Oxytocin Releases Atrial Natriuretic Peptide from Rat Atria In Vitro that Exerts Negative Inotropic and Chronotropic Action

Peptides, Vol. 18, No. 9, pp. 1377–1381, 1997 Copyright © 1997 Elsevier Science Inc. Printed in the USA. All rights reserved 0196-9781/97 $17.00 1 .00

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Oxytocin Releases Atrial Natriuretic Peptide from Rat Atria In Vitro that Exerts Negative Inotropic and Chronotropic Action ´ JO,‡ J. GUTKOWSKA,‡ A. L. V. FAVARETTO,* G. O. BALLEJO,† W. I. C. ALBUQUERQUE-ARAU J. ANTUNES-RODRIGUES AND S. M. MCCANN§1 Departments of *Physiology and †Pharmacology, School of Medicine of Ribeira˜o Preto, 14049-900 Ribeira˜o Preto-SP, Brazil ‡Centre de Recherche Hotel-Dieu de Montreal, 3850 St. Urbain Street, Marie-de-la-Ferre Pavillion, Montreal, Quebec H2W1T8, Canada §Pennington Biomedical Research Center, 6400 Perkins Road, Baton Rouge, LA 70818-4124 Received 3 March 1997; Accepted 1 July 1997 ´ JO, J. GUTKOWSKA, J. ANTUNES–RODRIGUES FAVARETTO, A. L. V., G. O. BALLEJO, W. I. C. ALBUQUERQUE–ARAU AND S. M. McCANN. Oxytocin releases atrial natriuretic peptide from rat atria in vitro that exerts negative inotropic and chronotropic action. PEPTIDES 18(9) 1377–1381, 1997.—Our previous experiments suggested that natriuresis induced by blood volume expansion, was brought about by oxytocin (OT)-stimulated atrial natriuretic peptide (ANP) release from the right atrium. We hypothesized that the ANP released might exert effects on the atrium itself and therefore carried out in vitro experiments to test this hypothesis. Heart rate and isometric tension were recorded from isolated rat atria mounted in an organ bath. Oxytocin exerted a dose-related, negative chrono- and inotropic effect with a minimal effective concentration (MEC) of 3 mM, 10-fold higher than required for ANP to exert comparable effects. The effects of OT were not blocked by atropine suggesting that they were not mediated via release of acetylcholine. Eight-bromoguanosine 39-59-cyclic monophosphate (cGMP) had similar effects to those of OT and ANP, suggesting that the effects of ANP were mediated by cGMP. When isolated ventricles, left or right atria, were incubated in vitro, OT had a dose-related effect to stimulate the release of ANP into the medium only from right atria with a MEC of 0.1 mM. A specific OT antagonist, F792 (1 mM), inhibited basal release of ANP and blocked the stimulatory action of OT on ANP release. The results support the hypothesis that OT, acting on its putative receptors in the right atrium, stimulates the release of ANP which then exerts a negative chrono- and inotropic effect via activation of guanylyl cyclase and release of cGMP. The ability of the oxytocin antagonist to reduce basal release of ANP from atria incubated in vitro supports the hypothesis that these effects could be physiologically significant. We hypothesize that blood volume expansion via baroreceptor input to the brain causes the release of OT which circulates to the heart and stimulates the release of ANP from the right atrium. This ANP then has a negative ino- and chronotropic effect in the atrium and possibly a negative inotropic effect in the right ventricle, left atrium and left ventricle, to produce an acute reduction in cardiac output that, coupled with its peripheral vasodilating actions, causes a rapid reduction in effective circulating blood volume. The ANP released would also act on the kidneys to cause natriuresis and ANP acts within the brain to inhibit water and salt intake leading to a gradual recovery of circulating blood volume to normal. © 1997 Elsevier Science Inc. Left and right atria

Ventricles

8 bromo cGMP

OT antagonist (F792)

INTRODUCTION

OXYTOCIN (OT) and vasopressin (VP) are the two major polypeptides secreted by the neurohypophysis. They are the two peptides formed in greatest abundance in the body and are synthesized in magnocellular neurons in the supraoptic and paraventricular nuclei (1,2). They are both transported along axons to the neural lobe where they are each stored in large amounts. Although, oxytocin has been mainly associated with milk ejection and uterine contractions in female mammals, the fact that it is found in 1

To whom requests for reprints should be addressed.

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Atropine

equivalent concentrations in the neurohypophysis in both sexes suggests that it also may have other physiological roles (1,2). In the CNS, OT-containing axons terminating in several brain stem nuclei involved in cardiovascular control have been described suggesting a potential role for OT in central cardiovascular regulation. Peripherally injected, OT has been shown to cause a fall in mean arterial pressure by unknown mechanisms (3). Furthermore, OT possesses potent natriuretic activity which is associated with increased atrial natriuretic peptide (ANP) release (4). Median

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eminence lesions, which block OT release, abolished the natriuretic effect of microinjection of hypertonic saline solutions into the third cerebral ventricle of conscious rats and of blood volume expansion induced by intraatrial injections of isotonic saline and the associated increases in plasma ANP concentrations (5). Furthermore, isotonic blood volume expansion was accompanied by concomitant increases in plasma OT and ANP, at a time when plasma VP was decreased. These results were interpreted to mean that blood volume expansion acting by baroreceptor input to the hypothalamus activated OT release that stimulated release of ANP from the right atrium. The ANP then circulated to the kidney and evoked natriuresis (4). Indeed, intravenous or intraperitoneal injection of OT to conscious male rats induced an increase in plasma ANP concentrations, observations that support the hypothesis that OT-induced natriuresis may be mediated by ANP released from the right atrium by OT and that this may also be the mechanism of the natriuresis induced by blood volume expansion. Furthermore, an OT antagonist blocked the increase in plasma ANP concentrations which followed suckling of dams by their litters (4). Therefore, we hypothesized that suckling or blood volume epansion triggers the release of OT from the neurohypophysis which circulates to the heart and stimulates the release of ANP from the right atrium. ANP would then induce natriuresis (6). Since the reduction in effective circulating blood volume that follows volume expansion occurs more rapidly than can be accounted for by natriuresis, it has been accepted that the ANP released by volume expansion also dilates blood vessels via activation of particulate guanylate cyclase and liberation of cyclic guanosine 39-59-monophosphate (cGMP), thereby producing a rapid reduction in effective circulating blood volume. It occurred to us that there might be an intracardiac mechanism as well. According to this hypothesis, volume expansion would cause release of OT that releases ANP in the right atrium by action on putative OT receptors. The ANP would act on its receptors in the right atrium to activate particulate guanylate cyclase, leading to generation of cGMP, which would induce a negative ino- and chronotropic effect, thereby decreasing right atrial output. The circulation of high concentrations of ANP to the remainder of the heart might exert a negative inotropic effect, further decreasing cardiac output. The experiments reported here support this hypothesis. METHOD

Procedure Atria were isolated from male Wistar rats (250 –350 g) and mounted in an organ bath containing Locke nutritive solution continuously bubbled with 95% O2–5% CO2 kept at 37°C for the recording of isometric tension. The atria were submitted to an initial tension of 0.5 g; after a period of 30 – 45 min, cumulative concentration-response curves for OT and ANP were obtained in spontaneously beating atria. In another set of experiments isolated ventricles, left or right atria were incubated in vitro at 37°C in a shaking incubator in 24 well plates containing 1 ml minimum essential medium (MEM), 0.1% BSA, 15 mM HEPES, pH 7.4. One atrial quarter or ventricle tip was placed in each well and after a preincubation for 15 min, medium was replaced with fresh medium containing 20 mg of EDTA, 20 ml of pepstatin A (500 mM) and 20 ml of phenymethysulfonylfluoride (PMSF, 1 mM) and incubated for a further 30 min in the presence or absence of OT (1 mM). In another series of experiments, F792 (1 mM), a specific OT antagonist (Ferring, Malmo, Sweden) was incubated with right atria for 15 min prior to and during incubation with OT. Aliquots of 100 ml were removed each 15 min for determination of ANP concentration by specific

FIG. 1. Effects of OT on the frequency (beats/min (BPM)) (A) and force (g) (B) of spontaneous atrial contraction. Data are expressed as mean 6 SEM from 4 experiments. *P , 0.01, **P , 0.001 compared to basal.

radioimmunoassay without extraction (7,8). This assay measures the circulating form of ANP as determined by HPLC. Materials ANP 1-28 was obtained from Peninsula Laboratories, Torrance CA, USA (cat. n°9103). All other reagents were obtained from SIGMA Chemical Co, St. Louis, MO. The OT antagonist (F792) was kindly provided by Ferring Research Institute AB (Malmo, Sweden). Statistics Data are presented as means 6 SEM. After analysis of variance, statistical significance of the differences between 2 means was determined by the Kruskal-Wallis U test. P , 0.05 was considered significant. RESULTS

Effect of OT on the Frequency and Force of Spontaneous Atrial Contractions OT caused a concentration-dependent reduction in both rate and contractile force of spontaneously beating isolated atria

OXYTOCIN INHIBITS ATRIAL FUNCTION BY RELEASING ANP

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(Fig. 4). The MEC was 0.1 mM, ten times less than in the functional experiments (1 mM, see Fig. 1). Preincubation for 15 min with the OT antagonist (F792) (1 mM) reduced the basal release of ANP (ng/ml/atria) significantly [(57 6 5) (24)1 control; 26 6 2 (12) F792 (P , 0.001)]. The antagonist blocked the action of OT (1 mM) and maintained the release below that of control atria during the entire 45 min incubation (Fig. 5). Oxytocin (1 mM) did not modify ANP release (pg/ml) from left atrial and ventricular tissue in vitro after 30 min of incubation (for left atria 290.3 6 44.6 (control) vs 303.2 6 44.6 (OT); for ventricular tissue 0.37 6 0.02 (control) vs 0.36 6 0.02 (OT) ventricle). DISCUSSION

These results show, for the first time, that OT has a direct inhibitory effect on both rate and force of contraction of rat atria which is unlikely to be due to acetylcholine release from parasympathetic nerves since atropine failed to influence this effect at doses that abolish the effects of acetylcholine. These negative

FIG. 2. Negative chronotropic (A) and inotropic (B) effect of OT on spontaneous atrial contraction in the presence or absence of atropine (ATR 0.1 mM). Data are expressed as mean 6 SEM from 4 experiments. †P , 0.05 compared to control group; *P , 0.001 compared to basal.

with a minimal effective concentration (MEC) in the micromolar range (Fig. 1). The maximum negative chronotropic effect (> 40%) was less than the negative inotropic one (> 50%). These effects were not influenced significantly by atropine (0.1 mM) at a concentration that blocks the action of muscarinic agonists (Fig. 2). Effect of ANP on the Frequency and Force of Spontaneous Atrial Contractions ANP also produced a concentration-related decrease in both force and frequency of atrial contractions with a MEC in the submicromolar range (Fig. 3). ANP was nearly 10-fold more potent to depress atrial functions than OT. Effect of OT on the Release of ANP from Right Atria Incubated In Vitro ANP was released from right atrial fragments in a time-dependent manner with constant release over the first 45 min. ANP release was increased by OT in a concentration-dependent manner 1

mean 6 SEM (no. of atria).

FIG. 3. Effects of ANP on the frequency (BPM) (A) and force (g) of spontaneous atrial contraction. Data are expressed as mean 6 SEM from 4 experiments. *P , 0.05 compared to control.

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FIG. 4. Dose-response curve of OT on the release of ANP from right atria incubated in vitro. Data are expressed as mean 6 SEM from 8 replicates from one experiment. *P , 0.05, **P , 0.005 compared to control.

chronotropic and inotropic effects of OT were shared by ANP, but it was almost 10-fold more potent than OT. The effects of ANP are likely to be mediated by guanylate cyclase activation since we also found in preliminary studies that the membrane permeant analogue, 8-bromoguanosine 39-59-cyclic GMP, exhibited similar actions. We hypothesized that the inhibitory effects of OT on atrial function were mediated by ANP directly released from the atria by OT acting on its putative receptors there. Indeed, we found that a concentration of OT that produced only minimal effect on cardiac function, produced highly significant increases in ANP release from incubated right atria which were completely blocked by F792, a specific OT receptor antagonist. One could argue that these results are only of pharmacological interest since the concentration of OT required to inhibit atrial function and to release ANP in vitro was much higher than that observed in plasma following blood volume expansion in the rat (4); however, in preliminary experiments with guinea pig hearts using the Langendorf preparation in which drugs are injected directly into the ascending aorta to perfuse the coronaries, we have obtained negative chrono- and inotropic effects with 0.5 and 5.0 nMole doses of ANP and OT, respectively. Our data show that in vitro, when OT is directly applied to the atria, it promotes an inhibitory effect on both ino- and chronotropic activities. Furthermore, the OT antagonist significantly decreased the basal release of ANP from the incubated atria which argues forcefully that OT plays a physiologically significant role in controlling ANP release in vitro. Petty et al. (3) reported that intravenously injected OT causes biphasic dose-dependent changes in mean arterial pressure (MAP), consisting of an initial pressor effect accompanied by bradycardia

FAVARETTO ET AL. and a decrease in cardiac output, followed by a prolonged fall in mean arterial pressure which reaches a maximum after 30 min and is accompanied by an increase in cardiac output. We suggest that in vivo OT has the same negative- and chronotropic action on the heart as in vitro producing a rapid decline in cardiac output that activates baroreceptor reflexes leading to the pressor effect observed (3). We have determined (Favaretto, et al., unpublished, 1997) that IP injection of OT had no significant effect on MAP until the dose was increased to 10 mg, a dose 10 times larger than that required to increase plasma ANP concentrations (4). This supports the idea that with lower doses of oxytocin, which are effective to release ANP, there would not be a dramatic baroreceptor activation and the decrease in cardiac output might be sustained in vivo. The dose of oxytocin required in vitro is higher than the concentrations that circulate in the blood even after volume expansion. There are two possible explanations for this. One is that the right atrium in vivo may be much more sensitive to the ANP-releasing action of OT than in vitro, or alternatively OT may be present in the atria either delivered to it by extrinsic nerves, such as the vagus, or synthesized in intraatrial ganglia and released from the terminals of these neurons (9,10). Several other peptides such as somatostatin, neuropeptide Y, dynorphin B and substance P have been found in vagal efferents to the atria and even in intracardiac ganglionic cells (9). This question can be answered by immunocytochemistry and by measurement of the atrial content and release of OT. Indeed, preliminary data from our laboratories indicated the presence of immunoreactive OT in right atrial homogenates (21.6 6 1.9 pg/atrium). The physiological significance of the direct release of ANP by OT is further supported by the ability of an OT antagonist to block

FIG. 5. Effect of OT (1 mM) and OT-antagonist (F792) (1 mM) on the release of ANP from right atria incubated in vitro. Data are expressed as mean 6 SEM from 12 replicates of each group from two experiments. *P , 0.05, **P , 0.001 compared to control; or †P , 0.001 compared to OT-stimulated.

OXYTOCIN INHIBITS ATRIAL FUNCTION BY RELEASING ANP suckling-induced ANP release (4). We are currently testing the ability of the more potent antagonist used here to block blood volume expansion-induced ANP release. Thus, the experiments reported here and previously support the hypothesis that blood volume expansion by baroreceptor input to the hypothalamus causes release of OT which circulates to the right atrium. There, it acts on OT receptors to induce release of ANP. The ANP acts via its receptors to activate particulate guanylate cyclase. The released cGMP inhibits the sinoatrial node, thereby decreasing its rate of discharge and slowing pulse rate. At the same time, the liberated cGMP exerts a negative inotropic effect. Thus, right atrial output is reduced. The elevated concentration of ANP reaching the right ventricle and ultimately the left atrium and ventricle may have a negative inotropic effect on these chambers causing a further reduction in cardiac output which,

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coupled with the vasodilatory action of ANP, would rapidly decrease blood volume ANP by its natriuretic action on the kidney, coupled with the action of ANP within the brain to inhibit water and salt intake, results in a gradual return to normal blood volume. ACKNOWLEDGEMENTS

These studies were supported by Fundac¸a˜o de Amparo a Pesquisa do Estado de Sa˜o Paulo (FAPESP Grant 94/3805-7) and Conselho Nacional do Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq Grant 521593/94-8) to J. Antunes-Rodrigues and A. L. V. Favaretto; National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-43900 and National Institute of Mental Health Grant MH-51853 (to S. M. McCann) and grants from the Heart and Stroke Foundation of Canada and the Kidney Foundation of Canada, Medical Research Council of Canada Grant MT10337 and MT-11674 (to J. Gutkowska).

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