0031-3998/04/5605-0756 PEDIATRIC RESEARCH Vol. 56, No. 5, 2004 Copyright © 2004 International Pediatric Research Foundation, Inc. Printed in U.S.A. Vasopressin Mechanism–Mediated Pressor Responses Caused by Central Angiotensin II in the Ovine Fetus LIJUN SHI, CATALINA GUERRA, JIAMING YAO, AND ZHICE XU Harbor-UCLA Medical Center and Research and Education Institute, Torrance, CA 90502 [L.S., C.G., J.Y. Z.X.]; and Soochaw University School of Medicine, Suzhou 215007, China [Z.X.] ABSTRACT AVP not only influences renal water excretion but also has receptor antagonist had no effect on either fetal or maternal profound cardiovascular effects in adults. Our recent studies have baseline BP. However, intracerebroventricular Ang II–increased demonstrated that central angiotensin induced fetal pressor re- BP was partially inhibited, although not completely abolished, by sponses accompanied with AVP release. However, little is the V1-receptor blockade. In contrast, fetal i.v. infusion of known of hormonal mechanisms in angiotensin-mediated fetal V2-receptor antagonist had no effect on the pressor responses blood pressure (BP) changes. The present study determined AVP induced by central Ang II. The results suggest that the central mechanisms in central angiotensin-mediated fetal pressor re- Ang II–mediated pressor responses at the last third of gestation is sponses. The V1-receptor antagonist or V2-receptor antagonist mediated partially by the AVP mechanism via V1 not V2 was infused intravenously into the ovine fetus at 90% gestation. receptors. (Pediatr Res 56: 756–762, 2004) Angiotensin II (Ang II; 1.5 ␮g/kg) was then injected intracere- broventricularly into the chronically instrumented fetus. Ang II Abbreviations produced a significant increase in fetal systolic, diastolic, and A-MAP, mean arterial pressure adjusted to amniotic pressure mean arterial pressure adjusted to amniotic pressure (A-MAP). Ang II, angiotensin II The enhanced fetal A-MAP was associated with intense c-fos AVP-ir, AVP-immunoreactivity expression in the central putative cardiovascular area: the para- BP, blood pressure ventricular nuclei (PVN). Double labeling demonstrated that a FOS-ir, c-fos immunoreactivity number of the AVP-containing neurons in the PVN were ex- i.c.v., intracerebroventricular pressing c-fos in response to central Ang II. Consistent with the MAP, mean arterial pressure activation of AVP neurons in the PVN, fetal plasma AVP was PVN, paraventricular nuclei markedly enhanced. Fetal i.v. V1-receptor antagonist or V2- RAS, rennin-angiotensin system The nonapeptide AVP, also known as antidiuretic hormone, has II (Ang II), are essential in the control of cardiovascular functions. multiple actions, including water reabsorption and vasoconstric- Central renin-angiotensin system (RAS) plays an important role in tion in the periphery. This peptide also plays a role in cardiovas- the regulation of arterial pressure and in the development of cular diseases, including hypertension and congestive heart failure certain forms of clinical and experimental hypertension (3,5,6). (1). In adults, plasma AVP has been reported to be elevated in Under certain conditions, the RAS and the AVP system operate as some forms of hypertension, and this elevation correlates best reciprocal mechanisms in the control of vascular resistance and with the severity of hypertension (2). Recent findings have dem- arterial pressure in adults (7). For example, intracerebroventricular onstrated that the development of cardiovascular regulatory mech- (i.c.v.) administration of Ang II causes a rise in blood pressure anisms starts before birth and that the in utero development of (BP) associated with AVP release (8–10). The pressor response cardiovascular regulation is critical for fetal cardiovascular ho- induced by the i.c.v. Ang II in adults is partially related to an meostasis (3,4). Several peptides, including AVP and angiotensin increase of AVP. In fetuses, previous study has shown that AVP plays a role in BP regulation during periods of hypovolemia (11). Received January 13, 2004; accepted April 15, 2004. The enhanced plasma AVP contributed to a rise of mean arterial Correspondence: Zhice Xu, Ph.D., Harbor-UCLA Medical Center, 1124 West Carson pressure (MAP) and decreased heart rate. I.v. infusion of the Street, RB-1, Torrance, CA 90502; e-mail: [email protected] vasopressin V1-receptor blockade abolished the increase of the Research described in this article is supported by March of Dimes Research Grant, External Research Grant from Philip Morris USA Inc., and UCLA Faculty Grant Program fetal MAP after application of AVP (12,13). Award to Z.X. Recent studies in our laboratory have demonstrated that DOI: 10.1203/01.PDR.0000141519.85908.68 i.c.v. Ang II increased fetal arterial pressure associated with an 756 FETAL V1-RECEPTOR AND ANG-RELATED PRESSOR 757 elevation of plasma AVP in the near-term ovine fetus (14). samples were replaced with an equivalent volume of heparin- This raised a question of whether the Ang-mediated fetal ized maternal blood withdrawn before the study. pressor response was mediated by hormonal mechanisms. Study 2: effect of i.v. V1- or V2-antagonist on the pressor Therefore, the present study sought to determine the AVP response caused by central Ang II. Animals (n ϭ 5) were mechanism in Ang-induced pressor responses and determine assigned for three experimental treatments on separate days which subtype of AVP receptors may be involved. AVP is randomly. Study began with a baseline (Ϫ30 to 0 min) fol- synthesized mainly in supraoptic nuclei and paraventricular lowed by a study period (0 to 90 min) after i.c.v. Ang II (1.5 nuclei (PVN) in the hypothalamus (3). Thus, we also sought to ␮g/kg) injection into the fetus, or, after the baseline (Ϫ60 to determine the hypothalamic AVP cellular activation under the Ϫ20 min) period, either [deamino-Pen1, O-Me-Tyr2, Arg8]- condition of i.c.v. administration of Ang II in the near-term vasopressin (a potent V1-antagonist; 10 ␮g/kg; Sigma Chem- 1 2 ovine fetus. ical Co., St. Louis, MO) or [adamantaneacetyl , O-Et-D-Tyr , Val4, aminobutyryl6, Arg8,9]-vasopressin (a potent V2- antagonist; 150 ␮g/kg; Sigma Chemical Co.), dissolved in 10 METHODS mL of 0.9% NaCl saline, was infused into the fetal vein catheter for 30 min (i.e. from Ϫ20 to 10 min) by an infusion Studies were performed in chronically instrumented fetal pump, Ang II (1.5 ␮g/kg) was then injected intracerebroven- sheep at 90% of gestation. Animals were housed indoors in tricularly into the fetus at 0 min. The doses of the drugs were individual steel study cages and were acclimated to a 12:12-h referred to in previous reports (16–18). Blood samples (4 mL light-dark cycle. Water and food (alfalfa pellets) were provided each) were withdrawn from the fetal and maternal arterial ad libitum. All surgical and experimental procedures have been catheters at Ϫ30, Ϫ10, 0, 10, 25, 40, and 60 min for measure- approved by the Animal Care Committee. ments of blood gases, hematocrit, plasma osmolality, plasma Surgical preparation. For the surgical procedures, ketamine electrolytes, and AVP. hydrochloride (20 mg/kg, i.m.) was injected in ewes. Anesthe- Throughout studies, maternal/fetal systolic and diastolic sia was maintained with 3% isoflurane and 1 L/min oxygen. pressure, heart rate, and amniotic pressure were recorded con- The uterus was opened over the fetal hindlimbs, and polyeth- tinuously. The fetal MAP was corrected against amniotic fluid ylene catheters were placed in the maternal (inner diameter: 1.8 pressure. Fetal and maternal BP was measured by means of a mm; outer diameter: 2.3 mm) and fetal (inner diameter: 1.0 Beckman R612 (Beckman Instruments, Fullerton, CA) physi- mm; outer diameter: 1.8 mm) femoral vein and artery. An ologic recorder with Statham (Garret, Oxnard, CA) P23 trans- intrauterine catheter was inserted for measuring amniotic fluid ducers. BP and heart rate were determined by computer anal- pressure. An intracranial catheter was placed in one of the fetal ysis of waveforms using a customized pattern recognition lateral ventricles and held in place with dental cement as algorithm. reported (15). Patency of the catheter at insertion was assessed Arterial blood values and AVP analysis. Arterial pH, blood by free flow of cerebrospinal fluid via gravity drainage. The gases (PO2,PCO2), and Hb were measured with a Radiometer placement of cannulae was finally verified with histologic BM 33 MK2-PHM 72 MKS acid-base analyzer system (Radi- analysis. The fetus was then returned to the uterus, and the ometer, Copenhagen, Denmark). Plasma osmolality was mea- uterus was closed using a double row of sutures. All catheters sured on an Advanced Digimatic osmometer (Advanced In- were led to the ewe’s flank. The ends of the catheters were held struments, Needham Heights, MA). Plasma Naϩ,Kϩ, and ClϪ in place in a small cloth pocket sutured to the skin of the ewe. concentrations were determined by a Nova analyzer (Nova During the initial 2 d of recovery, gentamicin (8 mg) and Biomedical, Waltham, MA). oxacillin (33 mg) were administered intravenously to the fetus, The blood samples were collected into iced tubes that con- and gentamicin (72 mg), oxacillin (1g), and chloramphenicol tained lithium heparin and centrifuged immediately for AVP (1 g) were administered intravenously to the ewe (15). All RIA. Plasma AVP concentrations were measured using Sep- animals were allowed 5 d for postoperative recovery. Pak C18 cartridge (Waters Associates, Milford, MA) extrac- Experimental protocol. On the testing day, sheep were tion (19). Sensitivity of the AVP antiserum is 1.5 pg/tube with allowed 60–100 min to acclimatize to the testing room. When intra-assay and interassay coefficients of variation of 6 and 8%, maternal and fetal heart rates and arterial pressures seemed to respectively. AVP recoveries average 70% in our experiments.