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Effect of melatonin on osmoregulation of gulls (Larus glaucescens) Kitamura, Nobu 2005

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EFFECT OF MELATONIN ON OSMOREGULATION OF GULLS (LARUS GLAUCESCENS) NOBU KITAMURA B.Sc, Okayama University, 2002  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS OF THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Zoology)  THE UNIVERSITY OF BRITISH COLUMBIA August 2005 © Nobu Kitamura, 2005  ABSTRACT Melatonin (MT) is largely known for its role in regulation of biological rhythms in many species. However, several studies suggest that MT may also have an osmoregulatory role. For example, Ching et al. (1999) showed that salt glands of Pekin ducks have M T receptors (MR) and that M R increased in number when ducks drank saline. They also found that elevating plasma MT to its night time level inhibited salt gland secretion (SGS). The gut and kidneys of Pekin ducks also have MR. Since Pekin ducks are not marine birds, I followed and extended the protocols of Ching et al. to examine effects of M T on simultaneous salt gland and kidney function of Glaucous-winged gulls (Larus glaucescens) that regularly have a high NaCl intake. I infused gulls during the day with 500mM NaCl and made timed SGS, urine, and plasma collections. In the first experiment, each bird received two treatments (given a week apart): 1) 500mM NaCl only (control treatment) and 2) plasma MT concentration ([MT] i) was initially elevated to and sustained at night time p  level during infusion of 500mM NaCl (MT treatment). Sample collections were made for an hour in each treatment. In the second experiment, after 60 minutes of control treatment [MT] i was elevated to and sustained at night time level for the second hour. p  When [MT] ] was elevated prior to salt loading (experiment 1), the response of the salt P  glands was opposite to the inhibition observed in Pekin ducks and M T increased SGS rate of gulls. However, when [MT] i was abruptly raised during ongoing SGS (experiment 2), p  its rate decreased during subsequent MT infusion, as it did in Pekin ducks. Kidney function was greatly affected by MT treatment in both experiments. GFR decreased when [MT] i p  was abruptly increased during ongoing SGS. Urine flow rate increased especially when [MT] was elevated prior to initiation of SGS and renal N a excretion increased in both +  pl  experiments. Simultaneous salt gland and renal excretion of N a exceeded excretion of +  water under both treatments and MT improved N a excretion. I concluded M T increased +  the efficiency of N a excretion in saline stressed gulls. +  Ul  TABLE OF CONTENTS Abstract  ii  Table of Contents  iii  List of Figures  iv  List of Symbols and Abbreviations  vi  Acknowledgements  vii  Preface  viii  Chapter 1. General Introduction  1  Melatonin and Osmoregulation  4  General Materials and Methods  6  Chapter 2. Effects of Melatonin on Salt Gland and Kidney Function in Gulls  9  Introduction  9  Materials and Methods  10  Results  10  Discussion  18  Chapter 3. Effects of Acute Melatonin Injection on Salt Gland and Kidney Function of Gulls  25  Introduction  25  Materials and Methods  25  Results  26  Discussion  34  Chapter 4. General Discussion  39  Literature Cited  43  iv  LIST OF FIGURES Figure 1.1. A diagram showing interactions of osmoregulatory organs in birds.  2  Figure 1.2. Apportioning of excretion (% of total output) of N a (upper graph) and water (lower graph) between salt gland and kidney of saline acclimated gulls during continuous hypertonic (500mM) saline infusion. 3 +  Figure 2.1. Effect of melatonin on salt gland secretion of saline-acclimated gulls.  12  Figure 2.2. Effect of melatonin on GFR and UFR function of saline-acclimated gulls.  13  Figure 2.3. Effect of melatonin on urine [Na ], [K*], and osmolality of salineacclimated gulls.  14  Figure 2.4. Effect of melatonin on renal reabsorption of N a , K , and H 2 O of salineacclimated gulls.  15  Figure 2.5. Effect of melatonin on total urine N a and K excretion of salineacclimated gulls.  16  Figure 2.6. Effect of melatonin on plasma constitution of saline-acclimated gulls.  17  Figure 2.7. Effect of melatonin on total N a and H 0 output (% infusate) of saline-acclimated gulls.  22  +  +  +  +  +  +  2  Figure 2.8. Apportioning of N a and water excretion (%) between salt gland and +  kidney of saline acclimated gulls.  23  Figure 3.1. Effect of acute melatonin on salt gland secretion of saline-acclimated gulls. 27 Figure 3.2. Effect of acute melatonin on GFR and UFR of saline-acclimated gulls.  28  Figure 3.3. Effect of acute melatonin on urine [Na ], [K ], and osmolality of salineacclimated gulls.  29  Figure 3.4. Effect of acute melatonin on renal reabsorption of N a , K , and H 0 of saline-acclimated gulls.  30  Figure 3.5. Effect of acute melatonin on total urine N a and K excretion of salineacclimated gulls.  31  +  +  +  +  2  +  +  Figure 3.6. Effect of acute melatonin on plasma constitution of saline-acclimated gulls. 32  V  Figure 3.7. Effect of acute melatonin on total N a and H 2 O output (% infusate) of saline-acclimated gulls.  36  Figure 3.8. Apportioning of N a and water excretion (%) between salt gland and kidney of saline acclimated gulls.  37  +  +  c  LIST OF SYMBOLS A N D ABBREVIATIONS All angiotensin II ADH antidiuretic hormone A VP arginine-vasopressin A VT arginin-vasotocin ECF extracellular fluid FW fresh water GFR glomerular filtration rate Hct hematocrit potassium concentration  [if]  MR melatonin receptor MT melatonin [MT] melatonin concentration [Na ] sodium concentration +  Osm osmolality RBC red blood cell RIA radioimmunological assay RPVrelative plasma volume UFR urine flow rate subscripts: pi plasma u urine sgs salt gland secretion  Vll  ACKNOWLEDGMENTS I would like to express my appreciation to my supervisor Dr. Maryanne Hughes for all her support and assistance throughout my study. I also wish to thank to Dr. Bill Milsom as my co-supervisor and my supervisory committee and Dr. Kim Cheng as my supervisory committee. I would like to thank to Dr. Darin Bennett for his assistance and help. I also thank to Allison Barnes, graduate secretary of the department for her assistance. I thank to Arthur Van der Horst for animal care. I thank to Sam Gopaul for animal care and his support during my study. 1 thank to Renee Schommer and Kim Swanson in radiation office at UBC hospital for their assistance. I thank to students who studied in this lab; Purdy Go, Greg Son, Sam Hsieh, and Juliana Kwan for their assistance and support, and students in Milsom's lab for their support during my study. Finally, I especially thank to my parents and my brother for their support, assistance, and understanding throughout my study. This research was supported by a grant from Dr. G.C. Hughes.  PREFACE This work was performed in the laboratory of Dr. Maryanne Hughes, Department of Zoology, University of British Columbia. Experimental design and execution of the research, as well as all data analyses and the writing of the manuscript was the responsibility of Nobu Kitamura under my supervision.  Maryanne R. Hughes, Ph.D. Department of Zoology University of British Columbia August 2005  1  CHAPTER ONE. GENERAL INTRODUCTION Birds living in saline environments face hyperosmotic stress. Interactions of their osmoregulatory organs: kidneys, salt glands, and hindgut (Fig. 1.1) maintain internal osmotic balance allowing them to tolerate this challenge (Peaker and Linzell 1975; Goldstein and Skadhauge 2000; Hughes 2003). Basic information on kidney function has been given by Goldstein and Skadhauge (2000). Primary functions of the kidney are to regulate body water and solutes and to excrete nitrogenous wastes. Kidneys of dehydrated birds can produce urine with sodium concentration ([Na ] ) two to three times that of +  u  plasma ([Na ] i). Avian kidneys possess mammalian-type nephrons that contain loops of +  p  Henle with high ability to concentrate urine and also reptilian-type nephrons which lack loops of Henle. The ratio of mammalian-type to reptilian type nephrons is smaller in birds than in mammals and birds produce less concentrated urine than do mammals. When marine birds are exposed to excessive salt load, their cephalic salt glands compensate their kidney function by secreting a concentrated NaCl secretion (salt gland secretion, SGS) and most N a and water is excreted by salt glands (Fig. 1.2). The salt +  glands, which, in gulls, are located on top of the skull, excrete an almost pure NaCl solution more concentrated than imbibed seawater. This process generates osmotically free water which can be used for other physiological processes and conserves water effectively (Schmidt-Nielsen 1960). Almost all sodium filtered by the kidney is reabsorbed along the renal tubules (Hughes 1995) and secreted via the salt glands. The specialized secretory tubules of avian salt gland cells are surrounded by blood capillaries (Fange et al. 1958). Their basal compartments have abundant mitochondria and vast absorptive surface (Ernst and Ellis 1969). Exposure to saline increases Na -K -ATPase +  +  activity of the basal membranes of these cells (Ernst and Ellis 1969), suggesting an increase in their NaCl transport capacity. When birds drink saline water, ingested water and osmolytes are quickly absorbed across the gut into the extracellular fluid (ECF) (Douglas 1970; Hughes and Roberts 1988). Plasma osmolality (Osm 0 and relative plasma volume (RPV) change simultaneously. P  These changes interactively initiate SGS of gulls (Hughes 1995; Bennett et al. 1997).  Figure 1 . 1 . A diagram showing interactions of osmoregulatory organs in birds.  3  100 r  i  r  ft  90 80 70 60 50 40 30  o  20  X LU + CD  10  3  100  3 O  90-  0)  80  3  70-  Q.  i  "5  3o  r  1  2  ~i  1  50-  30X LU  i_ OS  5  6  kidney salt gland  • •  kidney salt gland  r  60 -  40-  o  3 4 PERIOD  • •  201001  2  iii  3 4 PERIOD  5  6  Figure 1.2. Apportioning of excretion (% of total output) of N a (upper graph) and water (lower graph) between salt gland and kidney of saline acclimated gulls during continuous hypertonic (500mM) saline infusion. +  4  The input of the two variables is summed to initiate secretion and both are equally effective stimuli of SGS in Pekin ducks (Bennett et al. 1997). Blood volume expansion may contribute to initiate SGS in the goose (Zucker et al. 1977). A decrease in extracellular fluid volume (ECFV) may increase the osmotic threshold for SGS in ducks (Kaul and Hammel 1979). The increase in Osm i need not be due to elevated [Na ] i or [Cl"] i (Schmidt-Nielsen +  p  p  p  et al. 1958; Goldstein et al. 1986), suggesting that osmoreceptors respond to nonspecific increase in Osm i (Hughes 1989a). Possible stimuli for SGS in birds are also discussed by p  Hughes (1989b). In addition to the interaction of salt glands and kidneys in eliminating excess N a  +  loads, the hindgut may also play an important role in birds by modulating initial N a and +  water uptake and their retrieval from refluxed urine (Schmidt-Nielson et al. 1963; Anderson and Braun 1985; Braun 1999). Indirect experimental evidence supports an interaction between hindgut NaCl reabsorption and extrarenal NaCl secretion in Pekin ducks (Hughes et al. 1992; Hughes and Raveendran 1994) and in Mallard ducks (Hughes et al. 1999). Postrenal reabsorption of refluxed urine may vary in importance among bird species (Anderson and Braun 1985) and may not be very important in marine birds that have salt glands with high N a concentrating ability (Goldstein et al. 1986). +  Melatonin and Osmoregulation Melatonin (MT) (N-acetyl-5-methoxytryptamine) is a hormone synthesized and secreted mainly by the pineal gland, which is a small neuroendocrine organ located at the bottom of the third ventricle of the brain. Melatonin synthesis in vertebrates shows a diurnal (or circadian) pattern being greater at night and is very sensitive to light (Pang et al. 1996; Krause and Dubocovich 1997; Zawilska andNowak 1999; Kulczykowska 2002; Kulczykowska et al. 2001; Zawilska et al. 2002). Melatonin produced by the pineal gland is released into the blood circulation and metabolized in the liver and the kidney (Zawilska and Nowak 1999). Melatonin is also synthesized by the retina, harderian gland, and gut (Bubenik et al, 1978; Lee and Pang 1993; Zawilska and Nowak 1999; Bubenik 2002). Melatonin has a ubiquitous distribution with multifarious functions (Zawilska and Nowak 1999; Bubenik 2001). It is lipid-soluble and may cross membrane to act on cells of  5  the peripheral tissues (Pang et al. 1993). Melatonin receptors (MR) are membrane associated and members of a sub-family of G-protein-coupled receptors (Dubocovich 1995; Zawilska and Nowak 1999). They are classified into different subtypes according to their distribution sites and different regulatory roles (Dubocovich 1995). Although largely known as a regulator of biological rhythms, MT may also play an important role in osmoregulation. Most organs including salt glands (Ching et al. 1999) and kidneys (Song et al. 1992; Song et al. 1993a, b; Pang et al. 1996) of birds have MR. Unlike its role as a determinant of biological rhythm, MT's role in osmoregulation is not well understood. There are however a number of studies suggesting possible interactions between MT and osmoregulatory function in vertebrates. Melatonin might impact interactive function of salt glands and kidneys directly, or indirectly. For example, the reciprocal relation between plasma M T concentration ([MT] i) and GFR was reported in p  mammals (Tsuda et al. 1995). The direct MT inhibition of A D H release was also shown in suprachiasmatic nucleus-slices of rats (Isobe et al. 2001). GFR is also known to decrease during the night when [MT] i is high in song sparrows (Goldstein and Rothschild, 1993). p  Ching et al. (1999) found that elevation of [MT] i up to night time levels inhibited p  SGS rate and reduced its sodium concentration ([Na ]^), and almost significantly lowered 4  total N a secretion in Pekin ducks. They also showed that acute MT injection during +  ongoing SGS immediately reduced SGS rate and total N a secretion, and that these effects +  were sustained by subsequent MT infusion. Since the osmoregulatory capacity of Pekin ducks does not permit them to drink saline water concentrated more than 2/3 sea water (Bennett et al. 2003a), Ching et al's (1999) findings may not be generalized to marine bird species. I therefore repeated their two studies using a marine bird, the Glaucous-winged Gull (Larus glaucescens) to see if a more salt tolerant bird responds in the same manner under the same experimental condition. I extended their studies of effects of melatonin on salt gland function to include an examination of simultaneous kidney function. The aim of the present studies was to assess MT effects on salt gland and kidney function in gulls. Firstly, I hypothesized that 1) M T inhibits SGS rate and [Na "]^,., and, 4  therefore, total N a secretion of gulls; 2) M T decreases GFR and 3) M T decreases urine +  6  flow rate (UFR) of gulls. Secondary, I hypothesized that 1) acute M T injection immediately inhibits SGS rate and total N a secretion, but not [Na ] , and these effects are sustained by +  +  sgs  subsequent MT infusion; 2) acute MT injection immediately decreases GFR and this effect is sustained by subsequent MT infusion; 3) acute MT injection immediately decreases UFR and this effect is sustained by subsequent MT infusion of gulls.  GENERAL MATERIALS AND METHODS Experimental animals. The same birds were used in two experiments. They were held under natural photoperiod in partially covered outdoor enclosures with free access to drinking water at the University of British Columbia Animal Care Facility. They were fed herring ([Na ] = 82 mmol/Kg, [K ] = 124 mmol/Kg, [CI] = 69 mmol/Kg) (Goldstein et al. +  +  1986) and duck pellets (12.7% water, 17% protein, 2750 kcal/kg, and [Na*], [K ], and [CI], +  83, 153.5, and 99 mmol/kg, respectively) (Buckerfield's, Abbottsford, BC) ad libitum. Saline solution was presented in a large plastic wading pool and replaced at least once a day. They had been maintained for more than 2 years on approximately 225mM NaCl and given 300mM NaCl water for at least a week before the experiments. General experimental procedures. Each bird was fasted overnight, weighed, and banded. The lengths of its head, tarsus, and beak and depth of the beak were measured to help determine its sex. Its wings were gently bound to the body with a Velcro strap and venous catheters were inserted into the left leg for blood sampling and the right leg for I.V. infusion. It was placed with its keel down on a foam-lined restrainer with its head within a large funnel that directed SGS into a pre-weighed glass vial. A lubricated 1.5mL centrifuge tube with one side partially removed was inserted beneath the ureteral papillae to collect pure urine into a pre-weighed plastic vial (Hughes 1995; Bennett and Hughes 2003b). An initial 7ml blood sample was taken for both blood analyses and RIA (radioimmunological assay) for hormones (All, A V T , aldosterone, prolactin, and MT) for experiment 1. An initial 5mL blood sample was taken for both blood analyses and M T assay in experiment 2. Erythrocytes from large blood samples were resuspended in a volume of isotonic saline equivalent to the volume of the removed plasma and reinjected.  7  Ten min later each experiment was began. At the beginning of each experiment l u C i C-Inulin in 0.5mL of distilled water was i4  injected. To bring [MT] i to the night time level of approximately 78pg/mL (MR Hughes p  and DC Bennett, unpublished data) in experimental treatment, l.OmL isotonic saline containing 24x10" pg M T was injected. Continuous infusion at the rate of 0.37mL/min of a 3  500mMNaCl solution containing 0.014 uCi/mL of C-Inulin with (1.333xl0 pg/mL) or 14  3  without M T was applied during MT and control experiment, respectively. Time required to initiate SGS was recorded. During the experiment 10-minute samples of SGS, urine, and blood (0.5 to 0.7mL) were taken. A large blood sample (7 or 3 mL) was taken at the end of the experiment 1 and 2, respectively. S G S , urine, and plasma analyses. Each large blood sample was divided into a 1 mL aliquant placed in a 1.5mL centrifuge tube and a 6mL (or less) aliquant in a cold 7mL Vacutainer tube (Becton-Dickinson, Mississauga, ON, Canada) containing 0.07mL 15% KEDTA and 0.014mg K-sorbate. Triplicate microhematocrit tubes were immediately filled from the smaller blood sample and centrifuged with this small blood sample at 15,600 x g for 3min in an Eppendorf Model 5412 centrifuge. Hematocrit (Hct) was determined and plasma was decanted into a corresponding new tube. Duplicate determinations were made for osmolality of plasma (Osm i) and urine p  (Osm ) using a Wescor Model 5500C vapor pressure osmometer. Duplicate determinations u  for [Na ] and [K ] for both plasma and urine samples were made using a Model 943 +  +  Instrumentation Laboratory, Inc. internal standard flame photometer. The large aliquant was centrifuged for lOmin at 4°C at 3,000 x g in an Eppendorf Model 5403 centrifuge and the plasma was divided into four aliquots for RIA. These were frozen at -20°C until analyzed or shipped for RIA: 1) 0.5mL plasma for A V T and A l l assays into 1.5mL centrifuge tube containing 50uL, 0.025M 0-phenanthroline, an inhibitor of angiotensin converting enzyme (Dusterdieck and McElwee 1971); 2) 2.0mL of plasma were shipped on dry ice for M T RIA (Ching et al. 1999); 3) 0.5mL plasma for prolactin assay into 0.5mL centrifuge tube containing lOuL of sodium azide; 4) 0.5mL plasma was analyzed for aldosterone RIA in this laboratory (Coat-A-Count kit, Diagnostic Products Corporation,  8  Los Angeles). Subscripts "pi", "u", and "sgs" indicate plasma, urine, and salt gland secretion, respectively. Plasma and urine C-Inulin concentration analyses. Each plasma or urine sample 14  was centrifuged and its supernatant was transferred to the small tube, from which aliquots were transferred to scintillation vials each containing 2.0mL of Ready Value liquid scintillation cocktail solution (Beckman Coulter, Fullerton CA) and gently vortexed. Radioactivity (DPM) of each sample was determined in duplicate using a Beckman Coulter LS 6500 liquid scintillation counter. Statistics and calculations. Data are presented as means ± standard errors and statistically analyzed using SYSTAT 9 for Windows (SPSS Science, Chicago, IL). A P value of 0.05 or less was considered as significant. Overall MT effects on SGS, urine, and plasma variables were assessed by A N O V A by treat of residuals of linear regression of natural logarithm of values plotted against time in experiment 1. Overall M T effects on SGS, urine, and plasma variables were assessed by A N O V A by treat in experiment 2. Two group sample t test was used to compare control and M T treatments at corresponding sampling periods in experiment 1 (with pooled Variances). A paired sample t-test was used to investigate an immediate effect of melatonin, i.e. comparison of periods 60 and 70min in experiment 2. Time to initiate SGS by hypertonic saline infusion between control and experimental groups was assessed by A N O V A by treat in experiment 1. Relationships between variables were assessed by simple correlation. Relative plasma volume (RPV, %) is 100 - hematocrit (%). Total N a (and K ) +  +  excretion (uEq/lOmin) via the salt glands and kidneys in each 10 minutes sample is the product of secretion or urine [Na ] (mM) and flow rate (mL/lOmin). +  The following calculations are as described in Pitts (1968) and Goldstein (1993). Glomerular filtration rate (GFR) was calculated as: GFR (mL/min) = UFRx[lnulin] / [Inulin] ,. u  p  Renal tubular reabsorption of water (Rmo), N a (R +) and K (RK+) were calculated as: +  +  Na  RH2O  = (1 -UFR/GFR)  x  100 = (1- [inulin] i / [inulin] ) p  u  x  100 (%).  R,on = (1 - ((UFR x [ion] ) / (GFR x [ion] ))) x 100 (%). u  The UFR stands for urine flow rate.  pl  9  CHAPTER TWO. EFFECTS OF MELATONIN ON SALT GLAND AND KIDNEY FUNCTION IN GULLS.  LNTRODUCTON Birds have M R in osomoregulatory organs such as kidneys (Song et al. 1992; Song et al. 1993a, b; Pang et al. 1996), salt glands (Ching et al. 1999), and gut (Lee et al. 1993; Pang et al. 1996; Bubenik 2002). Binding of the MT agonist, [125I]iodomelatonin, to M R in the kidneys was highly specific in duck and chicken (Song et al. 1993a). The affinity (Kd) of renal M R does not vary diurnally, but the number of M R (Bmax) does (Song and Pang 1992; Song et al. 1993a), being as much as 53% (Song et al. 1992) and 52% (Song and Pang 1992) higher at mid-light than at mid-dark in Pekin ducks and in chicken, respectively. Binding sites of 2-[125I]iodomelatonin is 8-fold higher in the cortical region, which contain renal corpuscles, than that in the medullary region in guinea pig kidney (Song et al. 1993b). Receptors for MT are localized in the basolateral membrane of the renal cortical epithelium, especially the early proximal tubule (Song et al. 1997). In guinea pig kidney (and small intestine), M R are coupled to a G-protein, which may have a significant role in modulating activity of proximal tubule cells (Song et al. 1997). Marine birds are more tolerant to saline stress than non-marine birds (Bennett et al. 2003b). Saline acclimation of Pekin ducks increased Bmax of M R of salt gland cells (Ching et al. 1999). Gull's salt gland seems to have more M R than that of Canvasback, an estuarine bird (AMS Poon and M R Hughes, unpublished data). Therefore, it is possible that MT affects osmoregulatory functions of these organs of birds including gulls. For example, MT might alter plasma concentrations of osmoregulatory hormones such as A D H . Male Syrian hamsters given daily injections of M T increased posterior pituitary A D H content and decreased [ADH] i (Richardson et al. p  1992). This was associated with increased water consumption and UFR. Their [Osm] , u  [Na ] and [K ] decreased with no changes in N a and K excretion rates. Decreased GFR +  +  u>  +  +  u  also paralleled to an increase in [MT] i in mammals (Tsuda et al. 1995). It was also p  suggested that elevation of [MT] i may decrease GFR by reducing the renal blood flow in p  10  fish (Kulczykowska 2002). Until now there has been only one study investigating effect of M T on salt gland function in birds. Ching et al. (1999) found that exogenous M T decreased SGS rate and [Na ]sgs, and almost significantly reduced total N a secretion in Pekin ducks. +  +  I used their protocol to study a marine bird, the Glaucous-winged Gull to determine if marine birds, who are regularly exposed to high N a load, respond in the same manner as +  Pekin ducks when [MT] i is increased to its highest normal level (night time level). I p  extended their study to include effects of M T on simultaneous kidney and salt gland function. I hypothesized that 1) M T inhibits SGS rate and [Na ] , and, therefore, total N a +  sgs  secretion of gulls. I also hypothesized that 2) M T decreases GFR and 3) UFR of gulls.  MATERIALS AND METHODS In this experiment six gulls (one male and five females, mean body mass: 809±11 g) were used. Each bird received control and M T treatments with one week between treatments. At secretion (see chapter one, page 7) a 7mL blood sample was taken and rapidly centrifuged. Ten minutes later the red blood cells were reinjected into each gull, six 10-min sample collections of SGS and urine was begun. A small blood sample (0.5 to 0.7mL) was taken at the midpoint of each collection.  RESULTS Salt gland. Melatonin did not significantly affect time (min) to initiate SGS (P = 0.83; n = 6) between control (11.8 ± 2.4) and M T treatment (12.5 ± 1.9). Melatonin did however, produced significantly higher SGS rate (P = 0.04; Fig. 2.1. A) and total N a secretion (uEq) (P = 0.05; Fig. 2.1. B) during the fist half of the experiment +  (sample period from 10 to 30min), and difference between control and M T treatments diminished during the latter half of the experiment in both SGS rate (P = 0.89) and total N a secretion (P - 0.97). Melatonin did not affect [ N a ^ (P = 0.97; Fig. 2.1. C), nor +  4  +  11  [K ] +  sgs  (P = 0.31; Fig. 2.1. D).  Kidney. During M T infusion, GFR was not significantly lower than the control (P = 0.19; Fig. 2.2. A), but renal tubular reabsorption of H 0 (%) (Fig. 2.4. C) was significantly 2  decreased (P = 0.001) and UFR was significantly elevated (P = 0.001; Fig. 2.2. B). Renal tubular reabsorption of N a (Fig. 2.4. A) was significantly decreased by M T treatment (P < +  0.01) and [Na ] was significantly increased (P < 0.01; Fig. 2.3. A). As the result, total +  u  urinary N a excretion was maintained significantly higher during M T infusion (P = 0.001; +  Fig. 2.5.A). Although renal tubular reabsorption of K tended to be lower during M T +  treatment (P = 0.13; Fig. 2.4. B), [K ] was significantly decreased in experimental group +  u  (P < 0.01; Fig. 2.3. B). Total urinary K excretion (P = 0.21; Fig. 2.5. B) and Osm were +  u  unaffected (P = 0.78; Fig. 2.3.C) by M T treatment. Plasma. The relative plasma volume (RPV) (P < 0.001; Fig.2.6. A) and Osm i (P = p  0.03; Fig.2.6. B) were significantly lower during MT infusion than during control infusion, and Osm i was especially lowered in melatonin treatment during the second half hour. p  Melatonin treatment tended to maintain slightly lower [Na ] i (P = 0.14; Fig.2.6. C) without +  p  altering [K^pi (P = 0.51; Fig.2.6. D).  12  10  20  30  40  50  60  10  20  30  40  50  60  10  20  30  40  50  60  10  20  30  40  50  60  Time (min) Figure 2.1. Effect of melatonin on salt gland secretion of saline-acclimated gulls. Each gull was infused with 500mM NaCl solution only ( o , control treatment) or with melatonin (•, M T treatment). (A) SGS rate; (B) Total sodium (Na ) secretion; (C) SGS sodium concentration ([Na ]); (D) SGS potassium concentration ([K ]). The P values in the upper left and lower right quadrants in graph A and B indicate treatment effect during the first and second 30min periods, respectively. A P value in graph C and D refers to overall M T effect. +  +  +  13  Figure 2.2. Effect of melatonin on GFR and UFR of saline-acclimated gulls. Each gull was infused with 500mM NaCl solution only ( o , control treatment) or with melatonin (•, M T treatment). (A) Glomerular filtration rate (GFR). (B) Urine flow rate (UFR). A P value refers to overall treatment effect.  14  +  CD  CD C  10  20  30  P <  50  60  4  *  r  T  ~T  25  40  0.01  20  +  15  CD C  H10  20  30  40  50  60  10  20  30  40  50  60  TO O  E  </>  O  CD  c  Time (min)  Figure 2.3. Effect of melatonin on urine [Na ], [K*], and osmolality of saline-acclimated gulls. Each gull was infused with 500mM NaCl solution only (o, control treatment) or with melatonin (•, M T treatment). (A) Sodium concentration ([Na ]) of urine. (B) Potassium concentration ([K ]) of urine. (C) Urine osmolality. A P value refers to overall treatment effect. +  +  +  15  +  CO  o o co  X) CD CD  or  ^  1 0 0  ^  + o c o o  CO _o  CD CD  1  100 '  oCM  99  M—  98  X  o  c g  Q .  O CO XI CD CD  a:  1  P = 0.001 c  1  1  r  c  4  $  $  40  50  60  97 96 95  J  10  I  20  L  30  Time (min) Figure 2.4. Effect of melatonin on renal reabsorption of N a , K , and H 0 of salineacclimated gulls. Each gull was infused with 500mM NaCl solution only (o, control treatment) or with melatonin (•, MT treatment). (A) Reabsorption of N a of urine. (B) Reabsorption of K of urine. (C) Reabsorption of H 0 of urine. A P value refers to overall treatment effect. +  +  2  +  +  2  16  CT LU  300  1  "i  1  1 P =  c o  1  r  ^  0  50  60  0.001 A  ^—•  CD  o  200  X  111 + CO  100  CD  c  ro  o  0  J  10  CT LU  16.5  I  L  20  30  ~~\  1  c o CD  o  40  1  1  P =  0.21  r B  12.5  X LU  + CD  8.5  c  .JS  4.5  J  10  20  I  30  I 40  L  50  60  T i m e (min)  Figure 2.5. Effect of melatonin on total urine N a and K excretion of saline-acclimated gulls. Each gull was infused with 500mM NaCl solution only (o, control treatment) or with melatonin (•, MT treatment). (A) Total urine N a excretion. (B) Total urine K excretion. A P value refers to overall treatment effect. +  +  +  +  17  10  20  30  40  50  60  10  20  30  40  50  60  Time (min)  Figure 2.6. Effect of melatonin on plasma constitution of saline-acclimated gulls. Each gull was infused with 500mM NaCl solution only (o, control treatment) or with melatonin (•, M T treatment). (A) Relative plasma volume (RPV). (B) Plasma osmolality (Osm i). (C) Sodium concentration ([Na ]) of plasma. (D) Potassium concentration ([K ]) of plasma. A P value refers to overall treatment effect. +  p  4  18  DISCUSSION Salt glands and kidneys interact to maintain internal osmotic balance in birds. Both organs of birds examined (Pekin ducks and gulls) expressed M R (Song et al. 1993a; Ching et al. 1999; A M S Poon and M R Hughes, unpublished data). Ching et al. (1999) found that exogenous M T inhibited salt gland function in Pekin ducks. I repeated their study in gulls to assess the effect of night time [MT] i on salt gland function of a marine bird. I also p  examined effect of MT on simultaneous kidney function. In this experiment, I elicited SGS by infusion of a 500mMNaCl solution and made timed SGS and urine collections for one hour. A week later I repeated this procedure, but elevated [MT] i to the night time level during the experiment (MT treatment). p  Unlike Pekin ducks, gull's [Na ] +  sgs  was not affected by M T treatment (Fig. 2.1. C). I  found that MT treatment initially potentiated salt gland function of gulls by increasing SGS rate (Fig. 2.1. A) to significantly improve efficiency of N a secretion (Fig. 2.1. B). The +  SGS rate was positively correlated with the total N a secretion in both treatments (P < +  0.001 and P < 0.001, respectively). Therefore the increase in total N a secretion by M T +  treatment was due to increase in SGS rate. This was opposite to the effect elicited by MT in Pekin ducks, where MT treatment inhibited its function by decreasing SGS rate and its N a  +  concentration, and almost significantly decreased total N a secretion. The SGS rate is +  proportional to the salt gland blood flow in Pekin ducks (Kaul et al. 1983). A regulatory role of M T in modulating the contractile state of cerebral arteries via modulation of potassium channels is reported in rats (Geary et al. 1997). Physiological concentration of night time MT level may regulate cerebrovascular tone, stimulating vasoconstriction in rats (Viswanathan et al. 1997). Melatonin has both vasoconstrictor and vasodilatory effects (Scalbert et al. 1998), which may alter cerebral blood pressure, and therefore influence SGS rate in birds. Bringing [MT] i up to the night time level may have stimulated the blood flow p  to salt glands and elevated SGS rate in gulls. As the experiment progressed the difference between the two treatments was diminished (sample period from 40 to 60 min), and an M T effect was no longer detected. This suggests that the effect of MT on salt gland function may be transient. In gulls, the kidneys appeared to be very sensitive to MT. Previous studies showed that  19  MT decreased cardiovascular blood pressures in humans (Vazan et al. 2004) and in fish (Kulczykowska 1998). Plasma MT and A l l levels are significantly correlated in saline acclimated Pekin ducks (MR Hughes D A Gray and SF Pang, unpublished data) and A l l is reported to increase arterial blood pressure and heart rate in Pekin ducks (Gerstberger et al. 1984). The A V T increased blood pressure and melatonin reduced A V T induced hypertension in fish (Kulczykowska 1998). Therefore, M T may alter the blood flow to the kidney in gulls directly or indirectly. Although in the present study, 1 found that M T had no significant effect on GFR in gulls (Fig. 2.2. A). While M T did not affect GFR, M T decreased renal tubular reabsorption of water (Fig. 2.4. C) and increased UFR (Fig. 2.2. B). There was a high negative correlation between these variables (P < 0.001). Renal reabsorption of water of hummingbirds decreases linearly with increased water intake during night, and GFR is inhibited during this period (Bakken et al. 2004). Therefore, my results support the findings of Bakken et al (2004). Renal reabsorption of N a was significantly lowered by M T (Fig. 2.4. A) and both +  [Na ] (Fig. 2.3. A) and total urine N a excretion (Fig. 2.5. A) were significantly increased +  +  u  during M T infusion. The great elevation in [Na ] and in total urine N a excretion was due +  +  u  to decreased reabsorption of N a during M T infusion. The great increase in UFR also +  caused a significant increase in total urine N a excretion in M T treated birds. The present +  investigations suggest that MT elevates renal N a and water excretion of gulls by +  significantly elevating UFR. Melatonin may have inhibited the release of A V T (Richardson et al. 1992; Isobe et al. 2001; Juszczak et al. 2000) and thus significantly decreased renal reabsorption of H 0 in gulls. Melatonin may also have increased glomerular filtration of 2  N a and water as was demonstrated in mammals (Pishak and Kokoshchuk 1995). Binding +  sites of 2-[125I]iodomelatonin is 8-fold higher in the renal cortical region (Song et al. 1993b) and M R are localized in the basolateral membrane of the cortical epithelium, especially the early proximal tubule (Song et al. 1997). M R may have directly affected reabsoption of N a and water in the kidneys of gulls. +  Prolactin may have a positive direct effect on renal Na -K -ATPase in the medullary +  +  ascending limb and distal convoluted tubule in mammals (Bussieres et al. 1987). Prolactin reduces urine volume and renal N a excretion in mammals (Burstyn 1978). Long term M T +  20  treatment is reported to decrease serum prolactin levels in Syrian hamsters (Blask 1989). It may be possible that MT may have also influenced kidney function by altering the plasma prolactin level of gulls. No correlation (P > 0.05) was found between reabsorption of K (Fig. 2.4. B) and +  [K^u (Fig. 2.3. B) in MT treatment. The [K^u was significantly decreased by this treatment, and total urine K excretion stayed the same between treatments (Fig. 2.5. B). This may be +  due to dilution of urine K by increased UFR in M T treated birds. It is also possible that +  different rhythms of N a and K excretion were caused by urinary tubular secretion of K +  +  +  as was suggested by Koopman et al. (1989). They also reported that kidneys had higher N a  +  and water reabsorption rates during daytime in humans. M y findings in the present study support these investigations. The positive correlations between renal tubular reabsorption of N a and water and N a +  +  excretion and SGS rate regardless of treatments confirms the relationship between efficient N a and water uptake by the kidney and high N a excretion and SGS rates by salt glands. +  +  Although in the present study, decreased renal reabsorption of N a and water by M T during +  the first half of the experiment did not inhibit the SGS rate or alter its concentration in experimental group. While GFR had no effect on the SGS rate in the control treatment (P = 0.67), the GFR was positively correlated with the SGS rate (P < 0.01) in the M T treatment group, and this may suggest that changes in systematic blood flow affect both organs in the same manner. Present study also demonstrated that M T significantly influenced the plasma constitution of gulls. During M T infusion the RPV was significantly smaller (Fig. 2.6. A) and Osm i was significantly lower (Fig. 2.6. B) than those of control. When M T was p  infused with hypertonic saline, [Na ] i was slightly reduced (Fig. 2.6. C) and was not +  p  correlated with Osm i, although [Na ] and Osm i were positively correlated under the +  p]  p  p  control conditions. I found that combined N a and water excretion of salt glands and +  kidneys under control conditions were less than their excretion under M T conditions (Fig. 2.7), suggesting more N a and water accumulated in the body of control birds. The higher +  [Na^pi may have resulted in higher Osm i, where N a constitutes about half the total +  p  concentration, and accumulating water may have resulted in a larger ECFV, or larger RPV,  21  during the control infusion. Melatonin can cross red blood cell (RBC) membranes to mobilize intracellular calcium in humans (Hotta et al. 2003) and RBC have a volume regulatory capacity in frogs (Gusev et al. 1997). The volume of RBCs determines Hct (or RPV), and therefore, it is possible that MT treatment modulated the constitution of erythrocytes, causing ions and water movements between fluid compartments, thereby altered the RPV of gulls. Combined excretion of NaCl and water by salt glands and kidneys did not completely eliminate the infused saline load (Fig. 2.7) so that N a and water accumulated in bird's +  body during control and M T infusion. Total excretion of N a exceeded that of water in both +  treatments (Fig. 2.7), and osmotically free water was therefore produced, suggesting more water tended to be retained in the body than N a in both experimental groups. There was no +  significant difference between control and M T treatments in the way that salt glands compensated for the kidney's ability to excrete excess N a (P = 0.29) and water (P = 0.17) +  (apportioning of N a and water excretion (%) between salt gland and kidney) (Fig. 2.8. A , +  C). On the other hand in the kidneys, I found that significantly greater amount of N a  +  (PO.01) and water (P=0.001) were excreted during MT infusion (Fig. 2.8. B, D). Therefore, greater amounts of N a and water were excreted by the kidney function following M T +  treatment and this was probably due to elevated urine production. Not only Na , but also +  water was excreted at higher rates during M T infusion (Fig. 2.7), but gulls did not lose more water than was infused. When excess N a loads exceed its excretion capacity, Pekin +  ducks start losing its body water to get rid of excess Na , and [Na +  +  ] i p  and Osm i also p  increase (Bennett et al. 2003a). Melatonin treatment tended to slightly lower [Na ] j +  p  compared to control values in gulls. Birds with salt gland have higher water turnover rates compared to birds without salt glands and adult gulls have a high daily water flux (18% body mass) (Hughes et al. 1987). Present study indicated that M T may have elevated N a  +  and water turnover leading to a more effective excretion of excess N a without losing body +  water in gulls. In summary, I found that night time [MT] i potentiated salt gland function by elevating p  SGS rate, and thus total N a secretion in gulls. Therefore, the present results did not support +  my original hypothesis that M T inhibits SGS rate and [Na ] , and, therefore, total N a +  sgs  +  22  CD  "cc  1 0 5  CO .3  3 Q. O + CO  CO  o  I-  10  20  30  40  50  10  20  30  40  50  60  CD »  CO CO 13  3 Q.  o O  CN  X  15 o Time (min)  Figure 2.7. Effect of melatonin on total N a and H 0 output (% infusate) of salineacclimated gulls. Each gull was infused with 500mM NaCl solution only ( o , control treatment) or with melatonin (•, M T treatment). (A) Total N a output (% infusate). (B) Total H 0 output (% infusate). A P value refers to overall treatment effect. *P<0.05 (comparison between control and M T treatments at lOmin). +  2  +  2  23  Salt Gland  Kidney 1  ~i  20  100 h  P<0.01  1  r  B  3  . i f  15  x $ LU 2  10  +  CO  »  o  10  20  1  100 O  "55 1_  "3  30  40  50  1  1  P = 0.17  60 r  C  90  10 50  i  20  4  <W  60  30  40  50  1  1  1  1  r  P = 0.001  D"  40  Q .  3  O  80 -  o  <6  *H  30  CM  X CM  70  ro  20  <>  o  60 --  10  —' 50  0 10  20  30  40  50  60  J  10  20  30  I  I  40  50  L  60  Time (min)  Fig. 2.8. Apportioning of N a and water excretion (%) between salt gland and kidney of saline acclimated gulls. Each gull was infused with 500mM NaCl solution only (o, control treatment) or with melatonin (•, M T treatment). (A) N a excretion (% total N a output) by salt gland. (B) N a excretion (% total N a output) by kidney. (C) H 0 excretion (% total H 0 output) by salt gland. (D) H 0 excretion (% total H 0 output) by kidney. A P value refers to overall treatment effect. +  +  +  +  +  2  2  2  2  24  secretion of gulls. My hypothesis that M T decreases GFR was not supported since the MT effect was insignificant. Melatonin did affect kidney function by significantly elevating UFR compared to that of control group and greater amounts of N a and water were +  excreted via the kidneys. Therefore, my hypothesis that M T decreases UFR was not accepted. I found that a greater amount of the N a and water infused was excreted in MT +  treated birds compared to control birds. Since birds did not lose body water while excreting the excess Na , the data suggest that MT increased the efficiency of excreting excess N a to +  maintain internal osmotic balance in these birds.  +  25  CHAPTER THREE. EFFECTS OF A C U T E MELATONIN INJECTION O N SALT G L A N D A N D KIDNEY FUNCTION OF GULLS.  INTRODUCTION In Pekin ducks, acute injection of M T during ongoing SGS immediately reduced SGS rate and total N a secretion and they remained depressed during subsequent MT infusion +  (Ching et al. 1999). Since they did not investigate kidney function, I extended their study to examine the effects of M T on simultaneous salt gland and kidney function in gulls. I hypothesized that 1) acute M T injection immediately inhibits SGS rate and total N a  +  secretion, but not [Na ] , and these effects are sustained by subsequent MT infusion; 2) +  sgs  acute M T injection immediately decreases GFR and this effect is sustained by subsequent MT infusion; 3) acute M T injection immediately decreases UFR and this effect is sustained by subsequent M T infusion of gulls.  MATERIALS A N D METHODS Five gulls (one male and four females; mean body mass = 815 ±18 g) were used in this experiment. Each bird received sequentially control treatment (infusion of 500mM NaCl only) followed by M T treatment (injection of M T and infusion of 500mM NaCl containing MT). Six 10-min samples of SGS and urine were collected before (sample period from 10 to 60 min) and after (sample period from 70 to 120 min) the acute M T injection. Three and 10 minutes after the acute M T injection, a 3.0mL blood sample (for M T R1A, Hct, ion, and Osm analyses; see chapter 1, page 7) was taken (between sample period 60 and 70 min) and six 10-min collections of SGS and urine were begun, respectively. Small blood samples (0.5 to 0.7mL) were taken at the midpoint of the three SGS and urine collections before and after the acute M T injection. The immediate response to M T injection compares the last sample before and first sample after M T injection.  26  RESULTS Salt gland. The mean time to initiate SGS was 8.0 ± 2.0min (n = 5). The SGS rate was unaffected by acute MT injection (P = 0.29), but was significantly reduced (P < 0.001) during subsequent M T infusion (Fig. 3.1. A). Acute M T injection immediately increased [Na ] +  (mM) from 784.31 ± 15.03 to 823.17 ± 15.96 (P = 0.03) and [ N a ] ^ remained 4  sgs  significantly higher (P < 0.001) during M T infusion (Fig. 3.1. C). Acute M T injection did not affect total N a secretion (P = 0.72; Fig. 3.1. B), but tended (P = 0.07) to lower total +  N a secretion during continuous M T infusion. Neither acute M T injection (P = 0.36) or +  subsequent M T infusion (P = 0.77) affected [K ] +  sgs  (Fig. 3.1. D).  Kidney. Acute M T injection induced an immediate reduction in GFR (P < 0.01; Fig. 3.2. A) from 5.76 ± 0.56 to 4.68 ± 0.69 (mL/min) and GFR remained significantly reduced during M T infusion (P=0.04). Acute M T injection had no immediate effect on UFR (P = 0.54), but UFR tended to be higher during continuous M T infusion (P = 0.12; Fig. 3.2. B). Acute M T injection tended to increase [Na ] (P = 0.14; Fig. 3.3. A), but not urinary +  u  N a excretion (P = 0.72; Fig. 3.5. A). During continuous M T infusion, [Na^u and total +  urinary N a excretion (uEq/lOmins) were significantly higher than during control infusion +  (P < 0.01 and P = 0.02, respectively). Acute M T injection also tended to decrease [K*~\ (P u  = 0.15; Fig. 3.3. B) from 7.58 ± 1.08 to 6.06 ±1.13 (mM) and did decrease total urinary K  +  excretion (P = 0.04; Fig. 3.5. B). Both [K ] and total K excretion were significantly lower +  +  u  during M T infusion than during control infusion (P < 0.01 and P = 0.001, respectively). Neither acute M T injection nor subsequent M T infusion affected Osm (P = 0.35 and P = u  0.65, respectively; Fig. 3.3. C). Melatonin injection tended to decrease renal tubular reabsorption of N a (P = 0.08; Fig. +  3.4. A) and water, (P = 0.09; Fig. 3.4. C); continuous M T infusion tended to maintain lower reabsorption of N a (P = 0.09) and did decrease reabsorption of water (P = 0.04). Neither +  acute MT injection (P = 0.52) nor subsequent M T infusion (P = 0.48) affected renal reabsorption of K (Fig. 3.4. B). +  Plasma. Acute M T injection immediately increased RPV (P < 0.01) from 60.30 ± 0.77 to 63.01 ± 1.33 (%) and RPV remained elevated during continuous M T infusion (P < 0.001; Fig.3.6. A). Acute M T injection did not change Osm i (P=0.24; Fig.3.6. B), but, during p  27  Time (min)  Figure 3.1 Effect of acute melatonin on salt gland secretion of saline-acclimated gulls. After 60min of continuous 500mM NaCl infusion (o), plasma M T was abruptly raised and kept elevated for another hour (•). (A) SGS rate. (B) Total sodium (Na ) secretion. (C) Sodium concentration ([Na ]) of SGS. (D) Potassium concentration ([K ]) of SGS. *P<0.05 (an immediate effect of M T injection). A P value refers to comparison of control and MT infusion. +  +  +  28  7.5  1  T  A E  r  -i  1  1  1  r  P = 0.04  6.0  E U_  4.5  O 3.0  10  20  30  40  50  60 70  80  90  100  110  120  _l 10  I 20  I 30  I 40  I  L U 60 70  I 80  I  50  90  I 100  I 110  120  u  Time (min)  Figure 3.2. Effect of acute melatonin on GFR and UFR of saline-acclimated gulls. After 60min of continuous 500mM NaCl infusion ( o ) , plasma M T was abruptly raised and kept elevated for another hour (•). (A) Glomerular filtration rate (GFR). (B) Urine flow rate (UFR). **P<0.01 (an immediate effect of M T injection). A P value refers to comparison between control and M T infusion.  29  -I  10  J  I  I  I  L_l_l  20  30  40  50  60 70  1 80  I  I  I  90  100  110  ,  P <  0.01  U 120  B  1  _ J  I  I  I  L±J  I  I  I  I  10  20  30  40  50  60 70  80  90  100  110  120  10  20  30  40  50  60 70  80  90  100  110  120  _J  __l  850 800 750  o CD  c  ^3  650 600  T i m e (min)  Figure 3.3. Effect of acute melatonin on urine [Na ], [K ], and osmolality of salineacclimated gulls. After 60min of continuous 500mM NaCl infusion (o), plasma M T was abruptly raised and kept elevated for another hour (•). (A) Sodium concentration ([Na ]) of urine. (B) Potassium concentration ([K ]) of urine. (C) Urine osmolality. A P value refers to comparison between control and M T infusion. +  +  +  +  30  v°  100 i  i  i  i  ~  r "l  1  1  1  r  1  A + CD  z  99  g  9-  o to x>  98  P = 0.09  CO CD  100  20  30  i  i  1  40  50  60 70  r  1  I  L  80  90  100  110  120  1  1  1  1  _J  10  i  I  r  + c o 94  o  CO X) CO CD  92  P = 0.48 90  J  10  100  i  20  30  1—~i  40  50  1  1  60 70  r  J  L  80  •  90  c  U  100  110  120  1  o  CM  X t— o c o  99  o to  X) CO CD  P = 0.04 97 10  20  30  40  50  60 70  80  90  100  110  120  T i m e (min)  Figure 3.4. Effect of acute melatonin on reabsorption of N a , K , and H 2 O of salineacclimated gulls. After 60min of continuous 500mM NaCl infusion (o), plasma M T was abruptly raised and kept elevated for another hour (•). (A) Tubular reabsorption of N a o f urine (B) Tubular reabsorption of K o f urine. (C) Tubular reabsorption of H 2 O of urine. A P value refers to comparison between control and M T infusion. +  +  +  +  31  cr LU c o o X LU  T  1  A  r  1  P =  250 h  1  r  0.02  200  + CO CD  c  50 CO  10  -I—>  o  20  30  40  50  60 70  80  90  100  110  120  ~i  i  i  r  CT LU  ~i  1  r  c o  2  P =  0.001  10  o X  LU  +  ^ CD  5  — CO  Oh  c  o  j  10  I  20  i_  30  40  J  L-LJ  I  I  I  I  L  50  60 70  80  90  100  110  120  Time (min)  Figure 3.5. Effect of acute melatonin on total urine N a and K excretion of salineacclimated gulls. After 60min of continuous 500mM NaCl infusion (o), plasma M T was abruptly raised and kept elevated for another hour (•). (A) Total urinary N a excretion. (B) Total urinary K excretion. *P<0.05 (an immediate effect of M T injection). A P value refers to comparison between control and MT infusion. +  +  +  +  32  J?  o E in O co E  >  D.  in D.  10  20  30  40  50  70  IO  20  30  40  50  60 70  80  90  100  110  120  10  20  30  40  50  60 70  80  90  100  110  120  90  100  110  120  10  20  30  40  50  70  80  90  100  110  120  +  CD  ro E in  i?  CL-  Time (min)  Figure 3.6. Effect of acute melatonin on plasma constitution of saline-acclimated gulls. After 60min of continuous 500mM NaCl infusion (o), plasma M T was abruptly raised and kept elevated for another hour (•). (A) Relative plasma volume (RPV). (B) Plasma osmolality. (C) Sodium concentration ([Na ]) of plasma. (D) Potassium concentration ([K ]) of plasma. * * P < 0.01 (an immediate effect of M T injection). A P value refers to comparison between control and M T infusion. +  +  33  sustained M T infusion, overall Osm i tended to be lower than during control infusion (P = p  0.08). Acute MT injection tended to decrease [Na" "]^ (P = 0.06; Fig.3.6. C), but the overall 1  effect of MT infusion on  [Na ] i +  p  was not significant (P = 0.31). The increase in [K ] i  following acute M T injection was insignificant (P = 0.11; Fig.3.6. D) and overall unaffected by subsequent M T infusion (P = 0.55).  +  p  [K ] i +  p  was  34  DISCUSSION In this experiment, I assessed the acute effect of M T on simultaneous salt gland and kidney function during ongoing salt gland function. I induced SGS by infusion of a 500mM NaCl solution. After collecting SGS and urine samples for one hour, I injected M T and added M T to the infusate to maintain the night time level of [MT] i and continued to collect p  timed SGS and urine samples for a second hour. When [MT] i was abruptly increased to the night time level, SGS rate and total N a  +  p  secretion of Pekin ducks immediately decreased and remained depressed during subsequent M T infusion (Ching et al. 1999). When the same procedure was applied to gulls, the immediate responses were less drastic in gulls, but MT treatment did produce significantly lower maintained SGS rate (Fig. 3.1. A), just as it did in Pekin ducks. This may have been the result of reduced blood flow to the cerebral area in response to the elevated [MT] (Capsoni et al. 1995). Melatonin did not affect [Na ] +  immediate increase in [Na ] +  sgs  sgs  p]  in Pekin ducks, but it initiated an  in gulls that was maintained significantly elevated during the  MT infusion (Fig. 3.1 C). Despite the elevated [Na ] +  sgs  during the M T infusion, the total  N a secretion was largely influenced by the decreased SGS rate and so the efficiency of +  excreting N a (total N a secretion) by salt glands was reduced by M T infusion (Fig. 3.1. B). +  +  This result demonstrates that acute M T injection has similar effects on salt glands of Pekin ducks and gulls. Plasma M T and plasma A l l levels are significantly correlated in Pekin ducks (MR Hughes D A Gray and SF Pang, unpublished data) and A l l is reported to inhibit SGS rate in this species (Butler et al. 1998). Melatonin may have altered blood pressure in this region or influenced salt gland function via a hormone in the same way as it did in Pekin ducks in this experiment. I found that responses of the kidney to M T treatment were similar to most of those responses noted in the first experiment (Chapter two) in which  [MT] i p  was increased prior  to salt loading. The remarkable differences in response to M T treatment found in this experiment were that injection of MT during ongoing SGS immediately decreased GFR (Fig. 3.2. A) and total urinary K excretion (Fig. 3.5. B), where these effects were sustained +  during subsequent M T infusion. Abrupt increase in [MT] during ongoing SGS decreased p]  total urinary K excretion, but MT given prior to initiation of SGS did not (Fig. 2.5. B). +  35  While M T had no significant effect on GFR when it was applied prior to salt loading (Fig. 2.2. A), it significantly decreased its value when [MT] i was abruptly increased during p  ongoing SGS. As was discussed previously, acute M T injection may have altered the blood flow to the kidney maybe by decreasing cardiovascular blood pressure (Scalbert et al. 1998; Kulczykowska 2002; Vazan et al. 2004) or by modulating it via hormones (Gerstberger et al. 1984; Richardson et al. 1992; M R Hughes D A Gray and SF Pang, unpublished data). Despite a significant decrease in GFR following M T injection, UFR tended to stay elevated (P = 0.12) during M T infusion (Fig. 3.2. B). The UFR and renal tubular reabsorption of water were negatively correlated in M T treated birds (P < 0.01). Increases in both UFR and [Na ] combined to elevate total urine N a excretion. During control and +  +  u  MT infusion GFR and SGS rate were not correlated (both, P > 0.05). The UFR and SGS rate were negatively correlated before M T injection (P < 0.01), but no correlation was found between these variables after M T injection (P > 0.05), suggesting GFR was not a factor that contributed to the decreased SGS rate after M T injection. Although, UFR tended to increase during M T infusion , total urinary K excretion decreased significantly (Fig. 3.5. +  B) maybe due to decreased [K ] (Fig. 3.3. B). This may have been also due to changes in +  u  urinary tubular secretion of K (Koopman et al. 1989) before and after M T injection. +  Total N a and water excretion (% infusate) were significantly lower during M T +  infusion than during control infusion, but this response was transient. After about 30 minutes, total N a (Fig. 3.7. A) and water (Fig. 3.7. B) excretions (% infusate) returned to +  their control levels, suggesting that M T treatment improved these functions. It was demonstrated that excretion of infused N a exceeded excretion of infused water in both +  experimental groups (Fig. 3.7). These investigations support the results in experiment 1. There was no significant difference between control and M T infusions in apportioning of N a and water excretion (%) between salt glands and kidney to excrete excess N a and +  +  water (Fig. 3.8). Except for RPV, I found that plasma properties responded similarly to M T treatment in this experiment as in experiment 1. When [MT] i was brought up to the night time level p  during ongoing SGS, RPV immediately increased and remained elevated during subsequent M T infusion (Fig. 3.6. A). This contrasts with the first experiment, in which M T infusion  36  Figure 3.7. Effect of acute melatonin on total N a and H 0 output (% infusate) of salineacclimated gulls. After 60min of continuous 500mM NaCl infusion (o), plasma M T was abruptly raised and kept elevated for another hour (•). (A) Total N a output (% infusate). (B) Total H 0 output (% infusate). A P value refers to comparison between control and M T infusion. +  2  +  2  37  Salt Gland  Kidney  Time (min)  Fig. 3.8. Apportioning of N a and water excretion (%) between salt gland and kidney of saline acclimated gulls. Filled (•) and unfilled (o) circles indicate values for 500mM NaCl infusion with and without MT, respectively. (A) N a excretion (% total N a output) by salt gland. (B) N a excretion (% total N a output) by kidney. (C) H 0 excretion (% total H 0 output) by salt gland. (D) H 0 excretion (% total H 0 output) by kidney. A P value refers to comparison between control and MT infusion. +  +  +  +  +  2  2  2  2  38  significantly decreased RPV (Fig. 2.6. A). This may be partially the result of the 3mL blood sample taken shortly after the M T injection (between sample period of 60 and 70 min) since increase in RPV (%) was small (from 60.30 ± 0.78 to 63.01 ± 1.33). A drop in hematocrit by blood removal has also been reported; a 1 - 1.5% decrease in hematocrit (or increase in RPV) can be caused by about 6mL of blood removal (Gray et al. 1991). Unlike RPV, plasma concentration responded to M T treatment similarly to those seen in experiment 1 in this study, where Osm i tended to be lower with no significant changes in p  [Na^pi (Fig. 3.6. C) and [K ] i (Fig. 3.6. D) during M T infusion. Total N a and water +  +  p  excretion (% infusate) were especially lower during the first half an hour of M T infusion compared to control values (Fig. 3.7). Greater water accumulation may also have caused the slightly expanded ECFV in the experimental group. Since Osm i tended to be p  maintained lower and RPV was increased during M T infusion than during control infusion, water may have moved out of cells and reduced Osm i in M T treatment. p  In summary, I could not detect immediate changes in SGS rate and total N a secretion +  by acute M T injection, but overall, subsequent M T infusion significantly decreased SGS rate and tended to maintain a lower total N a secretion in gulls. Acute M T injection +  immediately increased [ N a ] ^ and subsequent M T infusion kept [Na ] 4  +  sgs  elevated.  Therefore, my hypothesis that acute M T injection immediately inhibits SGS rate and total N a secretion, but not [Na ] , and these effects are sustained by subsequent M T infusion +  +  sgs  was partially supported. Total N a secretion constantly increased during M T infusion, +  suggesting MT may have improved the efficiency of excreting N a by salt glands as the +  MT experiment progressed. The present study demonstrated that M T did greatly affect kidney function in gulls and it immediately decreased GFR, where this effect was sustained during continuous M T infusion. Therefore, this result supports my hypothesis that acute M T injection immediately decreases GFR and this effect is sustained by subsequent M T infusion. My hypothesis that acute MT injection immediately decreases UFR and this effect is sustained by subsequent M T infusion was not accepted since M T injection had no immediate effect on UFR and the subsequent M T infusion tended to maintain higher UFR in gulls. I found that M T treatment improved the efficiency of total N a excretion over time +  by both salt glands and kidneys without a loss of body water in gulls.  39  CHAPTER FOUR. GENERAL DISCUSSION There has been only one study (Ching et al. 1999) that investigated effects of M T on salt gland function of birds using Pekin ducks. Although Pekin ducks have functional salt glands, they are not marine birds. Ching et al. (1999) found that raising [MT] i of Pekin p  ducks to its night time level inhibited salt gland function by decreasing SGS rate, and, therefore, total N a secretion. They showed this in two types of experiments (with +  appropriate controls) in which SGS was activated by hypertonic saline infusion. In one experiment, [MT] i was elevated prior to initiation of secretion and in the other, after p  secretion had been ongoing for over 60 minutes. In these two experiments, the physiological states of the bird would have been different when [MT] i was elevated. I p  repeated these experiments on Glaucous-winged Gulls that are marine birds regularly exposed to saline in their natural environment. The protocols pursued were nearly identical in the two studies of Pekin ducks and gulls. I found that the responses of gulls differed somewhat from those of Pekin ducks. When [MT] i was raised to the night time level, there was a transient increase in both SGS rate p  and total N a secretion with no change in [Na ]^, but the effect of M T diminished as +  4  experiment progressed. This phenomenon was also seen in Pekin ducks. When [MT] i was p  acutely raised during ongoing SGS and sustained by continuous M T infusion, [Na ] +  sgs  was  unaffected in Pekin ducks, but immediately increased in gulls. These results suggest MT may affect salt glands of ducks and gulls differently, since M T has diverse effects on the whole animal. By altering blood pressure (Geary et al. 1997; Viswanathan et al. 1997; Scalbert et al. 1998) it could impact SGS rates. By acting on cells to modulate transmembrane transport of electrolytes and ions (Ligris et al. 1982) it could alter [Na ] . +  sgs  Simultaneous kidney function would have greatly influenced the response of the salt glands. GFR, and especially renal tubular ion and water reabsorption, directly impact SGS. GFR and tubular reabsorption dictate possible water and ion loss from the plasma and their recovery in the renal tubules, respectively. The effect of M T on kidney function was not examined in the study of Pekin ducks. I found M T had had significantly greater effects on the kidneys than on the salt glands of gulls. Melatonin decreased GFR only when [MT] i p  40  was abruptly elevated during ongoing SGS (Fig. 3.2. A). UFR of gulls was increased in experimental group in the first experiment (Fig. 2.2. B) and also tended to be maintained higher level during M T infusion in the second experiment compared to that of the control (Fig. 3.2. B). Elevated UFR greatly increased renal N a excretion in both experiments. +  Since effect of M T on renal function of Pekin ducks is unknown, renal responses of ducks and gulls to MT and their possible effects on SGS can not be assessed. The total excretions of N a and water were both at higher rates in M T treatment when +  [MT] i was elevated prior to initiation of SGS (Fig. 2.7). They also continued to increase p  during M T infusion after [MT] i was abruptly elevated during ongoing SGS (Fig. 3.7). This p  is consistent with the higher MT-induced water flux observed in mammals (Richardson et al. 1992). The N a excretion rate(% infusate) exceeded water excretion rate under both +  control and MT conditions (Fig. 2.7, Fig. 3.7), suggesting more water was retained in the body. My studies indicated that MT may elevate N a and water turnover and therefore N a +  +  and water excretion rates in gulls. Water flux of birds with salt glands is normally twice as high as birds without salt glands (Hughes 2003). Pekin ducks are reported to have unusually high water flux compared to the typical seabirds of the same body size (Bennett et al. 2003a). E C F V (% body mass) is larger in marine birds than in non-marine birds (Hughes 2003). A larger ECFV may provide a bird better capacity to equilibrate the osmotic changes to some extent (Bennett et al. 2003c). Therefore, the initial ECFV and water (and Na*) flux rates may contribute to different responses of ducks and gulls coping with osmotic challenges. Also, the proportionate volumes into which the saline and M T were infused likely differed between ducks and gulls. Whereas only males were included in the study of Pekin ducks, with one exception, the gulls were female. Previous studies showed that male and female Pekin ducks have different organs sizes (Hughes et al. 1995; Hughes and Bennett, 2004) and that ECFV is smaller in fresh water (FW) male than in FW female Pekin ducks (Hughes et al. 1989; Bennett et al. 2003c). The tolerance of saline may be different between sexes in this species (Hughes et al. 1989). Therefore, response of salt glands of female Pekin ducks to M R may be different from that of male Pekin ducks.  41  All gut segments of birds appear to have M R and its densities vary among them (Lee and Pang 1993, Lee. et al. 1995). Studies of mammals indicate that MT modulates the gut motility (Holloway et al. 1980), which should affect transit time for food and absorption of its contents (Martin et al. 1998), and that M T inhibits its N a uptake (Legris et al. 1982). +  Melatonin is a vasoconstrictor (Evans et al. 1992; Krause et al. 1995) and it might limit blood flow to the gut tissues and limit sodium uptake. The hindgut, especially the ceca, that are much larger in ducks than in gulls, plays an important osmoregulatory role by reabsorbing N a and water from refluxed urine (Schmidt-Nielson et al. 1963; Anderson and +  Braun 1985; Braun 1999). Pekin ducks do reflux urine to the hindgut, especially into the ceca (Hughes and Raveendran 1994), although N a and water reabsorption there have not +  been quantified. The Mallard, a wild ancestor of the Pekin duck, refluxes about 20% of the urine into the hindgut, mainly into the ceca, where about 20% of its N a and water are +  reabsorbed (Hughes et al. 1999). Ching et al. (1999) did not examine the simultaneous kidney function in Pekin ducks. Melatonin may have had affected N a and water retrieval +  from the hindgut of Pekin ducks, and, therefore, affected salt gland function of these birds. Melatonin from the pineal gland reaches cells via the plasma. The acute M T injection I applied during ongoing SGS would simulate a sudden release of MT, producing a sudden rise in [MT] i This may have modulated blood pressure directly or via another hormone, p  possibly A D H . Changes in GFR can easily be attributed to changes in blood pressure. However, changes in renal reabsorption of N a and water are less easily explained, since +  they occur at apical surfaces of renal tubular cells that may also receive MT from the lateral surfaces of adjacent cells. I can not distinguish in my studies whether plasma MT affected basal cell components of the N a uptake process or MT in the glomerular filtrate affected +  apical components of the process. Seasonal variations of the number of M R (Bmax) and Kd values in gut segments are reported in Pekin ducks and Bmax increases in summer with higher Kd values in posterior gut segments (ileum, rectum, and ceca; M R Hughes et al., unpublished data). Seasonal differences of [MT] in birds (Brandstatter et al. 2001) and GFR values of mammals pi  (Tsuda et al. 1995) are also reported. Ching et al. (1999) conducted their studies in winter (from October to January). My first experiment was conducted from May to September,  42  and the second experiment from October to May. The Different responses of salt glands to MT between experiment 1 and 2 in gulls and between Pekin ducks and gulls may have been also due to seasonal variation of M R in osmoregulatory organs in these birds. The duration of hypertonic saline infusion needed to activate SGS of Pekin ducks directly reflected initial [MT] and they were positively related (Ching et al. 1999). Data pt  for initial  [MT] i p  of the gulls is not yet available, but it probably varies among birds.  Although time to secretion was fairly uniform among gulls (after about lOmin infusion) regardless of treatment, the response of the salt glands and especially the kidneys, varied significantly among gulls. This may reflect  [MT] i p  of individual bird. Present studies  indicated that given the same environmental perturbation (in this case infusion of hypertonic saline), the physiological adjustments to cope with this stress differ among the individuals. Further studies are required to elucidate whether effects of M T on salt gland function differ among avian species, between sexes within a species, or among different seasons, and how effect of M T on renal function affects simultaneous salt gland function of Pekin ducks and gulls differently.  43  LITERATURE CITED Anderson G L Braun EJ (1985) Potrenal modification of urine in birds. Am J Physiol 8:R93-R98. Bakken B H McWhorter TJ Tsahar E Del Rio C M (2004) Hummingbirds arrest their kidneys at night: diel variation in glomerular filtration rate in Selasphorus platycercus. J Exp Biol 207:4383-4391. Bennett DC Gray D A Hughes M R (2003a) Effects of saline intake on water flux and osmotic homeostasis in Pekin ducks (Anas platyrhynchos). J Comp Physiol B 173:27 -36. Bennett DC Hughes MR (2003b) Comparison of renal and salt gland function in three species of wild ducks. J Exp Biol 206:3273-3284. 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