 ± 54*  32 ± 21*  246 ± 111  (3)  Gd3 +  2.0  80 ± 49*  31 ± 19*  189 ± 71  (2)  .-  Ni2 +  2.0  120 ± 23*  47 ± 9*  182 ± 48*  (3)  Zn2 +  2.0  160 ± 78*  62 ± 30*  377 ± 90*  (3)  Be'  2.0  176 ± 13*  68 ± 5*  109 ± 12*  (2)  Ba2 +  2.0  232 ± 40  90 ± 16  167 ± 34  (3)  Cd2 +  2.0  351 ± 42  136 ± 16  406 ± 157  (3)  Co'  5.0  248 ± 18  96 ± 7  233 ± 15  (3)  Sr+  5.0  272 ± 76  106 ± 29  325 ± 51  (3)  Ca'  5.0  257 ± 74  100 ± 29  204 ± 62  (4)  cTAL cells were magnesium-depleted, as given in the text, and the increase in [Mel assessed in the presence and absence of the various cations according to methods given in the text. Values are means ± S.E. * indicates significance (p < 0.01) from control.  45  0  500  1000  1500  2000^2500  3000  3500  TIME seconds  Fig.16. Specificity of Me+ influx pathway. Intracellular Mgt' concentration was assessed in normal or magnesium-depleted cTAL cells. LaC1, was added from a stock solution to a final concentration of 5.0 mM with 5.0 mM MgC1 2 . At the time indicated, Laa, was removed by washing 3 X in buffer solution.  46  approximated: Me  = La" = Gd' = Ni' = Zn" =Be 2+»Ba2 + = Co' = Cd 2+ = se+ = ce. Many  of these cations alter transmembrane voltage; no attempt was made to correct this sequence for other effects such as changes in voltage. These cations did not have any effect on basal [Mgl, in normal cells cultured in normal magnesium media in the timeframe of study used here (data not shown). In most cases, the inhibition with the inorganic cations was reversible as the refill rate returned to near normal values (Table 4). The notable exceptions were Ni" and Be' where the refill rates were 182 48 and 109 ± 12 nM.s', respectively.  IV.5.2. The effect of organic Ca' channel blockers on Me+ entry A number of organic Ca' channel blockers were tested for their ability to inhibit Mg' refill in Me-depleted cells (61-63). Nifedipine, a 1,4 dihydropyridine derivative, inhibited the Mg' influx pathway (Fig.17). This inhibition was fully reversible following removal of the agent. Bay K 8644, an analogue of nifedipine which increases the open-time of voltage-sensitive ca2+ channels, increased the rate of change of [Mel (Fig.17). Verapamil, a phenylalkylamine, and diltiazem, a benzothiazepine derivative, also inhibited Me+ refill but to a lesser extent compared to nifedipine. The apparent potency sequence of these channel blockers is in the order of: nifedipine > verapamil = diltiazem (table 5). These findings suggest that Mg' entry may be a channel with a close homology to Ca' channels. Further studies are required to establish this point. A number of other selective and semi-selective agents were used to characterize the specificity of Me+ entry into cTAL cells. Quinidine, a fast Na+ channel blocker, and tetrodotoxin, an inhibitor of Na+ channels were tested. These agents also have been  47  r.0  —  0.8 -  0  0  E  E  0.6 10 uM Boy K 8644  0.2 -  20 uM NIFEDIPINE  5.0 mM Mg 0  r^1^1^1 500^1000^1500^2000^2500^3000  TIME seconds  Fig.17. Effect of dihydropyridine antagonists and agonists on Mg' influx pathway. Refill was assessed by the change in [Mel as given in the text. Nifedipine, 20 /AM, and Bay K 8644, 10 AM, was added and removed, as indicated, to the buffer solution.  48  Table 5. Effect of Organic Ca 2 + Channel Blockers and Activators on Mg 2 + Influx Pathway  Blocker^Concentration^d([Mg2 +] )/dt^Inhibition^d([Mg2 +1)/dt (presence of blocker)^(removal of blocker) AM^nM.s'^% of Control^nM.s-1 (n) ;  Control  257 ± 37  100 ± 14  (11)  Nifedipine  42  117 ± 20*  45 ± 8*  243 ± 106  (5 )  Bay K 8644  10  366 ± 71*  142 ± 27*  107 ± 9*  (3)  Verapamil  20  165 ± 34*  64 ± 13*  201 ± 22  (3)  Diltiazem  70  194 ± 19*  75 ± 7*  227 ± 36  (3)  Pimozide  100  114 ± 40*  44 ± 15*  56 ± 49*  (4)  Mg2 + influx was determined in magnesium-depleted cells as illustrated in Fig.8. Values are mean ± S.E. *indicates significance (p < 0.01) from control values.  49  reported to inhibit Na+-Me+ exchange in red blood cells (64-66) and squid axons (67). Quinidine and tetrodotoxin had no effect on Me+ refill into Me-depleted cells (Table 6) suggesting that sodium ions are not directly involved with Me+ entry, for instance, through a putative Na+-Mg 2 + exchanger. Amiloride, an inhibitor of Na - channels, had no effect on Me+ refill but dichlorobenzamil, a potent inhibitor of Na+ transport and Na+-Ca" exchange (68,69) had modest effects on refill. The basis for this latter observation is unknown. On balance, these studies would suggest that Na+-Mg2 + exchange is not directly involved with Mg' entry into cTAL cells.  50  Table 6. Effect of Na+ channel antagonists on Me+ influx pathway  Concentration^d([Me],)/dt^Inhibition AM^nM.s-1^% of Control^(n)  Control  186 ± 10  Quinidine Tetrodotoxin Amiloride  100 ± 5  (5)  100  160 ± 63  86 ± 34  (2)  10  177 ± 17  95 ± 9  (3)  1000  186 ± 23  100 ± 12  (3)  73 ± 3*  (3)  Dichlorobenzamil 100  135 ± 6  Mg' influx was determined in magnesium-depleted cells as illustrated in Fig.4 except the external magnesium concentration was 1.0 mM in the refill solution. Values are mean ± S.E. * indicates significance (p < 0.05) from control values.  51  DISCUSSION Magnesium has long been recognized to be a necessary cofactor for numerous enzymatic reactions, but there is now considerable evidence to suggest that intracellular free Me may also be a key physiological regulator of cell activity (3,8,70,71). In the past, researchers had been reluctant to consider such a role for Me because it was believed that [Me] was in the order of 10 to 30 mM (3). During the last decade it has become ;  possible to measure [Me] accurately using techniques such as 31P and "F NMR, null;  point titrations, ion-sensitive microelectrodes, and fluorescent indicators (7). The results from these studies suggest that, although total intracellular Me concentration is indeed high, the majority of it exists in a bound form. The intracellular free Me concentration is actually 0.1 to 0.7 mM (3,70,71), one-hundredth of the total intracellular magnesium. Since the Michaelis constant (K.) values for Me activation or inhibition of many enzymes fall within this range (3), relatively small changes in [Me] could induce large effects on ;  cell activity. Further, 28Mg2 + influx into several types of cells is hormonally regulated, supporting the notion that changes in [Me] are physiologically relevant (8). The study ;  reported here indicates that Me is a dynamic cation which is closely regulated by intracellular Mg' concentration. The mechanism of magnesium transport across the plasma membrane is not very clear. The early studies suggested that magnesium transport across plasma membrane occurs via Na+-Me exchange, Mg2 +-Mg2 + exchange, Me-pump and Cl-dependent magnesium transport system in various type of cells (72-74). In our previous studies, we demonstrated the presence of magnesium specific transport pathways in MDCK cells and cardiac myocytes (46,52). The results in the present study indicate that these pathways are not  52  Na+-Mg" exchangers, but may be magnesium channels.  I. The role of intracellular free magnesium in regulation of magnesium transport across the plasma membrane. Calcium and magnesium are two of the most important intracellular cations. In most eukaryotic cells a diverse array of Ca" transporting systems functions to maintain the steep concentration gradient between extracellular Ca", estimated to be in the millimolar range, and intracellular Ca", which can vary between 0.1-1 AM, depending on the state of the cell. The major entry pathway for Ca" in many cell types is via plasma membrane Ca" channels. Ca" channels are normally closed; when opened, Ca" passively flows through the channels along the Ca" electrochemical gradient. Several million Ca" ions/sec can enter the cell through open Ca" channels. For magnesium, however, extracellular magnesium concentration is very close to the intracellular concentration in various cell types (8,71); [Me] is about 0.5 mM and [Me]. 0.3 mM respectively. The only driving ;  force for magnesium is the membrane potential which is much smaller than that of calcium. Presumably, this is one of the reasons why intracellular free magnesium concentration can not respond quickly to various stimuli. This is also a problem in the study of magnesium transport. The previous studies in our laboratory indicated that magnesium depletion can enhance magnesium transport process in MDCK cells (46) and cardiac myocytes (52). Magnesiumdepleted cTAL cells were used in the present study to demonstrate the characteristics of the magnesium influx pathway. After a period of incubation in magnesium-free medium, the intracellular free magnesium concentration was reduced by 50%, from 0.52 ± 0.02 mM  53  to 0.26 ± 0.01 mM ( Fig.6,7,8). [Mel in magnesium-depleted cTAL cells increased linearly with time when the cells were put back into magnesium-rich buffer, with a refill rate of 210 ± 25 nM/sec. The increase in [Mel levelled off when the [Mel was close to the normal level (Fig.9). This suggests that the level of free magnesium concentration plays an important role in the regulation of magnesium transport. This transport pathway is located in the plasma membrane as indicated in Fig.9. No magnesium refill could be found if depleted cells were kept in magnesium-free buffer instead of magnesium-rich buffer. The result of  g  28m 2+  uptake study also confirmed the effect of magnesium  depletion on magnesium uptake (Fig.12). The fact that the decrease in intracellular Me+ concentration enhances the Me+ refill rate indicates that magnesium influx is regulated by [Me] . The decrease in [Mel can ;  increase in magnesium influx through either enhancing the driving force or increasing the density of the entry pathways and/or lengthening the pathway's open time.  I.1. The membrane potential determines the driving force for magnesium movement Under the condition of magnesium depletion, [Mel reached the lowest level within 8 hr, at which point, magnesium refill rate was almost maximal. Following longer periods of magnesium-free medium incubation, no further significant changes could be observed in either [Mel or d([Me+1,)/dt (Fig.13). These results suggest that magnesium influx pathways are highly regulated by intracellular free magnesium concentration. One possible reason that magnesium influx pathways are regulated by [Mel is the magnesium equilibrium membrane potential. If the pathways dealt with in this model are channel proteins, the driving force for  54  Mg' ions should follow the Nernst equation:  RT [C]. V = In z F^[C] ;  equation 3.  where V is the equilibrium potential (volts); R, the gas constant (2 cal mold °K -1); T,the absolute temperature (°K); F, Faraday's constant (2.3 x 10 4 cal V-1 mold); z, the valence (charge) of the ion; and [C]. and [C] i, outside and inside concentrations of the ion, respectively. The flow of any ion through a membrane channel protein is driven by the electrochemical gradient for that ion. This gradient represents the combination of two influences: the voltage gradient and the concentration gradient of the ion across the membrane. When these two influences just balance each other, the electrochemical gradient for the ion is zero and there is no net flow of the ion through the channel. The voltage gradient (membrane potential) at which this equilibrium is reached is called the equilibrium potential for the ion. For any particular membrane potential V M, the net force tending to drive a particular type of ion out of the cell is proportional to the difference between VM and equilibrium potential for the ion: hence, for Na 2+ it is VM - V,,„ and for Me it is VM - VMg. Under our experimental condition where V M is -65 mV, [Me]. is 5 mM and [Me] ; is 0.5 mM in normal cTAL cells and 0.25 mM in Mg-depleted cTAL cells, Vms in normal and Mg-depleted cTAL cells is 30 and 39 mV respectively, based on equation 3. The driving force for Me is very high in both normal and Mg-depleted cTAL cells, but significant Me-influx was only found in Mg-depleted cells, presumabably because the actual current carried by each type of ion depends not only on this driving force but also on the ease with which that ion passes through its membrane channels, which is a function of the conductance of the channels (75). When V., was altered by  55  using different external Mg' concentrations, the membrane potential-dependent Mg' refill rate in normal cTAL cells was different from that in Mg-depleted cTAL cells (Fig.10). This observation indicates that magnesium depletion somehow induces the change of membrane conductance to Mg'. In order to further confirm the effect of membrane potential on magnesium transport, removal of membrane potential was done in magnesium-depleted cTAL cells. Transmembrane voltage can be abolished either through the use of ouabain and gramicidin D or through the employment of a depolarization solution. Under normal condition, the concentration of IC+ is typically 10 to 20 times higher inside cells than outside, whereas the reverse is true of Na+. These concentration differences are the basis of membrane potential and maintained by Na+-K+ ATPase in the plasma membrane. Na+-K+ ATPase operates as an antiport, actively pumping Nal- out and K+ into a cell against their electrochemical gradients. For every molecule of ATP hydrolysed inside the cell, three Na+ are pumped out and two K+ are pumped in. The specific pump inhibitor ouabain and K+ compete for the same site on the external side of the ATPase. Gramicidin D, a sodium ionophore, was used to ensure complete Na+ equilibrium across the plasma membrane. Fig.14 and Table 3 demonstrate that influx of Me+ is concentration-dependent and altered by the transmembrane voltage. Normal membrane voltage in Mg 2 +-depleted cTAL cells is supposed to be -65 mV. Three hrs after incubation with ouabain and gramicidin D , the membrane voltage is presumably reduced to zero. The magnesium refill rate increased as the external magnesium concentration was enhanced. When external magnesium concentration reached 5.0 mM, Me+ refill rate was saturated in both normal  56  and depleted cells. Magnesium refill rates in depleted cells were much lower than those in normal cells in any given external magnesium concentration. This observation supports the role of membrane potential in magnesium transport process. The properties of Me+ entry, concentration-dependence and saturation as indicated in Fig.14, are similar to those of an enzyme. Therefore, Michaelis-Menten kinetics can be employed to describe the characteristics of membrane channels. Channels modify the flux of ions the same way enzymes modify the flux of reactants ---- they stabilize the transition state between substrates and product, if we define the transition state of a channel as the state with an ion in the pore. The channel protein stabilizes this state (compared to what would happen without the protein) because it provides polarization charge to neutralize the permanent charge of the permeating ion ---- the channel protein has a high dielectric constant in the wall of its pore and so lowers the potential barrier to ion movement across the membrane. In the present study, the apparent J. was 210 ± 18 nM.s -1, and the K in was 0.27 ± 0.12 mM according to Michaelis-Menten kinetics (53,54). The kinetic parameters were altered by the transmembrane voltage; the saturation rate and apparent affinity decreased when the transmembrane voltage was abolished, apparent J. was reduced to 102 ± 12 nM.s 4 and K. increased to 6.16 ± 0.19 mM. The mechanism by which the magnesium depletion takes place is still uncertain. According to the Nernst equation, the resting cell membrane voltage of -65 mV constitutes a large driving force for Mg 2 + entry. If Me+ were allowed to distribute itself at equilibrium with this membrane voltage, intracellular Me+ concentration would be 45 mM in the presence of 0.3 mM extracellular Mg 2 +. In other words, if the cells approached the Me+ efflux as predicted by Nernst equation, extracellular Me+ concentration would be  57  lower than 0.0034 mM under the condition of 0.5 mM intracellular Mg'. However, magnesium depletion was still obtained in the presence of 0.005 mM external Me+ (data not shown), suggesting that some active transport systems might be involved in the formation of magnesium depletion. This point remains to be clarified.  1.2. Protein synthesis is involved in the regulation of magnesium entry pathways Another possible way in which intracellular magnesium concentration regulates magnesium influx pathways is the synthesis of entry pathway proteins. Theoretically, both the increase in density of the entry pathway proteins and the increase in open time of the entries can enhance magnesium influx across the plasma membrane. The response of cTAL cells to magnesium-free culture medium is relatively rapid and detectable within 12 hrs. It is interesting, however, that the increase in Me+ refill rate is not maximal until about 4-6 hrs. This suggests that in addition to increasing the entry rate there may also be recruitment of new or formed transporters into the apical membrane. To test this possibility we used a number of agents known to inhibit protein synthesis and translation steps. Cycloheximide, through its rapid action to block protein elongation, has been widely used to inhibit protein synthesis. Inhibition of the de novo protein synthesis by the use of cycloheximide resulted in diminution of the adaptive response, as assessed by the Me+ refill rate, by about 50% (Fig.15). The onset of the cellular effects of protein synthesis inhibition are directly related to the rapidity of the turnover of a specific protein. Cycloheximide resulted in rapid inhibition of the adaptive response; accordingly, the transporters may be rapidly turning over. To investigate the involvement of pretranslational steps in control of Me+ transport,  58  the inhibitors, actinomycin D and cordycepin, were used prior to adaptation. These agents resulted in 86% and 55% decrease in the adaptive response. Thus, total mRNA synthesis (69), RNA polymerase (76), nuclear posttranscriptional polyadenylation (68,69,55) and perhaps other steps (55), are involved in up-regulating Me transport activity. The present evidence would imply that both transcriptional and post-transcriptional processes are required in adding new transport units to the plasma membrane. Further studies will be necessary to characterize the turnover of the components known to be important in upregulation of Me transport. Magnesium refill rate can also be enhanced if the open-time of Me pathways is increased during magnesium depletion. To evaluate the state of the pathways in Mg' refill process, Bay K 8644, a Ca' channel activator which increases the open-time of voltage-sensitive Ca' channels, was used in this study. Bay K 8644 could not change basal [Me] level nor d([Me],)/dt when 5 mM Me was added into bathing solution in normal ;  cTAL cells (data not shown). When 10 AM Bay K 8644 was added into Me-depleted cTAL cell bathing solution d([Me] )/dt was increased by 1.5-fold (Fig.17, Table 5). The ;  different effects of Bay K 8644 on normal and Me-depleted cTAL cells are consistent with the conclusion that an increase in magnesium pathway density in plasma membrane plays a major role in the process of magnesium refill.  II. The specificity of magnesium influx pathway As mentioned above, the magnesium influx pathway in this experimental model seems to be a magnesium channel. The density of the channel proteins is critically regulated by the level of intracellular magnesium concentration. Channel proteins form water-filled  59  pores across membrane and show ion selectivity, permitting some ions to pass but not others. This suggests that their pores must be narrow enough in places to force permeating ions into intimate contact with the walls of the channel so that only ions of appropriate size and charge can pass. It is thought that the permeating ions have to shed most or all of their associated water molecules in order to get through the narrowest part of the channel. This both limits their maximum rate of passage and acts as a selective filter, letting only certain ions pass through. Thus, as ion concentrations are increased, the flux of ions through a channel increases proportionally but then levels off at a certain maximum rate. For a magnesium channel, however, any other ion with a similar size and charge to magnesium would have tendency to pass through. Various cations and some organic agents known to block calcium channels were tested in the present study.  II. I. The effect of inorganic cations on magnesium influx Mg" entry pathways, as assessed by the Mg" refill rate, are inhibited by a number of inorganic cations but not Ca" or Sr". The approximate potency sequence was: Mn" =La" = Gd3+ =Ni2+=Ze =Be2+> > >Ba2+=c02+=cd2+=sr2+=ca2+. Moreover, net 'Mg uptake but not "Ca, was enhanced by magnesium depletion. Finally, [Ca2 +]. was not altered by magnesium depletion nor by the process of Mg" refill. Accordingly, the Mg" influx pathway appears to be separate from Ca' channels in these epithelial cells. The Mg" entry into cTAL cells was different from similar entry pathways demonstrated in chick embryonic cardiac cells (52). These studies, performed under similar conditions as given here, showed an inhibitory potency sequence of inorganic cations of: Ca2+ =Se + -=-  Ni2+ = 032+ =Ba2+ > > >Zn2+ =Cd2+=Mn2+ (52). This sequence in cardiac embryonic  60  ventricular cells is quite different from that observed here in cTAL cells. This would suggest that there are a number of Me+ pathways which may be specific to different celltypes. That organ-specific Mg" pathways should exist is not surprising, as this has also been demonstrated for Ca 2 + channels (61-63,77). It should also be noted that Me+ does not share the Ca' channel in cardiac or vascular smooth muscle cells (77). Although large amounts of Me+ inhibit Ca 2 + movement through the L-type Ca' channel, Me+ is not measurably permeant (77). This is supported by the observation that Be', which readily crosses the Ca' channel (61-63) does not enter through the Mg 2 + pathway (deduced from data given here).  11.2. The effect of organic Ca" channel blockers on magnesium influx The organic channel blockers, nifedipine, verapamil, and diltiazem, inhibit Me+ entry into magnesium-depleted cTAL cells. These drugs appear to act at different sites along the length of the variously described Ca' channels particularly the L-type channels (6163). It would appear that these sites are also common to the Mg" pathways. Bay K 8644, an agonist of Ca' channels, also increases Me+ entry into cTAL cells. Further studies are required to determine if analogues of these or other agents may alter Mg 2 + entry without affecting Ca 2 + channels. Even though a Me+ channel has not been unequivocally demonstrated yet, experimental observations, mostly electrophysiological studies, are consistent with the movement of Mg' through membrane channels. For example, Preston (71) has measured Mg' currents in Paramecium, where Me+ takes a conductive membrane pathway that is impermeable  to K+ and Na+ but permeable to Mg'. The selectivity of this pathway is Me+ Me =  61 Co2 + >  se+ = Ba + > > Ca". The current is specific for Mg", but Mn 2  2  + and Co' can  substitute for Mg 2 + as charge carriers. Although it is unclear whether the current passes through channels or a carrier, the weak inhibition of the current by 1 mM amiloride which is known to block Na/Ca and Na/Mg exchange supports the conclusion of a Me+ channel. In the plasma membrane of toad retinal rods, Nakatani and Yan (78) have also observed currents attributable to the movement of Mg 2 +. As in Paramecium, this conductive pathway prefers divalent cations over monovalent cations. In the presence of physiological solutions the relative probabilities for Ca', Mg' and Na+ to permeate through this channel are 12:2.5:1. Working on single barnacle muscle cells with stable isotope techniques, Montes et al (79) found evidence supporting electrodiffusive leak of Mg" into cells. The permeability of this leak pathway (7 x 10 4cm/s) is similar to that of the Na+ leak into the cell. Presumably, leaks take place through membrane channels. Recently, the presence of channel-like Mg 2 + pathway was successfully demonstrated in cardiocytes and MDCK cells using the model of Mg' refill into Mg 2 +-depleted cells (52,46). The Mg2 + entry pathway described here may be a channel which has a close homology to the well known Ca' channels, but may differ in selectivity, as deduced from the following points: 1. Me+ influx is a passive transport process as indicated by concentrationand membrane potential-dependent fashion; 2. calcium channel blockers (nifedipine, verapamil, and diltiazem) inhibit Mg' uptake; 3. sodium does not appear to be involved with Mg' refill as a number of agents and maneuvers were without effect; 4. Mg' entry was not changed by Ca' at 5.0 mM, or Se+, Cc1 2 +, Co', and Ba 2 + at 2.0 mM but was inhibited by Mn2 +^...LezGezNi 241.--an 2 +A-.13e 2 + at 2.0 mM; 5. the study of protein synthesis inhibition provides an indirect evidence supporting the notion that Me+ influx takes place  62  through channels in the plasma membrane. In spite of the evidences obtained in this study, further experiments, especially electrophysiological and/or biochemical studies, are required to confirm the presence of magnesium channels in cTAL cells.  III. Summary 1. The intracellular free Mg' concentrations in cTAL cells vary between 0.35 and 0.65 mM with the mean value of 0.52 mM. 2. Magnesium-free medium incubation can induce magnesium depletion and thereby enhance the Me+ influx through the pathways in the plasma membrane. 3. The magnesium influx in magnesium-depleted cTAL cells is highly regulated by intracellular free Mg' concentration and needs de novo protein synthesis. 4. 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