0013-7227/91/1283-1317$03.00/0 Endocrinology Copyright © 1991 by The Endocrine Society
Vol. 128, No. 3 Printed in U.S.A.
Oxytocin Produces Natriuresis in Rats at Physiological Plasma Concentrations* JOSEPH G. VERBALIS, MICHAEL P. MANGIONE, AND EDWARD M. STRICKER Departments of Medicine and Behavioral Neuroscience, University of Pittsburgh, Pittsburgh, Pennsylvania 15261
ABSTRACT. Oxytocin (OT) is known to stimulate natriuresis in rats when administered in large doses that, produce high plasma levels. We examined the effects of physiological plasma OT levels on renal sodium excretion by infusing graded doses of OT sc in conscious adult male rats maintained on a sodiumdeficient diet. Our results demonstrate that OT causes a doserelated increase in urinary sodium excretion during the initial day of infusion. The lowest plasma OT levels associated with increases in urinary sodium excretion (5-6 pmol/liter) were well within the range of physiological OT secretion in rats. However, this natriuretic effect was not sustained during subsequent days of maintenance on a sodium-deficient diet, suggesting that the OT-induced natriuresis was limited in part by receptor desensi-
T
tization and/or a decreased exchangeable sodium pool in combination with secretion of opposing antinatriuretic factors such as aldosterone. Pretreatment with an OT receptor antagonist completely blocked the natriuresis produced by a 20 pmol/h infusion of OT, but urinary sodium excretion was not affected by a vasopressin V\ antagonist and was blocked only partially by a combined vasopressin Vx and V2 antagonist. Together with previous studies in rats demonstrating an inverse relation between pituitary OT secretion and sodium appetite, these results support the hypothesis that peripherally and centrally secreted OT act in concert in rats to produce a negative sodium balance by stimulating sodium excretion while inhibiting sodium ingestion. (Endocrinology 128: 1317-1322,1991)
within the range of physiologically stimulated OT secretion in rats (1, 13). The studies reported here were undertaken to assess the effects of exogenous OT on urinary sodium excretion in conscious rats using sc infusions at rates producing physiological plasma OT levels. OT was infused during adaptation to a sodium-deficient diet, a protocol that optimized our ability to detect changes in urinary sodium excretion by avoiding the large obligate natriuresis of rats maintained on standard sodium-rich laboratory chows (14).
HE NEUROHYPOPHYSEAL hormone oxytocin (OT) has a well recognized role to stimulate milk ejection in lactating females. In contrast, there is not yet any proven physiological function for circulating OT in males or nonlactating females. However, evidence in rats has suggested a possible involvement of this peptide in the regulation of sodium balance; OT is secreted along with vasopressin (AVP) in response to stimuli such as hyperosmolality and hypovolemia (1, 2), OT receptors are present in kidney tubular cells (3-5), administration of large doses of OT has long been known to cause increased renal sodium and chloride excretion (6-9), and pituitary OT secretion is correlated with inhibition of sodium appetite in rats (10-12). Although in early studies the doses of OT used to stimulate natriuresis were large, more recently OT has been shown to produce small increases in urinary sodium excretion for short periods in anesthetized rats at iv infusion rates that elevated plasma OT levels to 16 ± 2 /lU/ml (32 ± 4 pmol/liter),
Materials and Methods Animal protocols
Received August 28,1990. Address all correspondence and requests for reprints to: Dr. Joseph G. Verbalis, 930 Scaife Hall, University of Pittsburgh, Pittsburgh, Pennsylvania 15261. * This work was supported in part by NIH Grants DK-38094 and MH-25140 (MERIT Award). Parts of this study were presented in preliminary form at the Society for Neuroscience Meetings, Toronto, Ontario, Canada, 1988, and the American Society for Clinical Investigation, Washington, DC, 1989.
All studies used male Sprague-Dawley rats (Zivic-Miller, Allison Park, PA), weighing 250-300 g at the time of study. Rats were housed in individual plastic metabolic cages (Nalgene model 650-0100, Rochester, NY) in a temperature-controlled room (22 C), with lights on from 0700-1900 h. All animals were initially maintained on standard laboratory rat chow (Wayne Laboratories, Chicago, IL; Na+ content, 170.0 ± 3.4 mmol/kg) and tap water ad libitum. Three days before study, jugular venous catheters were inserted for blood sampling (15). On the morning of the first day of study (day 1), the food was switched to a sodium-deficient rat chow (Teklad 170840, Madison, WI; Na+ content, 3.9 ± 0.2 mmol/kg). On the morning of the next day (day 2), osmotic minipumps (Alzet model 2001, Alza, Palo Alto, CA) containing
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OXYTOCIN-INDUCED NATRIURESIS IN RATS
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140 mM NaCl or various concentrations of OT (2, 10, 20, or 100 nmol/ml) were implanted sc using inhalational anesthesia with methoxyflurane. The osmotic minipumps were left in place for the remainder of the study. Daily 24-h urine and plasma samples were collected at the end of each of the 4 test days. For some experiments, osmotic minipumps containing OT at a single concentration (20 nmol/ml) were inserted at the beginning of day 2, 3, or 4 in separate groups of rats. The pumps were left in place for the duration of the study, and daily 24-h urine and blood samples were collected as before. In additional experiments osmotic minipumps containing selected OT or AVP antagonists were implanted sc at the start of day 1, 24 h before insertion of a second osmotic minipump containing OT at a concentration of 20 nmol/ml. Peptide infusions Synthetic OT (Sigma, St. Louis, MO) was diluted in 150 mM NaCl to yield concentrations of 2, 10, 20, and 100 nmol/ml, which when loaded into the osmotic minipumps resulted in nominal OT infusion rates of 2, 10, 20, and 100 pmol/h, respectively. Subsequent RIA of the synthetic OT solution revealed actual infusion rates of 1.6, 7.8, 15.6, and 78.6 pmol/ h. Control animals received pumps containing only 150 mM NaCl. For receptor antagonist studies, OT or AVP antagonists were dissolved in 150 mM NaCl at concentrations that yielded an infusion rate of 1 nmol/h when released from the osmotic minipumps. The receptor antagonists used were: [Pen\Phe(Me)2,Thr4,Orn8]OT [OT antagonist (16); courtesy Drs. D. Smith and V. Hruby, University of Arizona, Tucson, AZ], [d(CH2)£1,Tyr(OMe)2,Arg8]AVP [AVP Vx antagonist (17); Bachem, Torrance, CA], and [d(CH2)6\DPhe2,Val4,Arg8,Des-Gly9]AVP [combined AVP Vx and V2 antagonist (18); Bachem]. Plasma and urine analysis Urine samples were analyzed for Na+ and K+ concentrations (Beckman Electrolyte II Analyzer, Brea, CA) and osmolality (Advanced Instruments 3C2 Osmometer, Needham Heights, MA). Total 24-h urinary Na+ excretion was calculated as the product of the urine volume (in milliliters) and the urinary Na+ concentration (in micromoles per ml). Blood samples (1.5 ml) were withdrawn through the jugular venous catheters and placed into iced heparinized glass tubes. The amount withdrawn was replaced immediately with an equal volume of 140 mM NaCl. After centrifugation (3000 x g at 4 C), the plasma Na+ concentration was measured (Beckman Electrolyte II Analyzer), and the remaining plasma was frozen to -20 C. Plasma OT levels were measured by RIA via methods described previously, using Posterior Pituitary Reference Standard (U.S.P, Rockville, MD) for the standard curve (15). For this report, the measured plasma OT levels were converted to molar concentrations using the relation 1 U = 2 nmol. Statistical analysis All results are reported as the mean ± SE. Statistical significance was determined by one-way analysis of variance for comparison of multiple groups, with post-hoc comparisons by
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the method of Newman-Keuls where appropriate. Correlation coefficients were calculated by the method of Pearson. Regression analysis was performed by the method of least squares (Tablecurve curve fitting software, Jandel Scientific, Corte Madera, CA).
Results Urinary sodium excretion ranged from 800-1400 /imol/ 24 h on the first day after initiation of the sodiumdeficient diet (day 1) in all rats. By day 2 urinary sodium excretion fell to 195 ± 2 1 /imol/24 h in the control rats (Fig. 1, • ) and then remained between 100-200 jumol/24 h for each day thereafter. The rats receiving sc infusions of OT demonstrated a dose-related increase in urinary sodium excretion on the first day of OT infusion (day 2; nominal rates of OT infusion in picomoles per h are shown below each bar in Fig. 1). However, on subsequent days of the continuous OT infusion (days 3 and 4) the urinary sodium excretion returned to levels equivalent to those in the control rats. Basal plasma OT levels (day 1) were similar in all animals and did not change significantly over the 4 days during which the control group was maintained on the sodium-deficient diet (Table 1, first row of each infusion rate). As expected, the sc OT infusions produced doserelated increases in plasma OT levels (r = 0.89; P < 0.001). The mean plasma OT levels for the OT-infused groups were statistically greater than the mean OT levels in the control rats at all nominal infusion rates of 10 pmol/h or greater (Table 1), the same doses associated with significantly stimulated natriuresis (Fig. 1, day 2). Plasma OT levels remained significantly elevated throughout the 3-day infusion period (Table 1), although the increased urinary sodium excretion did not persist on subsequent days of the continuous OT infusion (Fig. 1, days 3 and 4). Regression analysis of the relation between the measured plasma OT levels and urinary sodium excretion on the first day of OT infusion (day 2) in individual rats revealed that a logarithmic function best fit these parameters (urinary Na+, -152.13 + 746.25 X log10 [plasma OT]; r = 0.75; P < 0.001; n = 71; Fig. 2). In contrast, no significant relation was found between plasma OT levels and any of the other parameters followed throughout the infusion protocol [plasma Na+ concentration (Table 1, second row of each infusion rate), urinary K+ excretion, urine osmolality, urine volume, water intake, and body weight changes (not shown)]. Further inspection of Fig. 2 shows that increased urinary sodium excretion above basal excretion rates (195 ± 21 jumol/24 h) first became apparent in some of the OT-infused rats at plasma OT levels as low as 5-6 pmol/liter. When separate groups of rats were infused with OT beginning on different days after introduction of the
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OXYTOCIN-INDUCED NATRIURESIS IN RATS
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(12) (12)
(»)
1400-
FlG. 1. Urinary sodium excretion during the first day of the sodium-deficient (NaD) diet (day 1) and on each day of OT infusion (days 2-4). Each bar shows the results for groups of rats infused with OT at different rates (the numbers beneath the bars on days 2-4 indicate the nominal OT infusion rate for each group in picomoles per h). Shown are the mean ± SE. The numbers above the bars on day 1 indicate the number of rats studied at each infusion rate. *, P < 0.05; • * , P < 0.01 (compared to the 150 mM NaCl infusion group). 0
2 10 20 100
0
DAY 2
DAY 1
2 10 20 100
0
2 10 20 100
DAY 3
DAY 4
I NaD DIET I |OT INFUSION I
TABLE 1. Plasma OT levels and Na+ concentrations on each day of study OT infusion rate (pmol/h)
OT infusion Day 1 4.0 ± 0.6 142 ± 1 (20) 4.0 ± 0.8
Day 2
Day 3
5.0 ± 0.8 143 ± 1 (15) 6.2 ± 0.8
5.6 ± 0.8 143 ± 1 (14) 6.2 ± 1.0
Day 4
Mean for days 2-4
5.0 ± 0.8 4.8 ± 0.4 142 ± 1 141 ± 1 (20) (19) 6.0 ± 0.6 5.4 ± 1.0 144 ± 1 144 ± 1 143 ± 1 142 ± 1 144 ± 1 (9) (8) (11) (11) (9) 10 3.8 ± 1.0 9.0 ± 1.6° 12.4 ± 1.8° 10.2 ± 1.8" 15.2 ± 3.6° 142 ± 1 141 ± 1 142 ± 1 143 ± 1 142 ± 1 (11) (5) (11) (11) (5) 13.0 ± 0.8" 20 4.0 ± 0.4 10.4 ± 1.4" 12.2 ± 1.8° 15.0 ± 2.0° 143 ± 1 143 ± 1 142 ± 1 143 ± 1 143 ± 1 (20) (22) (15) (20) (18) 72.2 ± 8.2" 2.8 ± 0.8 100 66.8 ± 12.8° 64.2 ± 9.2" 72.6 ± 7.0" 141 ± 1 141 ± 1 144 ± 1 142 ± 1 141 ± 1 (12) (7) (12) (9) (8) For each OT infusion rate, the top row shows the measured plasma OT concentration (in picomoles per liter), and the second row shows the simultaneously measured plasma Na+ concentration (in millimoles per liter); the number of animals in each group is shown in parentheses in the bottom row. "P < 0.05 relative to controls (0 infusion rate). 0
sodium-deficient diet, each group demonstrated a significant increase in urinary sodium excretion for the initial 24-h period after the start of the OT infusion (Fig. 3). However, the magnitude of the induced natriuresis was significantly less when the OT infusion was started on day 4 of access to the sodium-deficient diet rather than earlier (P < 0.05 compared to values obtained when the OT infusion was begun on days 2 and 3). Although accurate metabolic sodium balances cannot be calculated because neither food intakes nor fecal sodium losses were measured during these studies., ap-
proximate sodium balances can be estimated with reasonable certainty, since these amounts are relatively small in rats on the sodium-deficient diet. Rats of similar body weight in our laboratory ingest 20 g/24 h sodiumdeficient diet, representing a sodium intake of approximately 80 jumol/24 h. Daily 1.5-ml blood samples were withdrawn, representing a net body sodium loss of approximately 125 /umol/24 h (assuming a mean plasma Na+ concentration of 142 mmol/liter and a hematocrit of 40-42%). However, because the blood withdrawn was replaced with 1.5 ml 140 mM NaCl (210 ixmol sodium),
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OXYTOCIN-INDUCED NATRIURESIS IN RATS
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1600 -
1400 -
1200 -
a
1000 -
800 -
600 -
400 -
200 -
100
10 Plasma Oxytocin (pmol/l)
FIG. 2. Relation between 24-h urinary sodium excretion and plasma OT levels on the first day of OT infusion. The solid line depicts the least squares regression equation that best fit the data (urinary Na+, -152.13 + 746.25 X log10 [plasma OT]; r = 0.75; P < 0.001; n = 71), and the dashed lines indicate the 99% confidence intervals of the calculated regression line. 1400-
(8) (8)
(8)
DAY 1
DAY 2
DAY 3
Endo•1991 Voll28«No3
fore, amounted to approximately 165 jumol/24 h, which is quite close to the measured urinary sodium excretion of 100-200 jtmol/24 h in rats on the sodium-deficient diet (Fig. 1). Application of these estimates to the results shown in Fig. 3 indicates that the groups of rats receiving the OT infusions on different days all had negative sodium balances of approximately 1 mmol before the OT infusions. The groups infused with OT on days 2 and 3 then developed an additional negative balance of approximately 0.5 mmol before becoming refractory to further stimulated natriuresis, while the rats infused with OT for the first time on day 4 of the sodium-deficient diet developed an additional negative sodium balance of only 0.2 mmol during the 24-h period of OT infusion. To identify the renal receptor type(s) responsible for the OT-induced natriuresis, groups of animals were pretreated with OT and AVP receptor antagonists for 24 h before initiation of an OT infusion of 20 pmol/h on day 2. All antagonists were infused continuously at a rate of 1 nmol/h beginning 24 h before initiation of the OT infusion, representing a 50-fold excess of antagonist to the infused OT. The OT receptor antagonist completely abolished all significant OT-induced sodium excretion (Fig. 4) without affecting urine osmolality (1768 ± 106 mosmol/kg H2O after infusion of the antagonist vs. 1554 ± 158 mosmol/kg H2O in control rats; P = NS). Although the AVP Vi antagonist had no effect on OT-stimulated natriuresis, a partial inhibition was seen in response to the combined AVP Vi and V2 antagonist, but not to the same degree as that seen with the OT receptor antagonist (Fig. 4).
DAY 4
FIG. 3. Urinary sodium excretion after institution of a sodium-deficient diet on day 1 and infusion of OT (20 pmol/h) beginning on day 2, 3, or 4 (as indicated by the arrows below the figure). Shown are the mean ± SE. The numbers above the bars on day 1 indicate the number of rats studied in each group. *, P < 0.05; • * , P < 0.01 (compared to 150 mM NaCl infusion group from Fig. 1).
the blood-sampling protocol actually resulted in a net sodium gain of approximately 85 /imol/24 h. The overall increase in body sodium resulting from the combined dietary sodium intake and the infusion protocol, there-
-OT
+OT (20 pmol/h)
FIG. 4. Urinary sodium excretion on the second day of the sodiumdeficient diet in rats pretreated for 24 h with OT or AVP receptor antagonists before infusion with OT (20 pmol/h). D, Control rats not receiving an OT infusion; • , rats receiving the OT infusion alone; 1, rats pretreated with the indicated antagonists before the OT infusion. The number of rats in each group is indicated above the bars. *, P < 0.05; **, P < 0.01 (compared to rats receiving the OT infusion without antagonist pretreatment).
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OXYTOCIN-INDUCED NATRIURESIS IN RATS
Discussion The studies reported here show that continuous sc infusions of OT cause relatively large dose-related increases in urinary sodium excretion in conscious rats maintained on a sodium-deficient diet. The increased natriuresis occurred at plasma OT levels well within the range of physiologically stimulated OT secretion. Although previous studies have reported natriuresis in response to administration of OT, for the most part those studies used doses that elevated plasma OT levels significantly in excess of concentrations generally achieved under physiological conditions. Our detection of natriuresis at lower plasma OT levels probably reflects an enhanced ability to detect modest increases in natriuresis at the markedly reduced levels of urinary sodium excretion in rats maintained on a sodium-deficient diet. The initial natriuretic effect of exogenous OT was relatively short-lived, however, and urinary sodium excretion returned to control levels by the following day despite maintenance of plasma OT levels within the same ranges as were achieved on day 2. Such blunting of the OT-induced natriuresis with prolonged infusions might be due to several potential causes. One factor could be desensitization of the receptor-mediated mechanism(s) by which OT stimulates urinary sodium excretion. This possibility is supported by the results shown in Fig. 3, because in these studies OT produced equivalent increases in urinary sodium excretion when infusions began on day 3 of sodium deficiency compared to day 2. However, these results could also be explained simply as a result of cumulative losses of body sodium in urine. Thus, rats given OT beginning on day 2 may have excreted little additional sodium on day 3 because they had accumulated a negative sodium balance of approximately 1.5 mmol during the 2 previous days, whereas rats given OT for the first time beginning on day 3 had a negative sodium balance of only approximately 1 mmol up to this point and, therefore, probably had a residual pool of exchangeable sodium sufficient to allow further stimulated natriuresis. However, this explanation cannot account for the blunted natriuresis seen when rats were given OT beginning on day 4, by which time they also had accumulated a negative sodium balance of only approximately 1 mmol. The latter results, therefore, suggest that after a more prolonged duration of sodium deficiency and/or increasing urinary sodium losses, the effects of OT to induce natriuresis are also antagonized by physiological mechanisms. The most likely candidate for such an antinatriuretic effect would be the adrenal steroid hormone aldosterone, which is known to be a mediator of sodium retention in response to sodium deficiency (19) and hypovolemia (20) in rats. Thus, although the use of a sodium-deficient diet in these experiments lowered
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basal urinary sodium excretion and thereby facilitated observation of a natriuretic effect of OT, it also probably limited the magnitude and duration of that effect, both because there was less exchangeable sodium to excrete, and because it stimulated elevated plasma levels of aldosterone. The OT-stimulated natriuresis appears to be mediated by specific renal OT receptors, because pretreatment with an OT receptor antagonist completely eliminated the natriuresis produced by infusion of OT at a rate of 20 pmol/h. It seems unlikely that the infused OT had significant effects on kidney AVP receptors, because urine osmolalities were not affected by infusion of the OT antagonist. Moreover, a potent AVP V\ antagonist had no effect on OT-stimulated natriuresis, and a combined Vi and V2 antagonist had only partial effects. These results were somewhat surprising in light of the fact that many AVP Vi receptor antagonists are also generally effective at blocking uterine OT receptors as well (21) and suggest the possibility that the renal OT receptors recently described in the kidney (3-5) may in some ways be biochemically and functionally different from OT receptors elsewhere. Further studies will be required to evaluate this possibility. Our findings demonstrate a robust urinary sodium excretion in response to physiologically relevant plasma OT levels, in agreement with numerous previous studies suggesting that OT might act as a natriuretic agent under certain physiological circumstances (22). One situation in which OT-induced natriuresis may be physiologically significant is during hyperosmolar volume expansion. Hyperosmolality is known to be a potent stimulus to pituitary OT as well as AVP secretion in rats (1, 2). Although it has long been known that hyperosmolalityinduced AVP secretion is responsible for the antidiuresis and water retention that contribute to osmoregulation under these circumstances, it has never been apparent why OT secretion should also be stimulated in rats during hyperosmolar conditions. The present studies demonstrating induced natriuresis of a significant magnitude at physiological plasma OT levels in conscious rats indicate a possible role for the secreted OT that would complement the water retention produced by the secreted AVP. In addition, some studies have suggested a synergistic effect of AVP and OT on natriuresis in rats (23, 24), and if present, this interaction might potentiate the natriuretic effects of OT during hyperosmolar conditions when both peptides are secreted. Furthermore, under conditions of a hyperosmolar volume expansion, OTinduced natriuresis would not be limited by a depleted exchangeable sodium pool or stimulated aldosterone secretion, as occurs during sodium deficiency. Recent data have also provided evidence that central secretion of both AVP and OT may produce behavioral
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OXYTOCIN-INDUCED NATRIURESIS IN RATS
effects that complement their physiological actions. Studies in dogs have suggested that centrally released AVP potentiates water ingestion during osmotic dehydration (25), which would complement the antidiuresis produced by pituitary AVP secretion. Similarly, in rats there is an inverse correlation between pituitary OT secretion and sodium appetite (10-12), and inhibition of NaCl consumption would complement an OT-mediated natriuresis to promote a negative sodium balance in hyperosmolar animals. In this regard, it is interesting to note that in several different models of sodium appetite in rats, induced hyperosmolality interrupts further NaCl ingestion in proportion to the magnitude of the stimulated OT secretion (11). Because recent studies have documented simultaneous central and peripheral secretion of AVP during induced hyperosmolality (26, 27), it seems likely that a similar coordination of central and peripheral secretion of OT could be the basis for mediating complementary physiological and behavioral effects on sodium balance in rats. In summary, our studies in conjunction with previous reports suggest that circulating OT stimulates urinary sodium excretion at physiological plasma levels in rats, and this effect appears to be mediated by specific OT receptors in the kidneys. Although the induced natriuresis is pronounced, it is limited and, therefore, is likely to be physiologically significant only under certain conditions, such as hyperosmolar volume expansion. The full physiological significance of these effects and the mechanisms that limit its duration and magnitude remain to be evaluated by further studies.
Acknowledgments The authors wish to thank Marcia Drutarosky for technical assistance, and Michele Dobransky and Marge Altvater for secretarial assistance.
References 1. Balment RJ, Brimble MJ, Forsling ML 1980 Release of oxytocin induced by salt loading and its influence on renal excretion in the male rat. J Physiol (Lond) 308:439-449 2. Strieker EM, Verbalis JG 1986 Interaction of osmotic and volume stimuli in the regulation of neurohypophyseal secretion in rats. Am J Physiol 250:R267-R275 3. Stoeckel ME, Freund-Mercier MJ, Palacios JM, Richard PH, Porte A 1987 Autoradiographic localization of binding sites for oxytocin and vasopressin in the rat kidney. J Endocrinol 113:179-182 4. Tribolle E, Barberis C, Dreifuss JJ, Jard S 1988 Autoradiographic localization of vasopressin and oxytocin binding sites in rat kidney. Kidney Int 33:959-965 5. Cantau B, Barjon JN, Chicot D, Baskevitch P, Jard S 1990 Oxytocin receptors from LLC-PKi cells: expression in Xenopus oocytes. Am J Physiol 258:F963-F972
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6. Fraser AM 1942 The action of the oxytocic hormone of the pituitary gland on urine secretion. J Physiol 101:236-251 7. Sawyer WH 1952 Posterior pituitary extracts and excretion of electrolytes by the rat. Am J Physiol 16:583-587 8. Jacobson HN, Kellogg RH 1956 Isotonic NaCl diuresis in rats, antidiuresis and chloruresis produced by posterior pituitary extracts. Am J Physiol 184:376-389 9. Chan WY 1965 Effects of neurohypophyseal hormones and their deamino analogues on renal excretion of Na, K and water in rats. Endocrinology 77:1097-1104 10. Strieker EM, Hosutt JA, Verbalis JG 1987 Neurohypophyseal secretion in hypovolemic rats: inverse relation to sodium appetite. Am J Physiol 252:R889-R896 11. Strieker EM, Verbalis JG 1987 Central inhibitory control of sodium appetite in rats: correlation with pituitary oxytocin secretion. Behav Neurosci 101:560-567 12. Strieker EM, Verbalis JG 1988 Hormones and behavior: biological basis of thirst and sodium appetite. Am Scientist 76:261-267 13. Balment RJ, Brimble MJ, Forsling ML 1982 Oxytocin release and renal actions in normal and Brattleboro rats. Ann NY Acad Sci 394:241-253 14. Strieker EM 1981 Thirst and sodium appetite after colloid treatment in rats. J Comp Physiol Psychol 95:1-25 15. Verbalis JG, McHale CM, Gardiner TW, Strieker EM 1986 Oxytocin and vasopressin secretion in response to stimuli producing learned taste aversions in rats. Behav Neurosci 100:466-475 16. Chan WY, Hruby VJ, Rockway TW, Hlavacek J 1986 Design of oxytocin antagonists with prolonged action: potential tocolytic agents for the treatment of preterm labor. J Pharmacol Exp Ther 239:84-87 17. Kruszynski M, Lammeck B, Manning M, Seto J, Haldar J, Sawyer WH 1980 [l-(|8-mercapto-j8,j8-cyclopentamethylenepropionic acid)2-(O-methyl)tyrosine]-arginine vasopressin and [l-(/3-mercapto-/?,/3-cyclopentamethylenepropionic acid) ] -arginine vasopressin, two highly potent antagonists of the vasopressor response to arginine vasopressin. J Med Chem 23:364-368 18. Jard S, Gaillard RC, Guillon G, Marie J, Schoenenberg P, Muller AF, Manning M, Sawyer WH 1986 Vasopressin antagonists allow demonstration of a novel type of vasopressin receptor in the rat adenohypophysis. Mol Pharmacol 30:171-177 19. Gross F, Brunner H, Ziegler M 1965 Renin-angiotensin system, aldosterone, and sodium balance. Recent Prog Horm Res 21:119167 20. Strieker EM, Vagnucci AH, McDonald Jr RH, Leenen FH 1979 Renin and aldosterone secretions during hypovolemia in rats: relation to NaCl intake. Am J Physiol 237:R45-R51 21. Manning M, Sawyer WH 1983 Design of potent and selective in vivo antagonists of the neurohypophyseal peptides. In: Cross BA, Leng G (eds) The Neurohypophysis: Structure, Function and Control. Elsevier, Amsterdam, pp 367-382 22. Forsling ML, Brimble MJ 1985 The role of oxytocin in salt and water balance. In: Amico JA, Robinson AG (eds) Oxytocin: Clinical and Laboratory Studies. Elsevier, Amsterdam, pp 167-175 23. Forsling ML, Brimble MJ, Balment RJ 1982 The influence of vasopressin on oxytocin-induced changes in urine flow in the male rat. Acta Endocrinol (Copenh) 100:216-220 24. Balment RJ, Brimble MJ, Forsling ML, Kelly LP, Musabayane CT1986 A synergistic effect of oxytocin and vasopressin on sodium excretion in the neurohypophysectomized rat. J Physiol 381:453464 25. Szczepanska-Sadowska E, Sobocinska J, Sadowski B 1982 Central dipsogenic effect of vasopressin. Am J Physiol 243:R372-R379 26. Pittman QJ, Veale WL, Lederis K 1982 Central neurohypophyseal peptide pathways: interactions with endocrine and other autonomic functions. Peptides 3:515-520 27. Demotes-Mainard J, Chauveau J, Rodriguez F, Vincent JD, Poulain DA 1986 Septal release of vasopressin in response to osmotic, hypovolemic and electrical stimulation. Brain Res 381:314-321
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Vol. 128, No. 3 Printed in U.S.A.
Oxytocin Produces Natriuresis in Rats at Physiological Plasma Concentrations* JOSEPH G. VERBALIS, MICHAEL P. MANGIONE, AND EDWARD M. STRICKER Departments of Medicine and Behavioral Neuroscience, University of Pittsburgh, Pittsburgh, Pennsylvania 15261
ABSTRACT. Oxytocin (OT) is known to stimulate natriuresis in rats when administered in large doses that, produce high plasma levels. We examined the effects of physiological plasma OT levels on renal sodium excretion by infusing graded doses of OT sc in conscious adult male rats maintained on a sodiumdeficient diet. Our results demonstrate that OT causes a doserelated increase in urinary sodium excretion during the initial day of infusion. The lowest plasma OT levels associated with increases in urinary sodium excretion (5-6 pmol/liter) were well within the range of physiological OT secretion in rats. However, this natriuretic effect was not sustained during subsequent days of maintenance on a sodium-deficient diet, suggesting that the OT-induced natriuresis was limited in part by receptor desensi-
T
tization and/or a decreased exchangeable sodium pool in combination with secretion of opposing antinatriuretic factors such as aldosterone. Pretreatment with an OT receptor antagonist completely blocked the natriuresis produced by a 20 pmol/h infusion of OT, but urinary sodium excretion was not affected by a vasopressin V\ antagonist and was blocked only partially by a combined vasopressin Vx and V2 antagonist. Together with previous studies in rats demonstrating an inverse relation between pituitary OT secretion and sodium appetite, these results support the hypothesis that peripherally and centrally secreted OT act in concert in rats to produce a negative sodium balance by stimulating sodium excretion while inhibiting sodium ingestion. (Endocrinology 128: 1317-1322,1991)
within the range of physiologically stimulated OT secretion in rats (1, 13). The studies reported here were undertaken to assess the effects of exogenous OT on urinary sodium excretion in conscious rats using sc infusions at rates producing physiological plasma OT levels. OT was infused during adaptation to a sodium-deficient diet, a protocol that optimized our ability to detect changes in urinary sodium excretion by avoiding the large obligate natriuresis of rats maintained on standard sodium-rich laboratory chows (14).
HE NEUROHYPOPHYSEAL hormone oxytocin (OT) has a well recognized role to stimulate milk ejection in lactating females. In contrast, there is not yet any proven physiological function for circulating OT in males or nonlactating females. However, evidence in rats has suggested a possible involvement of this peptide in the regulation of sodium balance; OT is secreted along with vasopressin (AVP) in response to stimuli such as hyperosmolality and hypovolemia (1, 2), OT receptors are present in kidney tubular cells (3-5), administration of large doses of OT has long been known to cause increased renal sodium and chloride excretion (6-9), and pituitary OT secretion is correlated with inhibition of sodium appetite in rats (10-12). Although in early studies the doses of OT used to stimulate natriuresis were large, more recently OT has been shown to produce small increases in urinary sodium excretion for short periods in anesthetized rats at iv infusion rates that elevated plasma OT levels to 16 ± 2 /lU/ml (32 ± 4 pmol/liter),
Materials and Methods Animal protocols
Received August 28,1990. Address all correspondence and requests for reprints to: Dr. Joseph G. Verbalis, 930 Scaife Hall, University of Pittsburgh, Pittsburgh, Pennsylvania 15261. * This work was supported in part by NIH Grants DK-38094 and MH-25140 (MERIT Award). Parts of this study were presented in preliminary form at the Society for Neuroscience Meetings, Toronto, Ontario, Canada, 1988, and the American Society for Clinical Investigation, Washington, DC, 1989.
All studies used male Sprague-Dawley rats (Zivic-Miller, Allison Park, PA), weighing 250-300 g at the time of study. Rats were housed in individual plastic metabolic cages (Nalgene model 650-0100, Rochester, NY) in a temperature-controlled room (22 C), with lights on from 0700-1900 h. All animals were initially maintained on standard laboratory rat chow (Wayne Laboratories, Chicago, IL; Na+ content, 170.0 ± 3.4 mmol/kg) and tap water ad libitum. Three days before study, jugular venous catheters were inserted for blood sampling (15). On the morning of the first day of study (day 1), the food was switched to a sodium-deficient rat chow (Teklad 170840, Madison, WI; Na+ content, 3.9 ± 0.2 mmol/kg). On the morning of the next day (day 2), osmotic minipumps (Alzet model 2001, Alza, Palo Alto, CA) containing
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OXYTOCIN-INDUCED NATRIURESIS IN RATS
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140 mM NaCl or various concentrations of OT (2, 10, 20, or 100 nmol/ml) were implanted sc using inhalational anesthesia with methoxyflurane. The osmotic minipumps were left in place for the remainder of the study. Daily 24-h urine and plasma samples were collected at the end of each of the 4 test days. For some experiments, osmotic minipumps containing OT at a single concentration (20 nmol/ml) were inserted at the beginning of day 2, 3, or 4 in separate groups of rats. The pumps were left in place for the duration of the study, and daily 24-h urine and blood samples were collected as before. In additional experiments osmotic minipumps containing selected OT or AVP antagonists were implanted sc at the start of day 1, 24 h before insertion of a second osmotic minipump containing OT at a concentration of 20 nmol/ml. Peptide infusions Synthetic OT (Sigma, St. Louis, MO) was diluted in 150 mM NaCl to yield concentrations of 2, 10, 20, and 100 nmol/ml, which when loaded into the osmotic minipumps resulted in nominal OT infusion rates of 2, 10, 20, and 100 pmol/h, respectively. Subsequent RIA of the synthetic OT solution revealed actual infusion rates of 1.6, 7.8, 15.6, and 78.6 pmol/ h. Control animals received pumps containing only 150 mM NaCl. For receptor antagonist studies, OT or AVP antagonists were dissolved in 150 mM NaCl at concentrations that yielded an infusion rate of 1 nmol/h when released from the osmotic minipumps. The receptor antagonists used were: [Pen\Phe(Me)2,Thr4,Orn8]OT [OT antagonist (16); courtesy Drs. D. Smith and V. Hruby, University of Arizona, Tucson, AZ], [d(CH2)£1,Tyr(OMe)2,Arg8]AVP [AVP Vx antagonist (17); Bachem, Torrance, CA], and [d(CH2)6\DPhe2,Val4,Arg8,Des-Gly9]AVP [combined AVP Vx and V2 antagonist (18); Bachem]. Plasma and urine analysis Urine samples were analyzed for Na+ and K+ concentrations (Beckman Electrolyte II Analyzer, Brea, CA) and osmolality (Advanced Instruments 3C2 Osmometer, Needham Heights, MA). Total 24-h urinary Na+ excretion was calculated as the product of the urine volume (in milliliters) and the urinary Na+ concentration (in micromoles per ml). Blood samples (1.5 ml) were withdrawn through the jugular venous catheters and placed into iced heparinized glass tubes. The amount withdrawn was replaced immediately with an equal volume of 140 mM NaCl. After centrifugation (3000 x g at 4 C), the plasma Na+ concentration was measured (Beckman Electrolyte II Analyzer), and the remaining plasma was frozen to -20 C. Plasma OT levels were measured by RIA via methods described previously, using Posterior Pituitary Reference Standard (U.S.P, Rockville, MD) for the standard curve (15). For this report, the measured plasma OT levels were converted to molar concentrations using the relation 1 U = 2 nmol. Statistical analysis All results are reported as the mean ± SE. Statistical significance was determined by one-way analysis of variance for comparison of multiple groups, with post-hoc comparisons by
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the method of Newman-Keuls where appropriate. Correlation coefficients were calculated by the method of Pearson. Regression analysis was performed by the method of least squares (Tablecurve curve fitting software, Jandel Scientific, Corte Madera, CA).
Results Urinary sodium excretion ranged from 800-1400 /imol/ 24 h on the first day after initiation of the sodiumdeficient diet (day 1) in all rats. By day 2 urinary sodium excretion fell to 195 ± 2 1 /imol/24 h in the control rats (Fig. 1, • ) and then remained between 100-200 jumol/24 h for each day thereafter. The rats receiving sc infusions of OT demonstrated a dose-related increase in urinary sodium excretion on the first day of OT infusion (day 2; nominal rates of OT infusion in picomoles per h are shown below each bar in Fig. 1). However, on subsequent days of the continuous OT infusion (days 3 and 4) the urinary sodium excretion returned to levels equivalent to those in the control rats. Basal plasma OT levels (day 1) were similar in all animals and did not change significantly over the 4 days during which the control group was maintained on the sodium-deficient diet (Table 1, first row of each infusion rate). As expected, the sc OT infusions produced doserelated increases in plasma OT levels (r = 0.89; P < 0.001). The mean plasma OT levels for the OT-infused groups were statistically greater than the mean OT levels in the control rats at all nominal infusion rates of 10 pmol/h or greater (Table 1), the same doses associated with significantly stimulated natriuresis (Fig. 1, day 2). Plasma OT levels remained significantly elevated throughout the 3-day infusion period (Table 1), although the increased urinary sodium excretion did not persist on subsequent days of the continuous OT infusion (Fig. 1, days 3 and 4). Regression analysis of the relation between the measured plasma OT levels and urinary sodium excretion on the first day of OT infusion (day 2) in individual rats revealed that a logarithmic function best fit these parameters (urinary Na+, -152.13 + 746.25 X log10 [plasma OT]; r = 0.75; P < 0.001; n = 71; Fig. 2). In contrast, no significant relation was found between plasma OT levels and any of the other parameters followed throughout the infusion protocol [plasma Na+ concentration (Table 1, second row of each infusion rate), urinary K+ excretion, urine osmolality, urine volume, water intake, and body weight changes (not shown)]. Further inspection of Fig. 2 shows that increased urinary sodium excretion above basal excretion rates (195 ± 21 jumol/24 h) first became apparent in some of the OT-infused rats at plasma OT levels as low as 5-6 pmol/liter. When separate groups of rats were infused with OT beginning on different days after introduction of the
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(12) (12)
(»)
1400-
FlG. 1. Urinary sodium excretion during the first day of the sodium-deficient (NaD) diet (day 1) and on each day of OT infusion (days 2-4). Each bar shows the results for groups of rats infused with OT at different rates (the numbers beneath the bars on days 2-4 indicate the nominal OT infusion rate for each group in picomoles per h). Shown are the mean ± SE. The numbers above the bars on day 1 indicate the number of rats studied at each infusion rate. *, P < 0.05; • * , P < 0.01 (compared to the 150 mM NaCl infusion group). 0
2 10 20 100
0
DAY 2
DAY 1
2 10 20 100
0
2 10 20 100
DAY 3
DAY 4
I NaD DIET I |OT INFUSION I
TABLE 1. Plasma OT levels and Na+ concentrations on each day of study OT infusion rate (pmol/h)
OT infusion Day 1 4.0 ± 0.6 142 ± 1 (20) 4.0 ± 0.8
Day 2
Day 3
5.0 ± 0.8 143 ± 1 (15) 6.2 ± 0.8
5.6 ± 0.8 143 ± 1 (14) 6.2 ± 1.0
Day 4
Mean for days 2-4
5.0 ± 0.8 4.8 ± 0.4 142 ± 1 141 ± 1 (20) (19) 6.0 ± 0.6 5.4 ± 1.0 144 ± 1 144 ± 1 143 ± 1 142 ± 1 144 ± 1 (9) (8) (11) (11) (9) 10 3.8 ± 1.0 9.0 ± 1.6° 12.4 ± 1.8° 10.2 ± 1.8" 15.2 ± 3.6° 142 ± 1 141 ± 1 142 ± 1 143 ± 1 142 ± 1 (11) (5) (11) (11) (5) 13.0 ± 0.8" 20 4.0 ± 0.4 10.4 ± 1.4" 12.2 ± 1.8° 15.0 ± 2.0° 143 ± 1 143 ± 1 142 ± 1 143 ± 1 143 ± 1 (20) (22) (15) (20) (18) 72.2 ± 8.2" 2.8 ± 0.8 100 66.8 ± 12.8° 64.2 ± 9.2" 72.6 ± 7.0" 141 ± 1 141 ± 1 144 ± 1 142 ± 1 141 ± 1 (12) (7) (12) (9) (8) For each OT infusion rate, the top row shows the measured plasma OT concentration (in picomoles per liter), and the second row shows the simultaneously measured plasma Na+ concentration (in millimoles per liter); the number of animals in each group is shown in parentheses in the bottom row. "P < 0.05 relative to controls (0 infusion rate). 0
sodium-deficient diet, each group demonstrated a significant increase in urinary sodium excretion for the initial 24-h period after the start of the OT infusion (Fig. 3). However, the magnitude of the induced natriuresis was significantly less when the OT infusion was started on day 4 of access to the sodium-deficient diet rather than earlier (P < 0.05 compared to values obtained when the OT infusion was begun on days 2 and 3). Although accurate metabolic sodium balances cannot be calculated because neither food intakes nor fecal sodium losses were measured during these studies., ap-
proximate sodium balances can be estimated with reasonable certainty, since these amounts are relatively small in rats on the sodium-deficient diet. Rats of similar body weight in our laboratory ingest 20 g/24 h sodiumdeficient diet, representing a sodium intake of approximately 80 jumol/24 h. Daily 1.5-ml blood samples were withdrawn, representing a net body sodium loss of approximately 125 /umol/24 h (assuming a mean plasma Na+ concentration of 142 mmol/liter and a hematocrit of 40-42%). However, because the blood withdrawn was replaced with 1.5 ml 140 mM NaCl (210 ixmol sodium),
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1600 -
1400 -
1200 -
a
1000 -
800 -
600 -
400 -
200 -
100
10 Plasma Oxytocin (pmol/l)
FIG. 2. Relation between 24-h urinary sodium excretion and plasma OT levels on the first day of OT infusion. The solid line depicts the least squares regression equation that best fit the data (urinary Na+, -152.13 + 746.25 X log10 [plasma OT]; r = 0.75; P < 0.001; n = 71), and the dashed lines indicate the 99% confidence intervals of the calculated regression line. 1400-
(8) (8)
(8)
DAY 1
DAY 2
DAY 3
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fore, amounted to approximately 165 jumol/24 h, which is quite close to the measured urinary sodium excretion of 100-200 jtmol/24 h in rats on the sodium-deficient diet (Fig. 1). Application of these estimates to the results shown in Fig. 3 indicates that the groups of rats receiving the OT infusions on different days all had negative sodium balances of approximately 1 mmol before the OT infusions. The groups infused with OT on days 2 and 3 then developed an additional negative balance of approximately 0.5 mmol before becoming refractory to further stimulated natriuresis, while the rats infused with OT for the first time on day 4 of the sodium-deficient diet developed an additional negative sodium balance of only 0.2 mmol during the 24-h period of OT infusion. To identify the renal receptor type(s) responsible for the OT-induced natriuresis, groups of animals were pretreated with OT and AVP receptor antagonists for 24 h before initiation of an OT infusion of 20 pmol/h on day 2. All antagonists were infused continuously at a rate of 1 nmol/h beginning 24 h before initiation of the OT infusion, representing a 50-fold excess of antagonist to the infused OT. The OT receptor antagonist completely abolished all significant OT-induced sodium excretion (Fig. 4) without affecting urine osmolality (1768 ± 106 mosmol/kg H2O after infusion of the antagonist vs. 1554 ± 158 mosmol/kg H2O in control rats; P = NS). Although the AVP Vi antagonist had no effect on OT-stimulated natriuresis, a partial inhibition was seen in response to the combined AVP Vi and V2 antagonist, but not to the same degree as that seen with the OT receptor antagonist (Fig. 4).
DAY 4
FIG. 3. Urinary sodium excretion after institution of a sodium-deficient diet on day 1 and infusion of OT (20 pmol/h) beginning on day 2, 3, or 4 (as indicated by the arrows below the figure). Shown are the mean ± SE. The numbers above the bars on day 1 indicate the number of rats studied in each group. *, P < 0.05; • * , P < 0.01 (compared to 150 mM NaCl infusion group from Fig. 1).
the blood-sampling protocol actually resulted in a net sodium gain of approximately 85 /imol/24 h. The overall increase in body sodium resulting from the combined dietary sodium intake and the infusion protocol, there-
-OT
+OT (20 pmol/h)
FIG. 4. Urinary sodium excretion on the second day of the sodiumdeficient diet in rats pretreated for 24 h with OT or AVP receptor antagonists before infusion with OT (20 pmol/h). D, Control rats not receiving an OT infusion; • , rats receiving the OT infusion alone; 1, rats pretreated with the indicated antagonists before the OT infusion. The number of rats in each group is indicated above the bars. *, P < 0.05; **, P < 0.01 (compared to rats receiving the OT infusion without antagonist pretreatment).
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OXYTOCIN-INDUCED NATRIURESIS IN RATS
Discussion The studies reported here show that continuous sc infusions of OT cause relatively large dose-related increases in urinary sodium excretion in conscious rats maintained on a sodium-deficient diet. The increased natriuresis occurred at plasma OT levels well within the range of physiologically stimulated OT secretion. Although previous studies have reported natriuresis in response to administration of OT, for the most part those studies used doses that elevated plasma OT levels significantly in excess of concentrations generally achieved under physiological conditions. Our detection of natriuresis at lower plasma OT levels probably reflects an enhanced ability to detect modest increases in natriuresis at the markedly reduced levels of urinary sodium excretion in rats maintained on a sodium-deficient diet. The initial natriuretic effect of exogenous OT was relatively short-lived, however, and urinary sodium excretion returned to control levels by the following day despite maintenance of plasma OT levels within the same ranges as were achieved on day 2. Such blunting of the OT-induced natriuresis with prolonged infusions might be due to several potential causes. One factor could be desensitization of the receptor-mediated mechanism(s) by which OT stimulates urinary sodium excretion. This possibility is supported by the results shown in Fig. 3, because in these studies OT produced equivalent increases in urinary sodium excretion when infusions began on day 3 of sodium deficiency compared to day 2. However, these results could also be explained simply as a result of cumulative losses of body sodium in urine. Thus, rats given OT beginning on day 2 may have excreted little additional sodium on day 3 because they had accumulated a negative sodium balance of approximately 1.5 mmol during the 2 previous days, whereas rats given OT for the first time beginning on day 3 had a negative sodium balance of only approximately 1 mmol up to this point and, therefore, probably had a residual pool of exchangeable sodium sufficient to allow further stimulated natriuresis. However, this explanation cannot account for the blunted natriuresis seen when rats were given OT beginning on day 4, by which time they also had accumulated a negative sodium balance of only approximately 1 mmol. The latter results, therefore, suggest that after a more prolonged duration of sodium deficiency and/or increasing urinary sodium losses, the effects of OT to induce natriuresis are also antagonized by physiological mechanisms. The most likely candidate for such an antinatriuretic effect would be the adrenal steroid hormone aldosterone, which is known to be a mediator of sodium retention in response to sodium deficiency (19) and hypovolemia (20) in rats. Thus, although the use of a sodium-deficient diet in these experiments lowered
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basal urinary sodium excretion and thereby facilitated observation of a natriuretic effect of OT, it also probably limited the magnitude and duration of that effect, both because there was less exchangeable sodium to excrete, and because it stimulated elevated plasma levels of aldosterone. The OT-stimulated natriuresis appears to be mediated by specific renal OT receptors, because pretreatment with an OT receptor antagonist completely eliminated the natriuresis produced by infusion of OT at a rate of 20 pmol/h. It seems unlikely that the infused OT had significant effects on kidney AVP receptors, because urine osmolalities were not affected by infusion of the OT antagonist. Moreover, a potent AVP V\ antagonist had no effect on OT-stimulated natriuresis, and a combined Vi and V2 antagonist had only partial effects. These results were somewhat surprising in light of the fact that many AVP Vi receptor antagonists are also generally effective at blocking uterine OT receptors as well (21) and suggest the possibility that the renal OT receptors recently described in the kidney (3-5) may in some ways be biochemically and functionally different from OT receptors elsewhere. Further studies will be required to evaluate this possibility. Our findings demonstrate a robust urinary sodium excretion in response to physiologically relevant plasma OT levels, in agreement with numerous previous studies suggesting that OT might act as a natriuretic agent under certain physiological circumstances (22). One situation in which OT-induced natriuresis may be physiologically significant is during hyperosmolar volume expansion. Hyperosmolality is known to be a potent stimulus to pituitary OT as well as AVP secretion in rats (1, 2). Although it has long been known that hyperosmolalityinduced AVP secretion is responsible for the antidiuresis and water retention that contribute to osmoregulation under these circumstances, it has never been apparent why OT secretion should also be stimulated in rats during hyperosmolar conditions. The present studies demonstrating induced natriuresis of a significant magnitude at physiological plasma OT levels in conscious rats indicate a possible role for the secreted OT that would complement the water retention produced by the secreted AVP. In addition, some studies have suggested a synergistic effect of AVP and OT on natriuresis in rats (23, 24), and if present, this interaction might potentiate the natriuretic effects of OT during hyperosmolar conditions when both peptides are secreted. Furthermore, under conditions of a hyperosmolar volume expansion, OTinduced natriuresis would not be limited by a depleted exchangeable sodium pool or stimulated aldosterone secretion, as occurs during sodium deficiency. Recent data have also provided evidence that central secretion of both AVP and OT may produce behavioral
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OXYTOCIN-INDUCED NATRIURESIS IN RATS
effects that complement their physiological actions. Studies in dogs have suggested that centrally released AVP potentiates water ingestion during osmotic dehydration (25), which would complement the antidiuresis produced by pituitary AVP secretion. Similarly, in rats there is an inverse correlation between pituitary OT secretion and sodium appetite (10-12), and inhibition of NaCl consumption would complement an OT-mediated natriuresis to promote a negative sodium balance in hyperosmolar animals. In this regard, it is interesting to note that in several different models of sodium appetite in rats, induced hyperosmolality interrupts further NaCl ingestion in proportion to the magnitude of the stimulated OT secretion (11). Because recent studies have documented simultaneous central and peripheral secretion of AVP during induced hyperosmolality (26, 27), it seems likely that a similar coordination of central and peripheral secretion of OT could be the basis for mediating complementary physiological and behavioral effects on sodium balance in rats. In summary, our studies in conjunction with previous reports suggest that circulating OT stimulates urinary sodium excretion at physiological plasma levels in rats, and this effect appears to be mediated by specific OT receptors in the kidneys. Although the induced natriuresis is pronounced, it is limited and, therefore, is likely to be physiologically significant only under certain conditions, such as hyperosmolar volume expansion. The full physiological significance of these effects and the mechanisms that limit its duration and magnitude remain to be evaluated by further studies.
Acknowledgments The authors wish to thank Marcia Drutarosky for technical assistance, and Michele Dobransky and Marge Altvater for secretarial assistance.
References 1. Balment RJ, Brimble MJ, Forsling ML 1980 Release of oxytocin induced by salt loading and its influence on renal excretion in the male rat. J Physiol (Lond) 308:439-449 2. Strieker EM, Verbalis JG 1986 Interaction of osmotic and volume stimuli in the regulation of neurohypophyseal secretion in rats. Am J Physiol 250:R267-R275 3. Stoeckel ME, Freund-Mercier MJ, Palacios JM, Richard PH, Porte A 1987 Autoradiographic localization of binding sites for oxytocin and vasopressin in the rat kidney. J Endocrinol 113:179-182 4. Tribolle E, Barberis C, Dreifuss JJ, Jard S 1988 Autoradiographic localization of vasopressin and oxytocin binding sites in rat kidney. Kidney Int 33:959-965 5. Cantau B, Barjon JN, Chicot D, Baskevitch P, Jard S 1990 Oxytocin receptors from LLC-PKi cells: expression in Xenopus oocytes. Am J Physiol 258:F963-F972
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6. Fraser AM 1942 The action of the oxytocic hormone of the pituitary gland on urine secretion. J Physiol 101:236-251 7. Sawyer WH 1952 Posterior pituitary extracts and excretion of electrolytes by the rat. Am J Physiol 16:583-587 8. Jacobson HN, Kellogg RH 1956 Isotonic NaCl diuresis in rats, antidiuresis and chloruresis produced by posterior pituitary extracts. Am J Physiol 184:376-389 9. Chan WY 1965 Effects of neurohypophyseal hormones and their deamino analogues on renal excretion of Na, K and water in rats. Endocrinology 77:1097-1104 10. Strieker EM, Hosutt JA, Verbalis JG 1987 Neurohypophyseal secretion in hypovolemic rats: inverse relation to sodium appetite. Am J Physiol 252:R889-R896 11. Strieker EM, Verbalis JG 1987 Central inhibitory control of sodium appetite in rats: correlation with pituitary oxytocin secretion. Behav Neurosci 101:560-567 12. Strieker EM, Verbalis JG 1988 Hormones and behavior: biological basis of thirst and sodium appetite. Am Scientist 76:261-267 13. Balment RJ, Brimble MJ, Forsling ML 1982 Oxytocin release and renal actions in normal and Brattleboro rats. Ann NY Acad Sci 394:241-253 14. Strieker EM 1981 Thirst and sodium appetite after colloid treatment in rats. J Comp Physiol Psychol 95:1-25 15. Verbalis JG, McHale CM, Gardiner TW, Strieker EM 1986 Oxytocin and vasopressin secretion in response to stimuli producing learned taste aversions in rats. Behav Neurosci 100:466-475 16. Chan WY, Hruby VJ, Rockway TW, Hlavacek J 1986 Design of oxytocin antagonists with prolonged action: potential tocolytic agents for the treatment of preterm labor. J Pharmacol Exp Ther 239:84-87 17. Kruszynski M, Lammeck B, Manning M, Seto J, Haldar J, Sawyer WH 1980 [l-(|8-mercapto-j8,j8-cyclopentamethylenepropionic acid)2-(O-methyl)tyrosine]-arginine vasopressin and [l-(/3-mercapto-/?,/3-cyclopentamethylenepropionic acid) ] -arginine vasopressin, two highly potent antagonists of the vasopressor response to arginine vasopressin. J Med Chem 23:364-368 18. Jard S, Gaillard RC, Guillon G, Marie J, Schoenenberg P, Muller AF, Manning M, Sawyer WH 1986 Vasopressin antagonists allow demonstration of a novel type of vasopressin receptor in the rat adenohypophysis. Mol Pharmacol 30:171-177 19. Gross F, Brunner H, Ziegler M 1965 Renin-angiotensin system, aldosterone, and sodium balance. Recent Prog Horm Res 21:119167 20. Strieker EM, Vagnucci AH, McDonald Jr RH, Leenen FH 1979 Renin and aldosterone secretions during hypovolemia in rats: relation to NaCl intake. Am J Physiol 237:R45-R51 21. Manning M, Sawyer WH 1983 Design of potent and selective in vivo antagonists of the neurohypophyseal peptides. In: Cross BA, Leng G (eds) The Neurohypophysis: Structure, Function and Control. Elsevier, Amsterdam, pp 367-382 22. Forsling ML, Brimble MJ 1985 The role of oxytocin in salt and water balance. In: Amico JA, Robinson AG (eds) Oxytocin: Clinical and Laboratory Studies. Elsevier, Amsterdam, pp 167-175 23. Forsling ML, Brimble MJ, Balment RJ 1982 The influence of vasopressin on oxytocin-induced changes in urine flow in the male rat. Acta Endocrinol (Copenh) 100:216-220 24. Balment RJ, Brimble MJ, Forsling ML, Kelly LP, Musabayane CT1986 A synergistic effect of oxytocin and vasopressin on sodium excretion in the neurohypophysectomized rat. J Physiol 381:453464 25. Szczepanska-Sadowska E, Sobocinska J, Sadowski B 1982 Central dipsogenic effect of vasopressin. Am J Physiol 243:R372-R379 26. Pittman QJ, Veale WL, Lederis K 1982 Central neurohypophyseal peptide pathways: interactions with endocrine and other autonomic functions. Peptides 3:515-520 27. Demotes-Mainard J, Chauveau J, Rodriguez F, Vincent JD, Poulain DA 1986 Septal release of vasopressin in response to osmotic, hypovolemic and electrical stimulation. Brain Res 381:314-321
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