uamnv V l 2H: 637 79— MICHIGANS ‘\ ”‘9“ 5““ l\\\\\\\\\\\\\\\\\\\l\l\ ll ljglllxllll l\\\ \ University 3 1293 005 This is to certify that the thesis entitled "Effects of Deoxycorticosterone Acetate (DOCA) on Salt Appetite" presented by Erkadius has been accepted towards fulfillment of the requirements for M.S. Physiology degree in ,/7ll Ki .7 Major professor Date &k?d_a’43r /é//3?/?j 0-7639 MS U is an Affirmative Action/Equal Opportunity Institution PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. DATE DUE DATE DUE DATE DUE MSU Is An Affirmative ActioNEquel Opportunity Institution . EFFECTS OF DESOXYCORTICOSTERONE ACETATE (DOCA) ON SALT APPETITE By Erkadius A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Physiology 1989 bo§0b4b ABSTRACT EFFECTS OF DESOXYCORTICOSTERONE ACETATE (DOCA) ON SALT APPETITE By Erkadius DOCA 5 mg/kg/ 12hr s.c. increased salt appetite more reliably and predictably than 25 mg DOCP i.m. Increased salt appetite was preceded by increased water intake, which was in turn preceded by increased urine volume. Taste preference threshold for saline was decreased from 0.03 M to less than 0.001 M, and the highest preferred concentration was increased from 0.1 to 0.3 M. Saline intake reached 61 and 57 ml/ 100g at 0.1 and 0.15 M, respectively, which was six times greater than control. Voluntary sodium intake was 8.56, 8.69, and 8.20 mEq/100g at 0.15, 0.2, and 0.3 M, respectively, eight times greater than control. These increases were accompanied by increased sodium excretion and urine volume. ANP 4.0 ug/hr i.v. for 4 days did not further increase the salt intake that was caused by DOCA. We conclude that DOCA increased salt appetite by decreasing taste pref- erence threshold and increasing the acceptance for higher concentrations, and that a higher dose of ANP might be needed to show further increase in salt appetite. AC KNOWI EIXTBWENTS «if gitw»WMwmuwwm.mnmw * ‘.' “WMHWWWMMFBM~ ‘ ' tgtwhohmefeuhfifi }$ - g3 MWMuMthpu-w ‘1\’Vl".“ L " tmummuauw mama-”naps: - A £ .4 A .1— __h thhmuWhfiW r Mafvmmfumfi. ACKNOWLEDGEMENTS I wish to acknowledge that without the participation of Dr. Rudy A. Bernard, my academic advisor, this thesis would never lnve been completed. His generosity, patience, and encouragement assisted and guided me through the whole process of conducting the research and writing this thesis, and enabled me to continue working for my degree when myfimncial supportwmnolongeravailable. I also wish to acknowledge the advice and guidance that I received frcrn Dr. John E. ChimoskeyandDr. Will'mm L. Frantzduringmycoursework, andtheircritieal roleas membersofmyguidancecrxnmitteeinthepreparationofthisthesis. I wish to acknowledge the support of my colleague and best friend, Karen J. .‘.1......., “ 7‘ ‘ fl—n_ _ “ and exewtion ofthe experimentsthatledtothisthesis. I owe her a debt of gratitude for all the energy, time, and thought she freely gave me to make mywork fruitful. Iarn also thankful to'l‘imothyJ. Prielnwho taught me thesurgiml techniques Dale Moreno, Bill Yant, and James Verlinde were others who offered valuable am'stance in conducting the experiments. I also would like to express my gratitude to Mrs. Marilyn Mooney of Okemcs, MI and Dr. Marcha P. Flint of Verona, NJ for having made my life in this country feel like home. Their support and encan'agement helped me through the difficult times and made my life more enjoyable while living away from my wife and son. May God bles you all. TABLE OF CONTENTS LIST OF FIGURES ............................................................................ vii INTRODUCTION .............................................................................. 1 REVIEW OF THE LITERATURE ........................................................... 3 Salt Appetite as a Regulatory Mechanism ........................................... 3 Salt Appetite in the Absence of Need ................................................ 4 Role of Renin-Angiotensin—Aldosterone System ................................... 5 Mnemlocorticoid Escape Phenomenon ............................................. 8 Role of ANP in Need-Free Salt Appetite ............................................ 9 MATERIALS AND METHODS .............................................................. 11 Experimental Design ................................................................... 11 Preliminary Experiments .............................................................. 11 Preference Experiment 12 ANP Experiment ....................................................................... 13 Data Collection ......................................................................... 15 Data Analysis ........................................................................... 16 RESULTS ...... .. .................................. 18 Preliminary Experiments .............................................................. 18 Preference Experiment 24 ANP Experiment ....................................................................... 39 DISCUSSION ................................................................................... 48 Determination of Dose and Route of 1‘ ‘ ' ' ‘ miun 48 Effects on Saline and Water Intake- 48 Preference Tests ........................................................................ 49 ANPExperiment ....................................................................... 51 Conclusion .............................................................................. 52 LIST OF REFERENCES ...................................................................... 53 LIST OF FIGURES l. FIDW O-IART FOR DATA ANALYSIS. I7 2. EFFECTOFTHREEDOSESOFDOCPONSALINEINTAKE .................. 19 3. IDNG TERM EFFECT OF DOCP ON SALINE INTAKE ......................... l9 4. EFFECTOFTHREEDOSESOFDOCPONWATERINTAKE. ................. 20 5. LONG TERM EFFECTOF DOCP ON WATER INTAKE. ........................ Z) 6. EFFECTOFDOCPANDDOCAONSALINEINTAKE ........................... 21 7. WOFDOCPANDDOCAONWA'IERINTAKE. .......................... n 8. EFFECTOFDOCAON SALINE INTAKE. 23 9. EFFECT OFDOCAON WATER INTAKE. 23 10. BODY WEIGHT BEFORE PREFERENCE TESTS ................................. 24 11. FOOD INTAKE BEFORE PREFERENCE TESTS. ................................ 7.5 12. WA'I'ERINTAKE BEFORE PREFERENCE TESTS. .............................. 25 I3. URINE VOLUME BEFORE PREFERENCE IBIS. .............................. 26 14. WATER BALANCE BEFORE PREFERENCE TESIS. ........................... x 15. SODIUM INTAKE BEmRE PREFERENCE TESTS. ............................. 27 16. URINARY SODIUM BEFORE PREFERENCE TESTS. .......................... 28 17. SODIUM BALANCE BEFORE PREFERENCE TESTS. .......................... 28 I8. URINARY POTASSIUM BEFORE PREFERENCE TESTS. ..................... 29 19. BODY WEIGHT DURING PREFERENCE TESTS. ............................... I) 20. FOOD INTAKE DURING PREFERENCE TESTS. ................................ I!) 21. SALINEINTAKE DURING PREFERENCE TESTS. ............................. 31 22. WATER INTAKE DURING PREFERENCE TESTS ............................... 31 23. TOTAL FLUID INTAKE DURING PREFERENCE TESTS. ..................... 32 24. SALINE AND WATER INTAKES OF DOCA GROUP. ........................... 32 25. SALINE AND WATER INTAKES OF CONTROL GROUP. ..................... 33 26. URINE VOLUME DURING PREFERENCE TESTS. ............................. 34 27. WATER BALANCE DURING PREFERENCE TESTS. ........................... 34 28. SODIUMINTAKEFROM SALINESOLUTIONS. 35 29. SODIUM INTAKE FROM SALINE, PIDT'IED IOGARI'IT-IMIQALLY. ...... 35 30. TOTAL SODIUM INTAKE DURING PREFERENCE TESTS. .................. 36 31. TOTAL SODIUM INTAKE, PLOTTED IDGARITHMICALLY .................. 36 32. URINARY SODIUM DURING PREFERENCE TESTS. .......................... 37 33. SODIUM BALANCE DURING PREFERENCE TESTS ........................... 38 34. URINARY POTASSIUM DURING PREFERENCE TESTS ...................... 38 35. BODY WEIGHT CHANGES IN ANP EXPERIMENT. ........................... 39 36. WATER AND SALINE INTAKE OF ANP EXPERIMENT. ...................... 40 37. TOTAL FLUID INTAKE AND URINE VOLUME .................................. 40 38. TOTAL SODIUM INTAKE AND URINARY SODIUM EXCRETION .......... 41 39. BODY WEIGHT DURING EXPERIMENTAL PERIODS. ........................ 42 40. TOTAL FLUID INTAKE DURING EXPERIMENTAL PERIODS. .............. 42 41. TOTAL SODIUM INTAKE DURING EXPERIMENTAL PERIODS. ........... 43 42. URINE VOLUME DURING EXPERIMENTAL PERIODS. ...................... 43 43. URINARY SODIUM DURING EXPERIMENTAL PERIODS .................... 44 44. URINARY POTASSIUM DURING EXPERIMENTAL PERIODS. ............. 44 45. WATER BALANCE DURING EXPERIMENTAL PERIODS ..................... 45 46. SODIUM BAIANCE DURING EXPERIMENTAL PERIODS. .................. 45 47. FLUID VARIABLES DURING SALINE TEST. 46 48. SODIUM VARIABLES DURING SALINE TEST. 47 H‘ITRODUCTION The cause and physiological significance of the increase in salt appetite that occurs during the administration of the mineralocorticoid desoxycorticosterone acetate (DOCA) or aldosterone has been widely investigated[15, 41, 76, 78, 80]. Adrenalectomized rats, whose source of mineralocorticoids has been removed, also develop an increased salt ap- petite, for which the explanation is a possible increase of angiotensin II in the brain[39]. This explanation is also true for other cases of salt appetite induced by sodium deficiency. But in intact rats treated with aldosterone or DOCA, renin and angiotensin concentrations are depressed and cannot be expected to participate in this model of salt appetite, which also differs from the others in that it does not result from lack of sodium. We wished to investigate the salt appetite of DOCA treated rats by doing salt prefer- ence tests, in which a wide range of concentrations of saline were studied to determine the most preferred concentration and the concentration at which aversion begins. We also wished to examine urine volume and sodium excretion during ingestion of the respective saline concentrations. To achieve this, before starting the preference tests, we maintained the rats on DOCA injections of 5 mg/kg body weight twice daily until the effect of injection on water intake, urine volume, sodium excretion, and body weight. had been stabilized. Recent discovery of atrial natriuretic peptide (ANP), whose synthesis and plasma level have been shown to increase during escape from the sodium retention effect of miner- alocorticoids in the kidney, and to remain elevated during mineralocorticoid administrate tion[7, 44, 49, 57], has led to the widely accepted theory that it plays an important role for initiating and maintaining the escape[45]. It is important to point out that dm’ing escape the sodium retaining effect of mineralocorticoid is sustained[24, 45, 59]. It is only in the kid- ney that the retention effect is overridden by ANP[45]. The coincidence that synthesis and plasma levels of ANP increase during mineralo- corticoid administration, and that salt appetite also increases during this time without any satisfactory explanation, led us to think that ANP might have a role in causing this appetite. To test this possibility, we conducted another experiment in which injection of DOCA 5 mg/kg twice daily for seven days, was followed by intravenous infusion of 4.0 pg ANP/hr. The effect on salt appetite was tested by giving the rats a choice of 0.5 M saline and distilled water to drink. Based on the assumption that ANP level had already increased during the injection of DOCA, administration of exogenous ANP would be expected to further increase its concentration, and contribute to a greater increase in salt appetite. REVIEW OF THE LITERATURE SALT APPETTIE AS A REGULATORY MECHANISM Sodium deficiency in nature and in the laboratory results in spontaneous increase in salt appetite. This can be seen in domesticated and wild animals in inland areas where there is lack of environmental sodium[26], and in the laboratory where sodium deficiency is pro- duced experimentally[39]. The first procedure used to produce sodium deficiency and stimulate salt appetite in the laboratory was adrenalectomy, which was reported by Richter in 1936[67]. He showed that adrenalectomized rats, which could not survive without receiving extra sodium in their food, could select the needed salt if they were given a choice of water and a salt solution to drink. He also showed later that increased salt appetite was mediated by a change in taste function, that is, preference threshold for salt solutions was decreased and suprathreshold responses were greater than normal[68]. Administration of adrenal cortical extract or DOCA to these rats returned their salt preference and intake to normal levels, thus confimring that the increased appetite was due to lack of salt-retaining hormones[69, 70]. For Richter[70], salt appetite was seen as an expression of need. He reported that adrenalectomy also increased the appetite for sodium phosphate, sodium iodide, potassium chloride, ammonium sulphate, and calcium lactate in proportion to their concentrations in normal blood serum. Fregly[33] in rats, and Denton[27] in sheep confirmed his report that adrenalectomy increased appetite for various sodium salts (sulfate, bicarbonate, and ni- trate), but they also reported that the appetite did not increase for non-sodium salts such as KCl, LiCl, CaClz, and MgClz. Adrenalectomy as the procedure used to produce sodium deficiency and stimulate salt appetite in the laboratory was followed later by other procedures. These include parotid fistula in sheep[26], intraperitoneal dialysis[30], subcutaneous injection of forma- lin[8l] and polyethylene glycol[73] in rats, administration of diuretics to rats[36, 38, 52] and sheep[26], and placing animals on a low sodium diet[22, 37, 61, 77]. These methods deplete the body of sodium by different routes, but they all act by decreasing preference threshold, and each can be linked to an effect on the renin c; ‘ ' ” ‘ sys- tem[39]. In all of these cases, salt appetite acts as an adaptive mechanism to meet the body's need for sodium. The adaptive nature of salt appetite was further demonstrated when rats made hy- pertensive by encapsulating their kidneys[1, 32, 34, 76] diminished their salt intake by re- ducing their preference for suprathreshold concentrations. SALT APPETITE IN THE ABSENCE OF NEED When Rice & Richter[66] and later, others[15, 41, 76, 78, 80] showed that admin- istration of DOCA and aldosterone to normal rats also increased their salt appetite, it was unexpected and considered paradoxical. Not only did the same drug produce opposite ef- fects, but salt appetite under these conditions is clearly not adaptive, since the animals are retaining sodium and have no need for more. The paradox of salt appetite in the presence of mineralocorticoid was further high- lighted by the finding that the drive and motivation for sodium were the same in adrenalec- tomized rats and in normal rats treated with DOCA[65]. Additionally, the preference threshold for salt solution, which is the concentration at which N aCl intake is significantly greater than water intake, was also diminished in both situations. In adrenalectomized rats, preference threshold for NaCl solution decreased from 0.055% to 0.003% [68], while in normal rats treated with DOCA, threshold was reduced from 0.024% to 0.005%[51]. Wolf[79] found that when adrenalectomized rats were injected with desoxycorticos- terone trimethylacetate (DOCT), they reduced their appetite for NaCl in a dose-dependent manner up to 11 mg/kg body weight. However, doses higher than this caused an increase of their appetite. He described this phenomenon as the restoration of sodium retaining ability at low doses of the mineralocorticoid, and stimulation of salt appetite at higher doses. Fregly[35, 41] reported a similar U-shaped dose-response relationship in adre- nalectomized rats treated with other mineralocorticoids, with the low point occuring with daily injection of 400 pg DOCA, 32 pg aldosterone, or 16 pg 9-a-fluorocortisol. These effects seemed to be specific for mineralocorticoids, since it did not occur with cortisone, estrone, or testosterone. The dose of aldosterone used was roughly the same as the aldo- sterone secretion rate measured from the left adrenal vein[39], showing that the appetite for saline is minimal at normal aldosterone concentration in the blood. These experiments showed that in normal rats, which do not have a need for addi- tional salt, increasing salt appetite can be invoked by increasing mineralocorticoids in their body. What causes this phenomenon, and what is the physiological meaning of it, are questions that still need to be answered. ROLE OF RENIN-ANGIO'IENSIN-ALDOSTERONE SYSTEM Aldosterone secretion is increased by administration of angiotensin II, high concen- tration of potassium, and low concentration of sodium in the systemic circulation as well as in the adrenal artery[13]. Decrease in plasma sodium concentration has been reported to in- crease plasma renin and angiotensin II concentrationsllZ], and subsequently, aldosterone concentration. In adrenalectomized rats, there is the possibility that increased appetite for sodium is caused by a high level of angiotensin II concentration, resulting from decreased plasma sodium concentration. When aldosterone is administered, plasma concentration of sodium increases and angiotensin H declines until the administered dose mimics normal al- dosterone secretion[39], and salt appetite returns to normal. In rats depleted of sodium by intraperitoneal dialysis, increased salt appetite is abolished by nephrectomy, suggesting that the kidney plays a role in increasing salt ap- petite. In those rats, intraperitoneal administration of renin reestablished the appetite within 20 minutes[20], showing that renin and angiotensin are responsible for increasing the ap- petite. Intracerebroventricular administration of angiotensin I as well as angiotensin II also increased the appetite of those rats, while the effect of angiotensin I administration was blocked by concomitant administration of SQ 20881, an angiotensin converting enzyme inhibitor[20]. This observation laid the foundation for the now widely accepted theory that angiotensin II acts in the brain to increase salt appetite. It is also important to note that in the sodium-deprived nephrectomized rats, aldos- terone concentration would have been elevated, but not angiotensin II because the renal source of renin had been removed. As was pointed out by Blair-West[13], decreased so- dium concentration alone in the adrenal artery can increase the secretion of aldosterone. The fact that salt appetite was abolished in these rats showed that aldosterone does not di- rectly increase salt appetite, although in normal rats it appears to do so. Rats fed low salt diet show a brisk appetite for saline solutions[22], and the result- ing low plasma sodium and volume reduction would be expected to increase both angio- tensin II and aldosterone concentrations in the plasma. It is not known whether increased angiotensin II in the plasma will lead to its increased concentration in the brain, but there is the possibility that increased angiotensin I in plasma will lead to increased angiotensin I and thus angiotensin II concentration in the brain[39]. Fregly[40] reported that captopril administration, which blocks the conversion of angiotensin I to angiotensin II and subsequently lowers aldosterone secretion, caused an increase in salt appetite. Administration of DOCA in this case reduced salt appetite in the previously described U-shaped dose-response manner, with the lowest intake occuring at 102 ug/d. This confirmed that low levels of mineralocorticoid can result in a high salt ap- petite, but in this study increased salt appetite could not be explained by an increase in plasma level of angiotensin II. This confusion was resolved when Epstein and colleagues[58] reported in sodium- depleted rats that while salt appetite was increased further by intravenous infusion of capto- pril at 0.04 mg/hr or lower, doses higher than 5 mg/hr decreased the appetite. The expla- nation for this was that at lower doses, captopril increased plasma angiotensin I, which then entered the brain and was converted to angiotensin H, which stimulated salt appetite. At higher doses, captoer also entered the brain and prevented this conversion, and no stimulation of salt appetite occurred. Angiotensin II in the brain has been reported to increase water intake when adminis- tered over short periods of time[6, 23, 28], and to increase salt appetite with chronic ad- ministration[6]. The increase in salt appetite was reported to be specific for sodium, and not secondary to increased water intake or natriuresis. Angiotensin H administered intra- ventricularly also has the ability to restore sodium appetite in sodium deprived, nephrec- tomized rats, as has been pointed out earlier[20]. While increasing mineralocorticoid concentration has been reported to increase salt appetite at the same time that it reduces plasma angiotensin II, Brooks[16] has reported that intraventricular infusion of angiotensin H can also reduce aldosterone secretion. She also reported that administration of SQ 20881, an angiotensin converting enzyme inhibitor, and administration of P-113, a competitive inhibitor to angiotensin H, had resulted in higher al- dosterone secretion. This led to the conclusion that there is a tonic inhibition of aldosterone secretion by brain an giotensin, which is released when its effect is blocked by an inhibitor, or when its concentration is decreased by the converting enzyme inhibitor. From the fact that increased brain angiotensin H and increased plasma aldosterone can individually or together work to increase salt appetite, and from the reports that they can inhibit each other, it is clear that angiotensin II and aldosterone play a complicated role in the mechanism of salt appetite. The question that remains to be explored is how high plasma mineralocorticoid levels stimulate salt appetite in the absence of high plasma or brain angiotensin levels. It has not been possible to stimulate salt appetite by injecting aldo- sterone orDOCA in the brain[17]. MINERALOCORTICOID ESCAPE PHENOMENON Prolonged administration of DOCA or aldosterone has been reported to result in transient retention of sodium in the kidney, which then ‘escapes’ from the mineralocorti- coid effect[5, 24, 25, 59]. This phenomenon has been reported in humans[5], rats[54, 59], dogs[25], and hamsters[31]. The observation that led to the idea of escape was described by Daughaday and MacBride[24] when they reported that after restoration of sodium balance following its ini- tial retention, plasma potassium and sweat sodium concentrations remain low, suggesting a sustained effect of the mineralocorticoid’s action. August[5] several years later observed that administration of large doses of aldosterone resulted in initial sodium retention and weight gain, which then returned approximately to control levels after three days. This escape was thought to be the explanation for the absence of marked edema and sodium re- tention in primary aldosteronism. An impoth observation made by Mohring & Mohring[59] in rats is a nyctohe- meral rhythm in the escape phenomenon. They reported that during the entire period of ob- servation, small amounts of sodium were continually retained during the day and escape occurred at night. This also showed that the mineralocorticoid effect was sustained throughout its administration, and that some regulatory mechanism overrode it during the night and resulted in the escape from its retention effect. This escape phenomenon has been a subject of intense interest to find what causes it. One of the leading hypotheses to explain it is that volume expansion due to sodium re- tention stimulates the release of atrial natriuretic peptide which acts in the kidney to increase sodium excretion[3, 18, 45, 55, 57, 62]. Recent experiments have shown that ANP synthesis and release can be induced by atrial stretch subsequent to volume expansion in rats[29, 56], pigs[49] and dogs[57, 71]. Its effects have been shown to increase glomerular filtration rate in rats[21], increase sodium excretion in humans[2] and in rats[53], inhibit renin secretion in dogs[44, 72] and rats[14], and inhibit aldosterone secretion in rats[l4, 19, 74] and in humans[4] During mineralocorticoid escape, ANP has been shown to increase in the plasma of humans[42, 82], dogs[44, 46, 57], pigs[49] and also rats[7]. The increase in the plasma was accompanied by increasing synthesis of ANP in the atria of the heart[7, 57]. Further, ANP concentration in the plasma was sustained during the time mineralocorticoid was ad- ministered[7, 44, 49, 57], and rapidly declined when the administration was stopped[44]. These observations, together with the report that in DOCA escape sodium reab- sorption decreases in proximal tubules as well as in inner medullary collecting duct[54], suggest that ANP plays a critical role in this phenomenon. Gonzalez-Campoy suggested two major effects of ANP in escape, viz. to block the antinatriuretic mechanisms and to promote the natriuretic pathways[45]. ANP may not play a significant role in blocking the antinatriuretic mechanism in primary aldosteronism or in DOCA treated animals because the concentration of mineralocorticoid is already high, and plasma renin concentration is al- ready depressed. In these cases, the ANP effect is directed more toward promoting the na- triuretic pathways such as increasing sodium excretion by reducing its reabsorption from the proximal tubule and the inner medullary collecting duct. ROLE OF ANP IN NEED-FREE SALT APPETITE The role of ANP in mineralocorticoid escape has led to the hypothesis that ANP may also play a causal role in the gradually increasing salt appetite that follows chronic ex- 10 posure to DOCA. The important role of volume expansion in triggering the ANP system has also suggested the hypothesis that other forms of need-free salt appetite may utilize the same mechanism. Need-free salt intake has been found in rats placed on a high Na diet[63, 64], in spontaneously hypertensive (SI-IR) rats [9], and in rats [10] and rabbits [11] receiv- ing ACTH. Since these procedures do not create Na deficiency, but may involve volume expansion to some degree, they may share the same ANP mechanism. Mooney & Bemard[60] showed that salt appetite increased in rats injected with DOCA, and in rats treated with different concentrations of hydrochlorothiazide (HCZ), but in rats treawd with DOCA and HCZ, the effect on salt appetite was not additive. This result showed that reducing volume expansion by HCZ reduced salt appetite, and that this was presumptively due to reduced plasma concentration of ANP. In human history, salt preference, as well as the desire to consume salty tastes in the absence of any known physiological need for sodium, have made salt a status symbol, and a reason to start a war to control its source[8, 26]. But in modern life, where salt has been more easily acquired, the preference is still exhibited by humans even when they are not sodium deficient[8]. Physiological and mental stress could be responsible for increas- ing secretion of ACTH and subsequent increase of DOCA and corticosterone from the zona fasciculata of the adrenal cortex[43, 50]. Whether the increase in salt appetite in the ab— sence of sodium need can be attributed to the increase in mineralocorticoids, and subse- quently to ANP, still needs further investigation. MATERIALS AND METHODS EXPERIMENTAL DESIGN This experiment was designed to examine the effect of long term administration of desoxycorticosterone (DOC) to rats on intakes of water and salt solutions over a wide range of concentrations. To achieve this, we first determined whether a long- or short-acting form of DOC should be used, and at what doses, during a series of preliminary experi- ments using DOC-pivalate (DOCP) alone, DOCP followed by DOC-acetate (DOCA), and DOCA alone. Since the effect of DOC on salt and water intake develops gradually, the preference tests were preceded by an adaptation period, during which water alone was offered. The preference tests were begun after water intake reached a steady state level. To determine whether administration of ANP will have an additive effect with en- dogenous ANP that was increased by DOCA administration, in enhancing salt appetite, another experiment was performed using ANP infusion in rats which had been injected with DOCA for several days. ANP delivery was designed to coincide with the first day of 0.5 M saline offering. PRELIMINARY EXPERIMENTS The experiments were conducted on adult male Sprague-Dawley rats (Sasco Inc., Omaha, NE) weighing between 300 to 450 grams on the first day of injection. Each animal was housed in a plexiglass cage (32 x 35 x 16 cm) with sawdust bedding and a detachable 11 12 metal top. Room temperature was maintained between 70 -72 'C and lights were kept on from 9 am to 9 pm. Two drinking bottles, one filled with distilled water and the other with 0.5 M saline were provided ad libitum along with a purified rat chow diet (Tekladm, Madison, WI) containing 0.19 mEq Na/gm. Position of the water and 0.5 M saline bottles was changed daily on a random schedule. An adjustment period preceded the drug injec- tions. The forms of DOC used were DOCP (Percorten® Pivalate, 25 mg/ml. CIBA) and DOCA (Desoxycorticosterone Acetate, Sigma) which was dissolved in sesame oil 7.5 mg/ml through heating with 95% alcohol. In the first experiment, DOCP (7.5 mg i.m.) was administered to six rats after an eight-day adjustment period, the dose was increased to 12.5 mg five days later, and a third dose of 25 mg (12.5 mg in each hindquarter) was given five days after the second dose. Four rats served as controls and received sesame oil at the same volume as the DOCP in— jections. This experiment was extended to 148 days because water intake took a long time to return to normal. All rats were sacrificed at the end of the experiment. In the second experiment, DOCP (25 mg/day, i.m.) was injected in eight rats for two consecutive days, followed by another 25 mg 14 days later. Fourteen days after this, DOCA was injected subcutaneously 5 mg/kg per day for four days, followed by 5 mg/kg per 12 hours for eight days. Eight rats served as controls and received sesame oil at the same volume as DOCP or DOCA. All rats were sacrificed after the experiment em The third experiment was performed on six rats which received DOCA 7.5 mg/kg every 12 hours, and six control rats which were injected with the same volume of sesame oil. PREFERENCE EXPERIMENT This experiment was performed on 20 male Sprague-Dawley rats divided into two equal groups. They weighed 297 to 324 grams when the injections of DOCA were begun 13 5 mg/kg every 12 hours. The control group were injected with a similar volume of sesame oil. Rats were housed individually in 24 by 18 cm (id. x height) plexiglass metabolic cages (Nalgene, cat. no. 650-0350) that had been modified to allow for attachment of a second drinking bottle. Cages were mounted in two tiers of four on stainless steel racks with locking swivel casters (Nalgene, cat. no. 350-0500). Standard rat chow (Tekladm, powdered) which contained 0.19 mEq Na /gm was offered ad libitum in a removable feed drawer. During the adaptation period, distilled water was offered in both drinking bottles. During the preference tests, salt solutions were offered in one of the bottles in order of in- creasing concentration, that is 0.001, 0.003, 0.01, 0.03, 0.1, 0.15, 0.2, 0.3, 0.5, and 1.0 M each for a two-day period, except for the first concentration, which was offered for three days. Position of the bottles was adjusted daily so that the saline bottle was never on the same side of the cage for the two days in which a given concentrations of saline was being tested. ANP EXPERIMENT The experiment was performed on male Sprague-Dawley rats weighing between 350 to 389 grams at the start of the experiment. The rats were divided into two groups of seven each, housed individually in the same metabolic cages and given the same diet as was used for the preference experiment. The course of the experiment was divided into three phases, that is, pre-DOCA, DOCA, and DOCA-ANP. Surgery was performed in the last two days of pre-DOCA phase to implant rninipumps for ANP delivery. During the DOCA phase DOCA alone was given to both groups, and ANP infusion accompanied DOCA in- jection in the experimental group during the DOCA-ANP phase. The salt intake test was performed in the DOCA-ANP phase. 14 Both groups were offered distilled water in both drinking bottles during the first two phases of the experiment. Half-molar saline was offered in one of the two bottles, on the left-hand side, in the third phase, to coincide with the scheduled beginning of ANP de- livery. ANP solution was prepared by diluting 3.5 mg ANP (Atriopeptin HI, Sigma®) in 0.9% saline to make 0.4 ml total. Alzet 2002 (Alza®) rninipumps were filled with 0.9% saline, and incubated in a waterbath at 37‘C for at least four hours before implantation. This incubation is needed to prime the rninipumps so they can begin delivery once they are put in the body and connected to the vein via PE tubing. PE 60 (Intramedic®) tubing was prepared by coiling it in 1 cm diameter by immersing it in boiled water for 30 seconds, and was filled to 194 mm length with 0.9% saline and 121 mm length with ANP solution. to give a pumping period of eight and five days, respectively. These lengths were chosen to allow pumping of saline during the second period when DOCA alone was given, to be fol- lowed by ANP infusion to coincide with presentation of 0.5 M saline. The end of the tubing containing ANP was connected to the minipump and the saline end was inserted into the jugular vein. With a pumping rate of 0.457 til/hr, and ANP concentration of 8.75 mg/ml, ANP was calculated to be delivered at a rate of 4.0 rig/hr beginning eight days after nrirripurrrps implantation. For the control group, PE 60 was filled with 0.9% saline, while the rest of the pro- cedure was the same as for the experimental group. Surgery was conducted on the experimental rats on one day, and on the control group the following day. All surgical equipment was autoclaved prior to the procedure. Rats were anesthetized using inhalation of methoxyflurane (Metofane®), they were then secured on the operating board, the neck hair was shaved, and the exposed skin was disin- fected with Betadine®. Guided by a dissecting microscope, the sldn of the neck was opened with a right paramedian incision. Fat tissue, fasciae and muscle were pushed aside, and the right jugu- 15 lar vein was isolated, cleaned, and the cranial part was closed. The middle section was punctured to allow for insertion of PE 60 tubing, which was inserted for 1 cm toward the heart and tied to the vessel. Skin in the left abdominal area was loosened to insert the minipump, Panalog® ointment was applied to the area, and the skin was closed with surgi- cal wound clips. DOCA was injected 5 mg/kg twice daily in both groups beginning two days after the initial surgery. All rats were sacrificed after four days of saline testing, then PE 60 at- tachment to the jugular vein was examined, and the rninipumps were dissected to see if they had delivered the solution. DATA COLLECTION For the preliminary experiments, intakes of saline and water were measured by daily weighing of the water and saline bottles using an electronic balance (BrainweighTM B1500, Ohaus) which was connected to an Apple® H computer running the “Bottle Weigh" program developed by Andrew Bernard. The program calculated daily intakes from each bottle, and salt preference as a percentage of saline intake to the total fluid intake. Body weight was measured manually using the same balance, and the values were entered together with bottle weigh results into an Apple® Macintosh Plus computer running Microsoft® Excel. For the preference and ANP experiments, the bottles, food containers, and individ- ual rats were weighed daily using the same balance. Urine volume was measured in a graduated cylinder, and a 100 pl sample was used to measure urinary sodium and potas- sium concentrations with a flame photometer (Instrumentation Laboratory, model 943). All data were entered into a Microsoft® Excel spreadsheet running on an Apple® Macintosh H computer, which calculated the intakes from each drinking bottle and food container, 16 sodium intake from food and salt solutions, sodium and potassium excreted in the urine, water balance, and sodium balance. DATA ANALYSIS Daily intakes by control and experimental animals in the preliminary experiment were compared using Student’s ‘t’ test on an Apple® Macintosh Plus computer running Microsoft® Excel. Data from the preference and ANP experiments were analyzed using an Apple® Macintosh H computer running Microsoft® Excel for data analysis, and Cricket Graph (Cricket® Software) for charting. Data entry consisted of each day's value for body weight, weight of food container, water and saline bottle, volume and Na & K concentra- tions of urine. From these data the program calculated daily weight gain, daily saline, wa- ter and total fluid intake, daily saline, food and total Na intake, and daily water and Na bal- ance (Figure 1). Water and sodium balance was calculated based on total intake of fluid minus urine volume, and total sodium intake minus urinary sodium excretion. For the preference tests, data from the two days on each concentration were com- bined to produce a daily average for each rat. For the the ANP experiment, data were also analyzed using averages of four-day periods of each rat on the period before surgery was performed, on the last four days before saline was offered, and during saline offerings. Student’s ‘t’ test was used to compare the daily values of all experiments, and also on the daily averages in the preference test, and on the four-day averages of the ANP ex- periment. Data are presented in charts comprising means and standard error of the means. FIGURE 1. FLOW CHART FOR DATA ANALYSIS. Yesterday's valmarehnpmedfiompreviousday'sslxeadslweadwsecondcolumnisme datacollectedonthecurrentday.andtheresultofanalysisisshownonthetwocolumns on the right. RESULTS PRELIMINARY EXPERIMENTS Effect of DOCP on Saline and Water Intake This first experiment showed that a dose of 25 mg DOCP caused a significant in- crease in daily intake of 0.5 M NaCl. Lower doses (7.5 and 12.5 mg in each 5-day pe- riod) did not produce a significant difference in saline intake between experimental and con- trol groups. DOCP at 25 mg produced significant increases on days 13 - 18 (Figure 2), and then saline intake gradually decreased to the control level (Figure 3). Water intake was increased on the first day after injection of 7.5 mg DOCP, and four days after injection of a 12.5 mg dose. The most significant increase in water intake was produced by the dose of 25 mg, beginning on day 12 and reaching the peak on day 18 (Figure 4). There was a gradual decline thereafter, but intake remained significantly differ- ent from control until day 137 (Figure 5), and the experiment was concluded on day 148. 19 FIGURE 2. EFFECT OF THREE DOSES OF DOCP ON SALINE INTAKE. Significant difference was found only on days 13 - l8 (" P<0.05). DOCP = Desoxycorti- costerone pivalate group, CTR = Control group. Vertical lines denotes the days when DOCP was injected intramuscularly. -5 10 25 40 55 70 85 100 115 130 145 DAY FIGURE 3. LONG TERM EFFECT OF DOCP ON SALINE INTAKE. DOCP was given on day 0, 5, and 10 at doses of 7.5, 12.5, and 25 mg i.m. respectively. 20 FIGURE 4. EFFECT OF THREE DOSES OF DOCP 0N WATER INTAKE. Significant difference was found on days 1 and 9 (" P<0.05), and remained significant (** P<0.01) from day 12 and beyond. -5 10 25 40 55 70 85 100 115 130 145 DAY FIGURE 5. LONG TERM EFFECT OF DOCP 0N WATER INTAIGS. DOCP was given on day 0, 5, and 10 at doses of 7.5, 12.5, and 25 mg i.m. respectively, and significant difference was seen between day 12 and 137 (P<0.01). 21 Effect of DOCP and DOCA on Saline and Water Intake In the second experiment 2x25 mg of DOCP produced a significant increase of 0.5 M saline intake on day 11 and beyond (Figure 6). Another 25 mg of DOCP on day 15 kept saline intake elevated without increasing it. Daily injections of DOCA (5 mg/kg) caused another sustained increase in saline intake, and twice daily injections, for a total of 10 mg/kg, increased it to more than six times that of control rats. Water intake increased gradually during the two weeks following the first dose of DOCP, and was stabilized for another two weeks after the second dose. DOCA, at 5 mg/kg/d did not change this intake significantly, but at 10 mg/kg/d increased it gradually to more than 2.5 times that of control rats (Figure 7). DAY FIGURE 6. EFFECT OF DOCP AND DOCA ON SALINE INTAKE. Two doses of25 mg DOCP were injected i.m. on days 0 and l, and one dose on day 15, followed by daily doses of DOCA s.c. 5 mykg on day 29-32, and 5 mg/kg/ 12hr on day 33-41. Saline intake increased significantly beginning on day 4 ( " P<0.05). No further increasein0.5MsalineintakewasobservedaftertheseconddoseofDOCP,butthefirst andseconddosesofDOCAincreamditevenmore. DOC=DOCP-andDOCA- treated group. 22 -5 0 5 10 15 20 25 30 35 40 DAY FIGURE 7. EFECT OF DOCP AND DOCA ON WATER INTAKE. Water intake began to increase significantly on day 2 (‘ P<0.05). The second dose of DOCPdidnotproduceaftrrtlrerinaeaseinwaterirrtake,buttheseconddoseofDOCA did. Effect of DOCA on Saline and Water Intake In the third phase of this preliminary experiment, DOCA was given 7.5 mg/kg ev- ery 12 hours to compare its effect on saline intake with the dose that was used in the previ- ous experiment. Intake of 0.5 M saline increased significantly beginning on day 2 and in- creased gradually until day 12 when the experiment was ended (Figure 8). Water intake in- creased significantly on day 1 and gradually increased until day 12 when the experiment was terminated (Figure 9). 23 FIGURE 8. EFFECT OF DOCA ON SALINE INTAKE. Intake of 0.5 M saline increased significantly two days after injection was begun C‘" P<0.001). DOCA was injected 7.5 mg/kg/12 hr beginning on day 0. 80 20 2 —-o— m . —0— cm 0 I I I I I I I I I I I I -1 o 1 2 3 4 s 6 7 s 9 10 11 12 my FIGURE 9. EFFECT OF DOCA 0N WATER INTAKE. Water intake increased significantly beginning one day after injection was begun ("* P<0.001). PREFERENCE EXPERIMENT Adaptation Period During the period before the salt preference tests were begun, no difference was observed between DOCA and control groups in body weight and in food intake (Figures 10 and 11). DOCA was injected subcutaneously 5 mg/kg every 12 hours. Water intake increased significantly on the first day after DOCA was injected, but no difference was observed between the two groups for eight days. The DOCA group drank significantly more water than the controls beginning on day 9 (P<0.05) and there- after (P<0.001) (Figure 12). Urine volume of the DOCA group was significantly higher than control on day 2 (P<0.05), and strongly differed from them starting on day 3 (P<0.001) (Figme 13). Water balance of the DOCA group was lower than that of controls on day 5, and between days 10 and 16 (Figure 14). FIGURE 10. BODY WEIGHT BEFORE PREFERENCE TESTS. DOCA was injected s.c. 5 mykg twice daily beginning on day 0. No significant differ- ence was found between these groups (P>0.05). Values are means i SEM. 25 FIGURE 11. FOOD INTAKE BEFORE PREFERENCE TESTS. No sigrtificant difference was found between these groups (P>0.05) in day-by-day compar- ison. ‘ 100g FIGURE 12. WATER INTAKE BEFORE PREFERENCE TESTS. Significmtdifferencebetween diesegmupsfirstappearedonday9ofDOCA injection (" P<0.05), and became more significant from day 11 ("* P<0.001). Water intake of the DOCA group on day l was significantly greater than on day 0 (§ P<0.001). 26 12 an.“ A N Y ' 8 § . I“ t ‘ ml/ 6'4 v7“ U 1008 , v ‘V 7" l I 4 on 2 —O— m '—0— CIR 0 I I r I I I I I fI I r I I rfi I I I I I -2 0 2 4 6 8 10 12 14 16 18 DAY FIGURE 13. URINE VOLUME BEFORE PREFERENCE TESTS. Onday2,theDOCAgroupbeganexcretingurineinsignificantlyhighervolumethanthe control group (‘I P<0.05), and maintained significantly higher thereafter 0"“ P<0.001). Urine volume of the DOCA group increased significantly (§ P<0.05) on the first day after injection. 3 ‘ + m ‘ + cm 0 I I I I I I I I T I I I I I I I I I I F FIGURE 14. WATER BALANCE BEFORE PREFERENCE TESTS. WaterbalanceoftheDOCAgroupwm significantly lowerthan thatofcontrols on day 5, and between days 10 and 16 (* P<0.05, “ P<0.01). 27 No difference was observed in food sodium intake (Figure 15), and also no signifi- cant difference was seen in urinary sodium output except on the first day after DOCA injec- tion when the experimental group was significantly lower than the controls (Figure 16). There was also a significant, but smaller difference in the opposite direction on day 6. There was avery significant increase in sodium balanceonday 1 anda smallerone on day 4 (Figure 17). No significant difference was observed in urinary potassium output (Figure 1 8). 1.5 1.2 J 0.9 mEq] ‘ 100g 4 0.6 d 0.3 . —'O— m 1 —0— CTR 0'0 I I I I I I I I I I I I II I I I I I I -2 0 2 4 6 8 10 12 14 16 1 8 DAY FIGURE 15. SODIUM INTAKE BEFORE PREFERENCE TESTS. No significant difference was found between these groups on day-to-day basis. 28 1003 1 0.6 0.3 t —9— m «4 _-._ CIR 0'0 I I I I r I I I I I I I I I I I I I I I I -2 0 2 4 6 8 10 1 2 14 1 6 1 8 FIGURE 16. URINARY SODIUM BEFORE PREFERENCE TESTS. Sodiumretentionwasobservedonday1,0nwhichdieDOCAgroupexcretedlesssodium than control group (" P<0.01). Sodium escape from retention began on day 2. 0.6 u FIGURE 17. SODIUM BALANCE BEFORE PREFERENCE TESTS. Significant difference was observed on day l (** P<0.01) and on day 4 (" P<0.05), and escape began on day 2. 29. 100g 05 ""*" [XXJA "‘."' (:FR Occur Irrrllrrr1rlllrllll -2 0 2 4 6 8 10 12 14 16 18 IINY FIGURE 18. URINARY POTASSIUM BEFORE PREFERENCE TESTS. No significant difference was noted between both groups (P>0.05). Preference Tests No significant difference was seen in body weight and food intake between the two groups (Figs. l9 & 20). The DOCA group drank significantly more saline than controls at all concentrations tested, reaching a peak at 0.1 M and declining thereafter, although the difference between 0.1 M and 0.15 M was not significant (Figure 21). Control rats drank significantly more water than the DOCA group at saline concentrations between 0.001 and 0.15 M, with no significant difference at 0.2 M, and significantly less above this level (Figure 22). Total fluid intake of the DOCA group was significantly higher than the con- trols at all concentrations of saline ofi'ered (Figure 23). Saline intake was significantly greater than water intake in the DOCA group from 0.001 M to 0.3 M (Figure 24). In the control group, saline intake was higher than water intake at 0.03 and 0.1 M, although it was also significantly higher at 0.003 M (Figure 25). 30 450 ‘ "0— m 1 --0- cm 430 I —-— 410 ‘ é GRAM tu- 390 l 370 , . 0.001 0.003 0.01 0.03 0.1 0.15 0.2 0.3 0.5 1.0 : NClConeentration(M) 350*I'r'r'r'r'rrrffifrrl 20 22 24 26 28 30 32 34 36 38 40 DAY ) FIGURE 19. BODY WEIGHT DURING PREFERENCE TESTS. No significant difference was nowd between the DOCA and control groups (P>0.05). 0.001 0.003 0.01 0.03 0.1 ’o.15 0.2 ’ 0.3 0.5 '1.0 ‘ NfilConcentration(M) 18 rI'rfI'I'I'I'I'I'ITI 20 22 24 26 28 30 32 34 36 38 40 DAY FIGURE 20. FOOD INTAKE DURING PREFERENCE TESTS. Significmtdifl'erencebetween DOCAmdcontrolgroupsoccurredonlyondays 33 and40 (* P<0.05). 31 100g o '— T V "V"" T T T—TVV" "I f 1 I I I .001 .01 .1 1 hdhhfifl FIGURE 21. SALINE INTAKE DURING PREFERENCE TESTS. TheDOCAgroupdrankmuchmoresalinethanthecontrolgroupatallconcentrationsof- fered(P<0.001). PeakintakewasobservedatOJMsalinemlthoughthedifferencebe- tween this intake and the intake at 0.15 M was not significant (P>0.05). 35 8 100g FIGURE 22. WATER INTAKE DURING PREFERENCE TESTS. TheDOCAgrmmdranklesswaterdnnmeconuolgroupbefaesalhremenuafion reached 0.2 M (P<0.001), at which point the difference was negligible, but drank signifi- cantly more water beyond this point (P<0.001) 100g 100g 32 60 50 40 q 30 20 10 i 0 I V I IIITVV" I ‘II" 11'1“" I I I ‘TIII' .001 .01 .1 1 hdhhil FIGURE 23. TOTAL FLUID INTAKE DURING PREFERENCE TESTS. The DOCA group drank significantly more total fluid than control at every concentration of saline (P<0.001). They drank maximally when saline concentration was 0.1 M although it didn’t differ significantly from 0.15 M (P>0.05). + w.m.§w .} 50, --v--- WATER \R // .; /’ if FIGURE 24. SALINE AND WATER INTAKES OF DOCA GROUP. Saline intake was significantly greater than water from 0.001 M to 0.2 M (P<0.001), and 0.3 M (" P<0.05). Beyond 0.3 M, water intake far exceeded that of saline (P<0.001). 33 Oil 12.5 10.0 1 7.5 mu 100g 5.0 2.5 --o-- WATER LN 0.0 I I ' I'VV—f" I U V VIVIVI ' V ' I'VVV' .001 .01 .1 1 M NaCl FIGURE 25. SALINE AND WATER INTAKES OF CONTROL GROUP. Saline intake was significantly higha at 0.003 M (* P<0.01) but no difference was ob— served at 0.01 M. Significantly higher preference was observed at 0.03 M (" P<0.05) and0.1 M C“ P<0.01). Nodifferenceinsalineandwaterintakewasobservedat 0.15 M, and beyond this concentration saline intake was significantly less than water in- take ("“‘“" P<0.001). Urine volumes of the DOCA group were greater than control at every concentration of saline offered. The maximum output was achieved at 0.15 M saline, which was not significantly different from 0.1 M (Figure 26). Water balance of the DOCA group was significantly higher than controls at 0.01, 0.03, and 1.0 M (Figure 27). Sodium intake from the salt solutions was higher in the DOCA group at all concen- trations offered (Figs. 28, 29), with the maximum intake occuring at 0.15, 0.2, and 0.3 M. Total sodium intake of the DOCA group was higher beginning at 0.01 M, and the maximum was again reached at 0.15, 0.2, and 0.3 M (Figure 30, 31). 100g 100g 34 . m A 45 o I I I I VIII'. I I I II"" T I II‘IT’V‘ .001 .01 .1 1 hdhhflfl FIGURE 26. URINE VOLUME DURING PREFERENCE TESTS. TheDOCAgroupexcretedfarmaeurinethancontrol irreverysalineconcentrationof- feted (P<0.001). Urine volume of DOCA group reached maximum at 0.15 M, although it didn’t differ significantly from 0.1 M (P>0.05). l2 ‘ . *g; ] .. 10 £3 o I ‘ ffr'tr" ' 1 f VTUIUI’ I I IIWTII] .001 .01 .1 1 FIGURE 27. WATER BALANCE DURING PREFERENCE TESTS. TheDOCAgroupretainedsignificantlymorewaterthancontrol whensalinewasoffered at 0.01 M, 0.03 M, and 1.0 M (" P<0.05). 35 100g 1 I IIIIVTI .001 .01 .l 1 FIGURE 28. SODIUM INTAKE FROM SALINE SOLUTIONS. Sodium intake of the DOCA group was significantly higher than that of control group (P<0.001) at all concentrations. The difference in sodium intake of the DOCA group from 0.15 M to 0.3 M saline was not significant (P>0.05). I r I I'vvt' V V I "V'U' .1 1 MNaCl FIGURE 29. SODIUM INTAKE FROM SALINE, PLOTTED LOGARTTHMICALLY. 36 10 mEq] 1008 q IO. 0' V V VVrVVV' I V V VVVVV' V I V VrVVV' .001 .01 .1 1 FIGURE 30. TOTAL SODIUM INTAKE DURING PREFERENCE TESTS. The DOCA group had significantly higher sodium intake at all concentrations of saline between 0.01 and l M (*" P<0.001). The DOCA group’s total sodium intake reached maximum at 0.2 M, although this was not significantly different from 0.15 M or 0.3 M (P>0.05). Anow denotes the beginning of significant difference. 1° 435;:2 1’ mEq] an / 100g L 1 I V I V V'VVVr V V V VVVVV' V V V V IVIV' .001 .01 .1 l hdbhil FIGURE 31. TOTAL SODIUM INTAKE, PLOT'TED LOGARTTHMICALLY. 37 Urinary sodium output of the DOCA group was significantly higher (P<0.001) than that of the control group at all concentrations offered except 0.001 M. The peak was achieved at 0.3 M, which was not significantly different from excretion at 0.15 and 0.2 M (Figure 32). The DOCA group had significantly higher sodium balance at saline concentra- tions of 0.003 M and 0.03 M ( but very much lower at 0.3 M (Figure 33). The DOCA group excreted significantly less potassium than the control group at saline concentrations of 0.001 to 0.03 M and at 0.5 M (Figure 34). 12 100g 0' V V I 'VV'V' V T V VVVVV' V I V'VVVV" .001 .01 .1 1 LJPBKH FIGURE 32. URINARY SODIUM DURING PREFERENCE TESTS. The DOCA group excreted significantly greater amounts of sodium than control at all concentrations except 0.001 M (*** P<0.001). Sodium excretion reached maximum at 0.3 M, but this did not differ significantly from 0.15 M or 0.2 M (P>0.05). 38 -1 V V V VVVVV' V V T VVVVV' V V V VfVVV‘ .001 .01 .1 ' l hdlflfifl FIGURE 33. SODIUM BALANCE DURING PREFERENCE TESTS. The DOCA group had significantly higher balance at 0.003 M and 0.03 M (" P<0.05), but very much lower at 0.3 M (“'" P<0.001). 100g 0.5 "'."' [XXJK “”9"“ CTR 0.0 1 V V VVVVV' V V VVVVVVT V V V V VVVV' .001 .01 .1 I hdhhfin FIGURE 34. URINARY POTASSIUM DURING PREFERENCE TESTS. The DOCA group excreted significantly less potassium than control group at many differ- ent concentrations of saline ("' P<0.05. " P<0.01). 39 ANP EXPERIMENT After the rats were sacrificed, examination showed that the PE 60 attachment to the jugular veins was in good condition, and that rninipumps had worked as expected. This confirmed the calculations that ANP was delivered at the rate of 4.0 rig/hr beginning on the day that 0.5 M saline choice was initiated. No difference was noted in body weights between experimental (EXP) and control (CI'R) group before surgery was performed, that is, on day -2 for EXP and day -l for CI'R. However, it was significantly different shortly thereafter and during the rest of the experiment (Figure 35). There were no differences either between the two groups in water and 0.5 M saline intake (Figure 36), urine volume (Figure 37), total sodium intake or uri- nary sodium excretion (Figure 38). 450 1 $ 2:»* L" '5 4 : \ r . 4 ",9" ----~ -~-- * fl 1 375.: A. ,5/ aflua..‘. 1 ‘ r. m r '1' ‘ cm I I I I I I I I I I I r I I I -5 -4 - -2 -1 0 I 2 3 4 5 6 7 8 9 10 11 DAY 0'1 3”. q- 0‘ FIGURE 35. BODY WEIGHT CHANGES IN ANP EXPERIMENT. NodiffereneewasobservedbetwmexperimentalmXPhndeonuol (CTR)groups beforesurgery(day -2),butsignifieantdifferencewasnotedshortlyaftasrugeryand for the rest of the experiment (" P<0.01, " P<0.05). 100g 100g 25 1 2°: 15 ‘ 1 M 5: : Pre-DOCA DOCA mm o. r T I I -9 -8 -7 -6 -5 -4 -3 -2 -1 0 I 2 3 4 5 6 7 8 9 1011 IJKY FIGURE 36. WATER AND SALINE INTAKE OF ANPEXPERIMENT. Nodiffaencewasnotedbetweeneomolmdexperimentalgrouponanyday. WTR:waterintake,SALll~lE-=O.5Msalineintake. 0 I I T T r I T I IIIIIIIrII -9-8-7-6-5—4-3-2-101234567891011 FIGURE 37. TOTAL FLUID INTAKE AND URINE VOLUME. Nodiffaeneewasnotedbetweenexpaimartalandcmuolgroupsmanyday. T'Flstotalfiuidintake,UV=urinevolume. 41 s «I + TNaI-EXP —o— T'NaI-CTR M 6 . —-o— UNI-EXP 4 -—o— um-m (44 may 4 100g 1 nocxnnp 1 IIIIIrIII 01234567891011 FIGURE 38. TOTAL SODIUM INTAKE AND URINARY SODIUM EXCRETION Nodiffaeneewasnotedbetweenexpuimtalandcmuolgroupsmmyday. TNaI=totalsodiumintake,UNa=minarysodiumexcretion. Comparisons of four-day averages before surgery (Pre-DOCA), before (DOCA), and during the 0.5 M saline test (DOCA-ANP) are presented in the following figures: no difference in body weight was found before surgery, but a significant difference was found during DOCA injection and during DOCA and ANP administrations and the saline test (Figure 39); there were no differences in water and saline intakes (Figure 40), in sodium intakes from food and saline solution (Figure 41), in mine volume (Figure 42), in urinary sodium and potassium excretion (Figure 43 and 44, respectively), in water balance (Figure 45), and in sodium balance (Figure 46). ANP was designed to be delivered on day 7 in the experimental group, the day on which the saline test was begun. 42 FIGURE 39. BODY WEIGHT DURING EXPERIMENTAL PERIODS. Signifieontdifferencewas observedduringDOCAand DOCA-ANPplnses (‘ P<0.05). Pre-DOCA = before srn'gery was started. DOCA = during injections of DOCA 5 mykg twice daily, DOCA + ANP = during injection of DOCA and offer- ing of saline to both groups. 100g FIGURE 40. TOTAL FLUID INTAKE DURING EXPERIMENTAL PmODS. Nodifferencewmnotedbetweenexperimentalandcmuolgroupsin anyperiod. SALINE=salineintakeintheperiodwhensalinewasoffered 100g mu 1003 43 / FIGURE 41. TOTAL SODIUM INTAKE DURING EXPERIMENTAL PERIODS. No difference was noted between experimental and control groups in any period. SALINE = sodium intake from saline in the period when saline was offered. FIGURE 42. URINE VOLUME DURING EXPERIMENTAL PERIODS. No difference was noted between experimental and control group at any period. 6 . I EXP I CIR 4 mEq! 100:; . 2 0 -r PREDCXIA m FIGURE 43. URINARY SODIUM DURING EXPERIMENTAL PERIODS. Nodiffamcewasmtedbetweurexperimentalandcmuolgroupatanyperiod mFA’ 100g FIGURE 44. URINARY POTASSIUM DURING EXPERIMENTAL PERIODS. Nodifi'erarcewrsmtedbetweurexperimenmlmdcmuolgroupatanyperiod mU 100g 100g 45 FIGURE 45. WATER BALANCE DURING EXPERIMENTAL PERIODS. Nodifi’ermcewasmtedbetweenexpaimentalandconuolgroupatanypa‘iod FIGURE 46. SODIUM BALANCE DURING EXPERIMENTAL PERIODS. Nodiffermcewasmtedbetweenexperimamlandcmuolgmrmatanyperiod 45 Comparisons between experimental and control groups during the saline test, when ANP was also being infused in the experimental group, are shown for fluid variables (water intake, saline intake, total fluid intake, urine volume, and water balance) and sodium variables (food sodium, saline sodium, total sodium intake, urinary sodium excretion, and sodium balance) in figures 47 and 48, respectively. No difference was noted in any of those variable between the two groups. mu 100g VMAJER SAJINE '“JEAL URBWE BALANCE FIGURE 47. FLUID VARIABLES DURING SALINE TEST. Nodifferencewasformdinanyofthevariablesmeasmed. Water=waterintake, saline=salineintake,total=totalfluidintake,urine=urinevolume,balance=water balance. 47 100g m SALINE TOTAL URINE BALAPKIE FIGURE 48. SODIUM VARIABLES DURING SALINE TEST. Nodifferencewasformdinanyofthevariablesmeasmed. Food=foodsodium. safine=salinesodium,uxal=mtalsodiuminmke,uflm=minarysodiumoutput, balance=sodiumbalance. DISCUSSION DETERMINATION OF DOSE AND ROUTE OF ADMINISTRATION Intramuscular injection of DOCP gives a very slow absorption rate, in humans, with an estimated 1.5 - 3.5% of the injected dose being absorbed daily[75]. In the prelim- inary experiments we found that intramuscular DOCP increased 0.5 M saline intake up to 30 ml/day, while subcutaneous DOCA increased it to 45 ml/day when given two weeks after DOCP injection, or to 37 ml/day when given to fresh rats. Water intake reached the maximum of 108 milday with DOCP while DOCA increased it to 133 ml/day. Daily injec- tions of DOCA at 10 mg/kg divided into two doses caused a larger increase in both water and saline intakes than 15 mg/kg, but this was probably due to the residual effect of previ- ously injected DOCP. We decided to use 10 mg/kg DOCA daily given in two doses, because it gave us a more predictable result and higher intake than DOCP, and this smaller dose gave the same or better result than 15 mg/kg/d. EFFECTS ON SALINE AND WATER INTAKE In the preliminary experiments (Figures 2 to 9), we observed that increased saline intake was always preceded by increased water intake. DOCP at 7.5 mg and 12.5 mg in- creased water intake only on one day and four days after each injection, respectively, but no difference was noted in saline intake. DOCP at 25 mg resulwd in increased water intake beginning two days after injection and lasting for more than 100 days, while saline intake 48 49 became significantly elevated three days after injection and remained elevated for six days. Injection of 50 mg DOCP in two days resulted in increased water intake beginning two days after the first injection, and saline intake began to increase after four days. Also, in— jection of DOCA 15 mg/kg/d increased water intake on day 1, while saline intake began to increase on day 2. During the adaptation period preceding the preference tests, before any saline was given, water intake began to increase on day 9 (Figure 12), but urine volume increased be- ginning on day 2 (Figure 13). Both water intake and urine volume increased significantly on the first day following the initiation of DOCA injections. Sodium retention was noted on day l and escape the next day, and there was no difference in sodium excretion noted between the DOCA and control groups in subsequent days (Figure 16 and 17). These results agree with Green, who had suggested that polydipsia in DOCA ad- ministration is caused by a rise in tissue sodium content due to sodium retention[47], and that an augmented salt appetite was discernible somewhat later usually during the first week, together with a correlated increase in sodium output[48]. Davis[25] reported that, in dogs, the onset of polyuria preceded the development of polydipsia. He also found that DOCA increased glomerular filtration rate and renal plasma flow, and decreased filtration fraction. From the temporal relation in these experiments, we found that in our experiment DOCA administration first reduced sodium excretion, which was followed by escape on the second day, and increased minary output on the same day. This was later followed by in- creased water intake and later by increased saline intake. PREFERENCE TESTS Saline solutions were offered after water intake had stabilized for several days, indi- cating that the effect of DOCA had reached a plateau. The treated rats had greater saline in- 50 take than the control rats at all concentrations offered, from 0.001 M to 1.0 M. At 0.001 M, intake was twice as much as the controls, at 0.1 and 0.15 M, it was six times greater, and even at 1.0 M, when both group showed an aversion, the DOCA group still drank more than twice that of the controls (Figure 21). At 0.1 and 0.15 M, saline intake of the experimental was 61 and 57 ml/100g, respectively, compared to control intakes of 11 and 8 ml/100g; total fluid intakes were 62 and 60 ml/100 g for the DOCA group, and 16 ml/100g for the controls. Preference threshold, defined as the concentration at which significantly more saline than water is drunk could not be determined in this experiment because at the lowest con- centration offered saline intake was already eight times higher than water intake. This indi- cates that preference threshold was considerably below 0.001 M. The highest concentra- tion at which they still drink more saline than water was 0.3 M. Compared to the controls, whose preference ranged from 0.03 M to 0.1 M (Figure 25), the DOCA group clearly expanded their preference, which ranged from less than 0.001 M to 0.3 M (Figure 24). Richter showed that the point of divergence of saline and water intake curves of normal rats occurred at 0.009 M[68], while Herxheimerfil], using the excess intake of saline greater than 1/8 of total intake as a significant value, found a preference threshold at 0.004 M, which was reduced to 0.001 M by a 15 mg DOCA implant. The present result suggests that rats treated with DOCA not only will increase their intake of salt at lower con- centrations, but they will also be more tolerant of higher concentrations. Total sodium intake of the DOCA group reached maximum when saline was offered at 0.15, 0.2, and 0.3 M, without any significant difference between them (Figure 30). It is interesting to note, however, that saline intake at these concentrations, combined with water intake, would result in the total molarity of fluid ingested of 0.143, 0.158, and 0.163 M, concentrations that are very close to isotonicity. Voluntary sodium intakes of the DOCA group from the salt solutions at 0.15, 0.2, an 0.3 M were 8.56, 8.69, and 8.20 mEq/100g, respectively (Figure 28 and 29), about eight times greater than the control 51 group’s or the sodium contained in the food. The control group’s voluntary intakes were 1.09, 1.20, and 1.20 mEq/100g at saline concentrations of 0.1, 0.15, and 0.2 M. Sodium intake from food ranged from 1.10 to 1.40 mEq/100g in both groups before saline was offered (Figure 15), and 0.92 to 1.22 mEq/100g during preference tests, without any significant difference between them. Urine volume increased steadily together with increasing fluid intakes, and reached maximum at 0.1 and 0.15 M (Figure 26), which were the concentrations when total fluid intakes reached maximum (Figure 23). Urinary sodium output reached maximum at 0.15, 0.2, and 0.3 M (Figure 32), also at the same concentrations when total sodium intake reached maximum. These results show that increasing appetite for saline solutions, with the resulting increase in water and sodium intakes, was dealt with appropriately by the kid- neys. The negative sodium balance which occurred at 0.3 M (Figure 33) may be the result of the kidney’s effort to excrete the accumulated sodium in the body. During the adaptation period preceding the preference tests, no difference was found in urinary potassium excretion between the DOCA and control group (Figure 18), which is consistent with the return of sodium excretion during escape to control values. However, during the preference tests, the DOCA group excreted significantly less potas- sium than control when saline was offered at concentrations of 0.001, 0.003, 0.01, 0.03, and 0.5 M (Figure 34). The reason for this is not clear. ANP EXPERIMENT Daily administration of 10 mg/kg DOCA during the period before the 0.5 M saline test resulted in greater water intake and urine volume than in the period before surgery was performed, and no difference between control and experimental groups (Figures 40 & 42). The saline test, together with infusion of 4 ug AN P/hr, increased total fluid intake and urine volumes compared to the period when DOCA was given alone, but again, no differ- 52 ence was found between experimental and control groups. No difference was noted either between the two groups in saline intake (Figures 36 & 48), urinary sodium and potassium excretion (Figures 43 & 44), or total sodium intake (Figures 41 & 48). These results showed that no further increase of natriuresis. diuresis, and saline in- take was caused by this dose of ANP. The hypothesis that ANP increase occurring during DOCA escape was responsible for the increased salt intake cannot be proven wrong, how- ever, because there is a possibility that plasma ANP was not sufficiently elevated by the in— fused dose to cause a greater effect, or that salt intake was at a maximum. These possibili- ties can be tested by giving a higher dose of ANP to the experimental group along with a second booster dose of DOCA to the controls. CONCLUSION Administration of DOCA subcutaneously at 5 mg/kg every 12 hours gave a reliable and predictable increase in salt appetite, water intake, and urine volume, compared to 5 mg/kg/d, or to single doses of DOCP intramuscularly. Increasing the dose to 7.5 mg/kg/ 12hr also gave good and predictable results. Saline intake was increased to the maximum at concentrations of 0.1 M and 0.15 M, which was up to eight times greater than basal level. Preference threshold was reduced to below 0.001 M, and salt aversion did not occur until concentration of saline of- fered exceeded 0.3 M. Voluntary sodium intake was up to eight times greater than that of the control during the preference tests or the basal intake preceding the preference tests. While increased salt appetite in response to sodium need can be attributed to higher angiotensin 11 concentration in the brain, but not directly to increased mineralocorticoid levels, the release of ANP during mineralocorticoid escape offers a possible explanation for increasing salt appetite during DOCA administration. Infusion of 4.0 ttg ANP/hr for four days was not sufficient to increase ANP level enough to increase salt appetite further. 10. 11. 12. LIST OF REFERENCES Abrams, M., A. I. C. DeFriez, D. C. Tosteson and E. M. Landis. 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