Pressor and bradykardie effects of centrally administered relaxin in conscious rats

A]H 1995; 8:375-381

Pressor and Bradycardic Effects of Centrally Administered Relaxin in Conscious Rats Ren-Hui Yang, Stuart Bunting, J. Michael Wyss, Kathleen H. Berecek, Lin Zhang, and Hongkui Jin

The current study tested the hypothesis that centrally administered relaxin elevates arterial pressure in conscious rats and that this hypertensive effect is mediated, at least in part, by central or peripheral vasopressin. Injection of human relaxin (0.068 or 0.34 ttg in 200 nL artificial cerebrospinal fluid) into the right lateral ventricle of conscious, unrestrained Sprague-Dawley rats caused significant dose-related increases in arterial pressure and decreases in heart rate. The pressor and bradycardic responses to intracerebroventricular injections of relaxin were significantly blunted by pretreatment with either intracerebroventricular or intravenous injection of a vasopressin receptor (VI) antagonist, suggesting that the cardiovascular effects of central relaxin are mediated, at least in part, by VI receptors in the brain and perhaps also by vasopressin released into the peripheral circula-

tion. Neither intracerebroventrlcular injection of the vehicle alone nor intravenous injection of relaxin (0.34 p,g) altered arterial pressure or heart rate. In contrast to the above, intravenous injections of relaxin (40 f1g/kg) elicited pressor and tachycardic responses that were not blunted by pretreatment with either intracerebroventricular or intravenous injection of the VI receptor antagonist. Together, these data suggest that in the central nervous system relaxin contributes to the regulation of cardiovascular function and that the mechanisms for the cardiovascular effects of central and peripheral relaxin are distinct. Am J Hypertens 1995;8: 375-381 KEY WORDS: Relaxin, blood pressure, heart rate, intracerebroventricular injection, rats, VI receptor antagonist.

elaxin is a heterodimeric member of the in- oping and mature rats, suggesting that relaxin may sulin family of polypeptide hormones that playa role in the central regulation of several normal is best known for its actions on the mam- functions in addition to pregnancy.' Further, recent malian reproductive system. Whereas re- autoradiographic studies have demonstrated that laxin mRNA is expressed in the pregnant ovary, in specific, high-affinity relaxin binding sites are present situ hybridization experiments demonstrate that re- in discrete brain regions of male and female rats, inlaxin mR..NA is also expressed in the brains of devel- cluding the circumventricular organs (subfornical organ and organum vasculosum of the lamina terminals, areas that are involved in the regulation of blood Received December 7, 1993. Accepted November I, 1994. pressure, heart rate, and fluid balance) and the neuFrom the Department of Cardiovascular Research, Genentech, rosecretory magnocellular hypothalamic nuclei (i.e., lnc., South San Francisco, California; and Departments of CellBiology OMW) and Physiology (KHB, LZ), University of Alabama at paraventricular and supraoptic nuclei, areas that regBirmingham, Birmingham, Alabama. ulate vasopressin and oxytocin release)." In vivo Address correspondence and reprint requests to Hongkui [in, studies in anesthetized, lactating rats demonstrate MD, Department of Cardiovascular Research, Mailstop 42, Genentech, Inc., 460 Point San Bruno Blvd., South San Francisco, CA that administration of relaxin into either the lateral 94080. ventricle or dorsal portion of the third ventricle

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© 1995 by the American Journal of Hypertension, Ltd.

0895-70611951$9.50 0895-7061(94)00208-5

376 YANG ET AL

causes significant increases in arterial prE:!ssure, heart rate (HR), and vasopressinreleaser' but none of these effects of centrally administered relaxin on arterial pressure or HR have been tested in conscious animals. The present study was designed to elucidate the effects of intracerebroventricular injection of relaxin on arterial pressure and HR in conscious, unrestrained rats. Male rats were used to avoid influences of fluctuating ovarian steroid concentrations on the endogenous relaxin system and to provide a uniform steroidal milieu. Our results demonstrate that intracerebroventricular injection of relaxin causes pressor and bradycardic responses while intravenous injection of relaxin increases both arterial pressure and HR in the conscious rat. To test whether t! .e effects of intracerebroventricular relaxin are mediated by vasopressin receptors (VI) in brain or the periphery, rats were pretreated with an intracerebroventricular or intravenous injection of a VI receptor antagonist and the subsequent pressor and bradycardic responses to an intracerebroventricular injection of relaxin were measured.

MATERIALS AND METHODS Sprague-Dawley (SD) rats were obtained from Charles River laboratories (Wilmington, MA) at 9 weeks of age. Allrats were maintained three per cage at constant humidity (60 ± 5%), temperature (24 ± I'C), and light cycle (6 AM to 6 PM). All rats were maintained on a standard rat diet (Ralston Purina 5001, Richmond, IN) and were given free access to food and water and were adapted to the diet and housing condition for 1 week prior to the initiation of the experimental protocols. Bodyweights were 332.5 ± 4.5 g. ' Twodays beforean acute experiment, each rat was anesthetized with sodium pentobarbital (60 mg/kg, intraperitoneally), and a catheter (polyethylene PE-lO fused with PE-SO) filled with a heparin-saline solution (50 U/mL) was implanted into the abdominal aorta through the right femoral artery. For the intracerebroventricular experiments rats were then placed into a stereotaxic apparatus, the skin overlying the middle of the skull was incised, and a smallhole was drilled through the appropriate portion of the skull. A guide cannula (26-gauge stainless steel needle) was lowered to a position 1.0 mm dorsal to the intended site of microinjection in the right lateral ventricle (0.8 mm anterior to bregma, 1.4mm lateral to midlineand 4.0 mm ventral to the surface of the brain), and fixed to the skull with dental acrylic. A 32-gauge obturator (stainless steel wire) was inserted into the guide cannula after implantation. Forty-eight hours after surgery, the arterialcatheter was connected to a model C1'-01 pressure transducer (Century Technology Company, Inglewood, CA)

AJH-APRIL 1995-VOL. 8, NO, 4, PART 1

coupled to a polygraph (Model 7; Grass Instruments, Quincy, MA). Mean arterial pressure (MAP) and HR were measured simultaneously. After a 40-min stabilization period, the obturator was removed from the guide cannula and replaced with an inner cannula (32-gauge stainless steel tubing) filled with the agent to be administered. The tip of the inner cannula extended 1.5 mm beyond the guide cannula. The inner cannula was attached to a O.S-J.LL Hamilton syringe through polyethylene tubing (PE-20) filled with saline. A smallair bubble was made between the saline and the injection solution. Rats were randomly injected with either 0.068 J.Lg or 0.34 J.Lg of human synthetic relaxin (Genentech, Inc., South San Francisco, CA) in 200 nl, of artificial cerebrospinal fluid (ACSF) or ACSF vehicle alone. The results of a pilot study demonstrated 1) that for intracerebroventricular injection of relaxin the dose of 0.34 /J-g was the lowest dose that caused maximal changes in MAP in conscious SO rats and 2) that this dose of relaxin did not alter MAP or HR when injected intravenously. Each rat received only a single injection on a given day. Only rats received ACSF vehicle injections were studied on a second day. All microinjection experiments were carried out in conscious freely moving rats. In parallel experiments, human relaxin (40 ug/kg) was intravenously injected in conscious SD rats, and its effects on MAP and HR were monitored. This dose of relaxin was shown in a pilot study to produce reliable and significant alterations in MAP and HR in conscious SO rats. For these experiments, the arterial catheter was implanted as indicated as above, and a second catheter was implanted into the right femoral vein for drug administration. A third series of experiments tested the hypothesis that the action of relaxin on arterial pressure and HRis mediated by the central or peripheral VJ receptor. Rats were pretreated with either an intracerebroventricular or intravenous injection of d(CH2)5Tyr(Me)AVP, a specific VI receptor antagonist, or with an intracerebroventricular or intravenous injection of the vehicle, and the responses to a subsequent injection of relaxin were monitored. Surgical placement of arterial and venous catheters, and intracerebroventricular cannula implantation were performedas above. d(CH2)STry(Me)AVP (0.3 ug/kg) (Sigma Chemical Co., S1. Louis, MO) or ACSF vehicle was injected into the lateral ventricle, or d(CH2)5Tyr(Me)AVI' (10 J.Lg/kg) or saline vehicle was injected into the femoral vein. Ten minutes later, relaxin (0.34 ""g) was injected into the lateral ventricle. MAP and HR were monitored continuously before and after both injections. Previous studies have shown that intravenous administration of d{CH2)5TYl'(Me)AVP at the dose of 10 ""g/kg Hocks the pressor response to intravenous injection of AVP (100 ng/kg) while intracerebroventricular injection of

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this VI receptor antagonist at the dose of 0.3 fJ..g/kg completely abolished effects of intracerebroventricular AVP (0.5 ng/kg/min for I h) in conscious rats." In a fourth series ofexperiments, d(CH~5Tyr(Me)A VP (0.3 ILglkg) or ACSF vehicle was injected into the lateral ventricle, or d(CH2)5Tyr(Me)AVP (10 fJ..glkg) or vehicle was injected into the femoral vein in conscious SO rats. Ten minutes later, relaxin (40 ILg/kg) was intravenously injected. MAP and HR were monitored before and after injections. To confirm that the pretreatment with the VI antagonist was suffident to block pressor responses to intravenous AVP, d(CH2)5Tyr(Me)AVP (10 ug/kg) or saline wasinjected into the femoral vein, and 10 min later, AVP (100 ng/kg) was injected intravenously. A fifth seriesof experiments examined the effect of centrally administered relaxin on vasopressin release. Human relaxin (0.34 ILg) or ACSF vehicle was injected into the lateral ventricle in conscious rats. Thirty minutes before and 6 min after the injection, blood was collected from the femoral arterial catheter into heparinized tubes on ice. Our preliminary study has shown that pressor and bradycardic effects are evident5 to 7 min after intracerebroventricular injection of relaxin. Previous studies from other investigators have demonstrated that plasma levels of vasopressin are substantially elevated 6 min after central administration of relaxin in rats." Plasma was separated by centrifugation at 4°C. Plasma samples were stored at - 80°e. Plasma vasopressin was measured by radioimmunoassay. At the conclusion of the experiments in rats implanted with the intracerebroventricular cannula, 200 nL of 1% methylene blue in distilled water was injected into the lateral ventricle through the cannula. The rat was then anesthetized with sodium pentobarbital (100 mglkg, intraperitoneally), decapitated, and the cannula was removed from the brain. The brain was removed from the skull and sectioned at 30 ILm on a cryostat-microtome (Slee Medical Equipment Ltd., London, England). All sections were mounted and stained with 1% thionin for verification of the microinjection. The success rate implantation of intracerebroventricular cannula was 90.4%. All experimental procedures were approvedby Genentech's Institutional Animal Care and Use Committee before initiation of the study. Statistical Analysis Results are expressed as mean ± SEM. One-wayanalysis of variance (ANOVA) was performed to assess differences in MAP and HR at the same timepoint between groups and to compare changes over time within eachgroup. Significant differences were then subjected to post hoc analyses using the Newman-Keuis method. The data of plasma vasopressin before and after injection in the same group were compared by a paired t test, and the

date" in two groups were compared b: :!. .., ilt'~irl: d t trst. Significance was defined as P < .L.' RESULTS Effects of Intracerebroventricular Injection of Relaxin In conscious SD rats, injection of relaxin into the lateral ventricle resulted in dose-related pressor and bradycardic responses (Figure 1) that began almost immediately after the injection, had a latent period of 30sec, reached rnaximallevels within 15 to 20 min, and returned to baseline by 90 min postinjeclion. The maximal changes in MAP and HR in response to the O.34-ILg injection of relaxin was + 11.8 ± 0.9 mm Hg and -43.6 ± 4.5 beats/min, respectively. NeitherIntracerebroventricular injection of the vehicle nor intravenous injection of 0.34 ILg of relaxin altered MAP and HR significantly (Figure 2). Effects of Intravenous Injectionof Relaxin Intravenous injection of 40 ILglkg relaxin caused a modest, significant elevation in MAP and a marked increase in HRin conscious rats (Figure 3). The maximal increase in MAP and HR in response to the intraventricular injection of relaxin at this dose was lOA ± 2.5mm Hg and 79.2 ± 6.3 beats/min, respectively. Thus, the 0-0 r ...

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pressor response to intravenously injected relaxin ~ was equal in magnitude to the response to the intra- ~c cerebroventricular injection of relaxin, while the ta- .~ chycardic response to the intravenous injectionof re- g' laxir, was opposite to the bradycardicresponse to in- .,g tracerebroventricular injectionof relaxin. Intravenous U injections of the vehicle did not alter MAP and HR significantly (Figure 3). Effects of the Vl Receptor Antagonist on Responses to Relaxin Injection of d(CH2)5Tyr(Me)AVP (0.3 ~g/ kg) into the lateral ventricle did not alter MAP or HR significantly. Resting MAP and HR were 115.8 ± 4.3 mm Hg and 364 ± 9 beats/min before the injectionof the antagonist and 116 ± 5 mm Hg and 369 ± 9 beatsl min 10 min after the antagonist injection (ie, immediately prior to the injection of relaxin). Similarly, intravenous injection of the antagonist (10 /-"glkg) did not cause significant alterations in MAP and HR (ll(j ± 5 compared to 115 ± 6 mm Hg; 371 ± 7 compared to 370 ± 10 beats/min before and 10 min after the injection of the antagonist, respectively). Pretreatment with an intracerebroventricular injection of d(CH2)5Tyr(Me)AVP (0.3 ....glkg) significantly attenuated the bradycardic and pressor responses to a subsequent intracerebroventricular injection of ~e-

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EFFECTS OFCENTRAL RELAXIN IN RATS 379

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sor and bradycardic responses to intracerebroventricular relaxin were delayed by a latent period of 30 sec, which mayrepresent the time for relaxin to reach the relaxin receptors near the dorsalwall of the third ventricle, The present study also demonstrates that pretreatment with an injection of a VI receptor antagonist (either intracerebroventricular or intravenous) significantly inhibits the pressor and bradycardic responses of conscious rats to intracerebroventricular administration of relaxin, suggesting that the cardiovascular responses to centra, administration of relaxin are mediated, at least in part, by centrally released vasopressin acting on VI receptors in the brain. Further, following central administration of relaxin, plasma levels of vasopressin were significantly elevated when the pressor and bradycardic effects were evident. This finding also provides evidence tuat release of vasopressin into the peripheral circulation may contribute to pressor and bradycardic responses to centrally administered relaxin.

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relaxin; plasma levels of vasopressin were significantly elevated (from 32.0 ± 12.8 to 100.3 ± 4.1 pgl mL, P < .01) (Figure 6). In contrast, control injection of ACSF did not significantly affect plasma vasopressin. After injection, plasma levels of vasopressin were significantly higher in relaxin- than vehicletreated animals (Figure 6). DISCUSSION Thepresent study demonstrates that injections of human relaxin into the lateralventricle cause significant dose-related increases in MAP and decreases in HR in conscious SDrats. Neither injections of an equal volume of the vehicle into the lateral ventricle nor injections of an equal dose of relaxin into the femoral vein alters MAP and HR. These data demonstrate that centrally administered relaxin has significant effects on the central nervous system and suggest that relaxin mightparticipate in the central control ofarterial pressure and HR in normal rats, likely via specific, high-affinity binding sites that are located in brain areas involved in the regulation of arterial pressure and HR, eg, the circumventricular organs." The pres-

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380 YANG ET AL

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Our finding that central administration of human relaxin increases MAP and that intravenous pretreatment with the VI receptor antagonist significantly attenuates this response in conscious rats is consistent with the observation of Mumford et al, who have demonstrated that in the anesthetized, lactating, Wistar rat, administration of porcine relaxin (1 IJ,g) into the lateral ventricle or into the dorsal portion of the third ventricle causes a significant rise in arterial pressure and an increase in plasma vasopressin concentration." Further, they also demonstrated that in these rats an injection of relaxin into the ventral por~ tion of the third ventricle produces only a short-term rise in arterial pressure, whereas an injection of re~ laxin into the fourth ventricle does not alter blood pressure or HR. Interestingly, lesions of the subfornical organ of the brain abolish the pressor response to relaxin injected into the dorsal portion of the third ventricle, whereaslesions of the anteroventral wallof the third ventricle (AV3V region, including the organum vasculosum of the lamina terminalis and the nudeus medianus) selectively abolish the pressor response to relaxin injected into the ventral area of the third ventricle." These data along with the finding of high concentrations of relaxin binding sites in two of the circumventricular organs (the subfornical organ and the organum vasculosum of the lamina terminaliS,2 suggestthat the pressor effect ofcentrally administered relaxin are mediated, at least in part, by these two circumventricular organs. In addition, Mumford et al and Parryet al have reported that central administration of a specific angiotensin II receptor antagonist can block the pi'essor response to centrally administered relaxin in anesthetized rats, suggesting that centralangiotensin may mediate the pressor effect QJ central relaxin.v" It has been speculated that central relaxin may act via the circumventricular or-

gans to activate brain angiotensin mechanisms and indirectly stimulate vasopressin release. Our finding that central administration of relaxin causes significant dose-related decreases in HR in conscious, unrestrained rats contrasts with the previous observation of Mumford et al, who indicated that injection of relaxin (1 jJ.g) into the lateral ventricle causes a significant increase in HR in anesthetized rats." It is likely that the apparent inconsistency is relatedprimarily to effects of anesthesia and lactation on the central regulation of HR in the Mumford et al study." Previous studies of the effects of acute intravenous administration of relaxin on arterial pressure and HR in normotensive rats have yielded inconsistent results. Intravenous administration of porcinerelaxin (5 !J.g) causesan initial, transient «1 min) fall, followed by a sustained risein arterial pressure in anesthetized female WiEtar rats," and in anesthetized female SD rats, intravenous injection of porcine relaxin (1.25 to 10 IJ,g) increases arterial pressure and HR in a dosedependent fashion." However, Ward et al have reported that in conscious, late-pregnantSD rats, intravenous injection of human relaxin (0.1 and 2 mglkg) does not alter arterial pressure or HR. 9 Together, these data suggest that in normotensive rats, the effrets of acuteintravenous administration of relaxin on arterial pressure and HR are significantly influenced by anesthesia, by the species of relaxin used, and by pregnancy, Serum relaxin is elevated by more than twofold in prepartum rats compared to nonpregnant rats'? and this increase like!y downregulates relaxin receptors. Thus the difference be-tween our findings and those of Ward et al9 may relate to a downregulation of relaxin receptors in the pregnant rat. The mechanism by which intravenously injected relaxin causesincreases in MAP and HR remains ambiguous. It has been reported that intravenous injection of relaxin results in an increase in plasma vasopressin levels," and that pretreatment with a VI receptor antagonist attenuates the pressor response to intravenous relaxin in anesthetized rats.8 However, peripheral administration of relaxin has not been shown to alter plasmavasopressin levelsin conscious rats." Our observation thai pretreatment with the VI receptorantagonist did not affect the intravenous relaxin-induced pressor and tachycardic responses in conscious rats suggeststhat the effects of intravenous relaxin are not due to the release of vasopressin into the peripheral circulation or into the brain. Local administration of relaxin in the mesenteric vasculature does not affect mesenteric arterioles.'! and relaxin binding sites are not present in the heart ventricle of rats.12 Thus, the pressor effect of intravenously administered relaxin is not the result of a directeffect on peripheralarteries or on the ventricle. However, sev-

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eral studies suggest that the tachycardic effects of intravenous relaxin maybe attributed to a direct cardiac effect. Specific, high-affinity relaxin binding is present in the atria of both male and female rats,12 and in isolated heart preparation from rats, relaxin increases HR by a direct action on the atria. 13,14 Thus, while the mechanism(s) underlying the pe~ ripheral action of relaxin remain undefined, the present results dearly demonstrate that the central vasopressin systemis not the likely primarytarget of peripheralrelaxin. In contrast, centrally administered relaxin dearly acts through a central nervous system mechanism that involves the activation of central VI receptors, and also through vasopressin release into the peripheral circulation.

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larly administered al-adrenoceptor agonists in the conscious rat. J Hypertension 1985;3:61~620. Parry LI, Summerlee AJS: Central angiotensin partially mediates the pressor action of relaxin in anesthetized rats. Endocrinol 1991;129:47-52. Mumford AD, Parry LJ, Summerlee AJS: Central angiotensin II blockade suppresses the pressor effect of centrally administered relaxin in anesthetized rats. Eur J Neurosci 1989(suppl 2):66. Jones SA, Summerlee AJS: Relaxin increases blood pressure and vasopressin levels in anesthetized rats (abst). J PhysioI1980i381:37P. Parry LL Poterski RS, Summerlee AJS, Jones SA: Mechanism of the haemotensive action of porcine re· laxin in unaesthetized rats. J Neuroendocrinol 1990;2: 5~58.

9. Ward DG , CroninMJ, Baertschi AI: Lack of cardiovas-

ACKNOWLEDGMENTS The authors are grateful to Dr. Phyillis L. Osheroff, Dr. Joffre Baker, and Dr. Michael J. Cronin for their support.

to.

REFERENCES 1. Osheroff PL, Ho W~H: Expression of relaxin mRNA 11. and relaxin receptors in postnatal and adult rat brains and hearts. J BioI Chern 1993;268:1519~15199. 2. Osheroff PL, Phillips HS: Autoradiographic localization of relaxin binding sites in rat brain. Proc l.]atl 12. Acad SciUSA 1991;88:6413-6417. 3. Mumford AD, ParryLJ, Summerlee AJS: Lesion of the subfornical organ effects the haemotensive response 13. to centrally administered relaxin in anesthetized rats. I EndocrinoI1989;122:747-755. 14. 4. Hiwatari M, [ohnston CI: Involvement of vasopressin in the cardiovascular effects of intracerebroventricu-

cular and vasopressin responses to human relaxin in conscious, late-pregnant rats. Am I Physiol 1991;261 (Heart Circ Physiol 30):H206-H211. Sherwood 00, Crnekovic VE, Gordon WL, Rutherford JE: Radioimmunoassay of relaxin throughout pregnancy and during parturition in the rat. EndocrinoI1980;107:691-698. Bigazzi M, Mese AD, Petrucci F: The local administration of relaxin induceschangesin the microcirculation of the rat mesocaecum. Acta EndocrinoI1986;112:296299. Oshercff PL, CroninMJ, Lofgren JA: Relaxin binding in the rat heart atrium. Proc Natl Acad Sci USA 1992; 89:23B4-2388. Kakouris H, Eddie LW, Summers RJ: Cardiac effects of relaxin in rats. Lancet 1992;339:107fr1078. Ward DG, Thomas GR, Cronin MJ: Relaxin increases rat heart rate by a direct action on the cardiac atrium. Biochem Biophys Res Commun 1992;186:999-1005.