- Email: [email protected]
In utero development of central ANG-stimulated pressor response and hypothalamic fos expression
Developmental Brain Research 145 (2003) 169 – 176 www.elsevier.com/locate/devbrainres
Research report
In utero development of central ANG-stimulated pressor response and hypothalamic fos expression Zhice Xu *, Lijun Shi, Fang Hu, Rodney White, Lauren Stewart, Jiaming Yao Department of Obstetrics and Gynecology and Division of Vascular Surgery, Harbor-UCLA Medical Center and Research and Education Institute, 1124 West Carson Street, RB-1, Torrance, CA 90502, USA Accepted 24 July 2003
Abstract Central renin – angiotensin system (RAS) is as important as the peripheral RAS in the control of the cardiovascular homeostasis in the adult. However, previous fetal studies on angiotensin II (ANG II)-induced cardiovascular responses focused exclusively on the peripheral side. Thus, few data exist characterizing the in utero development of central angiotensin-mediated pressor responses. The present study determined cardiovascular responses to central application of ANG II in the chronically prepared near-term ovine fetus, and determined the action sites marked by c-fos expression in the fetal hypothalamus following intracerebroventricular (icv) injection of ANG II in utero. ANG II significantly increased fetal systolic, diastolic, and mean arterial pressure (MAP) within 5 min after injection of this peptide into the brain. Adjusted fetal MAP against amniotic pressure was also increased by icv ANG II, associated with increased c-fos in the central putative cardiovascular area—the paraventricular nuclei (PVN). Application of ANG II also induced intense c-fos expression in the supraoptic nuclei (SON), accompanied by a significant increase of fetal plasma vasopressin (AVP) levels, while maternal blood pressure (BP) and plasma AVP concentration were not changed. These results indicate that the central ANG II-mediated pressor response is functional at the last third of gestation, acting at the sites consistent with the cardiovascular neural network in the hypothalamus. D 2003 Elsevier B.V. All rights reserved. Theme: Endocrine and autonomic regulation Topic: Cardiovascular regulation Keywords: Angiotensin II; Pressor response; c-fos; Fetal brain development; AVP
1. Introduction Recent progress has been made in demonstrating that the development of regulatory mechanisms for cardiovascular homeostasis may start during fetal life and that the in utero development of cardiovascular mechanisms is important for fetal and ultimately adult blood pressure (BP). There are numerous studies that have demonstrated the importance of renin – angiotensin system (RAS) in regulation of BP [1,17,18]. A major function of angiotensin (ANG) in the healthy fetus appears to be maintenance of fetal arterial pressure under conditions of fetal stress, such as hemorrhage or hypoxia [13,14]. However, investigation of the development of the brain RAS and its role in the control of BP in
* Correspondence author. Tel.: +1-310-222-8179; fax: +1-310-2224131. E-mail address: [email protected] (Z. Xu). 0165-3806/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0165-3806(03)00226-8
utero has been limited. Although it is well known that the RAS plays an important role in the central nervous systems mediated cardiovascular responses, it is noted that previous work performed in the fetus focused exclusively on actions of ANG at the peripheral side in the control of BP at the developmental stages [7,8,12,16,20]. However, in the course of conducting experiments in our laboratory on the capacity of ANG II to stimulate fetal swallowing activity, we frequently saw cardiovascular responses after injection of this peptide into the lateral ventricle of the near-term ovine brain in utero [27] This observation prompted these initial experiments investigating the cardiovascular action of fetal intracerebroventricular (icv) injection of octapeptide ANG II. Administration of either ANG II or its precursors into the brain produces a pressor response, induces drinking behavior, and causes the release of adrenocorticotropic hormone and vasopressin (AVP) [2,9,22]. In the adult, there is significant information regarding the effects of central ANG [9]. Intracerebroventricular ANG II has been repeat-
170
Z. Xu et al. / Developmental Brain Research 145 (2003) 169–176
fied with histological analysis after sacrificing the animal. An intrauterine catheter was inserted for measuring amniotic fluid pressure. The fetus was then returned into the uterus, and the hysterotomy was closed in two layers. All catheters were externalized to the maternal flank and placed in a cloth pouch. Catheters were filled with heparinized saline, and the animals were treated with antibiotics [27], and given 5– 6 days for postoperative recovery.
edly shown to produce a reliable pressor response in adult animals. Furthermore, administration of ANG II icv also induces neuroendocrinological responses (e.g., vasopressin release) [3,20]. However, as mentioned above, few data exist regarding effects of central action of ANG on the cardiovascular system in utero. As a result, knowledge concerning the development of central mechanisms for ANG-regulated cardiovascular homeostasis is very limited. The present study determined the effects of central ANG II on fetal pressor responses in utero and determined whether central ANG can activate the brain pathways in the near-term fetus. Information gained increases our understanding of the ontogeny of central ANG mechanisms for cardiovascular homeostasis and provides information to the development of central RAS-related cardiovascular neural network in the early stages of life.
2.2. Cardiovascular experiments Studies began with a baseline ( 120 – 0 min) followed by study period (0 – 100 min). There were two groups (control: n = 5; experimental: n = 5). Beginning at time 0, ANG II (1.5 Ag/kg, 1 ml) (Sigma) in isotonic saline was injected icv into the fetus over 5 min. For the control animals, isotonic saline was injected. Throughout the study, maternal and fetal systolic and diastolic pressure, amniotic pressure, and heart rate were monitored continuously. The fetal mean arterial pressure (MAP) was corrected for amniotic cavity pressure. Fetal and maternal BP was measured by means of a Beckman R612 (Beckman Instruments, Fullerton, CA) physiological recorder with Statham (Garret, Oxnard, CA) P23 transducers. BP and heart rate was determined by computer analysis of waveforms utilizing a customized pattern recognition algorithm.
2. Material and methods 2.1. Surgical preparation Time-dated pregnant ewes (Nebeker) with singleton fetuses (130 F 3 days gestation at test) were used. Experimental protocols have been approved by the Institute Animal Care Committee. Ketamine hydrochloride (20 mg/ kg, i.m.) was used for anesthesia and general anesthesia was maintained with 5% isoflurane and 1 l/min oxygen. The uterus was exposed by a midline abdominal incision, and a small hysterotomy was performed to provide access to a fetal hindlimb and head. Polyethylene catheters were placed in the maternal and fetal femoral vein and artery and threaded to the inferior vena cava and abdominal aorta, respectively. An intracranial cannulae was placed in the fetal lateral ventricle and held in place with dental cement. The coordinates were anterior –posterior: + 0.1 cm in front of the bregma; medial – lateral: 0.8 cm from the middle line; and ventral: 1.8 cm below the dura. Patency of the catheter at insertion was assessed by free flow of cerebrospinal fluid via gravity drainage. The placement of cannulae was veri-
2.3. Endocrine experiments Maternal and fetal blood samples were collected and assessed for hematocrit, pH, PO2, and PCO2, remaining blood was centrifuged and plasma osmolality, sodium, chloride, and potassium concentrations were measured as reported [27]. Plasma samples were used for AVP radioimmunoassay. The sensitivity of the AVP antiserum is 0.8 pg/tube with intra-assay and interassay coefficients of variation of 6% and 9%, respectively. Plasma AVP recoveries average 70%. Blood PO2, PCO2, pH, hemoglobin (Hb) were measured with a Radiometer BM 33 MK2-PHM 72 MKS acid – base analyzer system (Radiometer, Copenhagan). Plasma osmo-
Table 1 Effects of icv ANG II (1.5 Ag/kg) on maternal (upper panel) and fetal (lower panel) cardiovascular responses 60 min Systolic pressure (mm Hg) Diastolic pressure (mm Hg) MAP (mm Hg) Heart rate (beats/min)
100 F 14 62 F 8 80 F 11 106 F 7
Systolic pressure (mm Hg) Diastolic pressure (mm Hg) MAP (mm Hg) Adjusted MAP (mm Hg) Heart rate (beats/min)
56 F 7 43 F 6 49 F 6 42 F 1 153 F 5
60 min
* p < 0.01.
30 min 98 F 14 63 F 5 81 F 11 108 F 10 30 min 55 F 5 44 F 5 49 F 4 43 F 1 149 F 13
5 min
15 min
30 min
60 min
96 F 14 63 F 8 79 F 11 103 F 6
103 F 8 64 F 6 83 F 6 102 F 9
103 F 11 64 F 6 83 F 8 104 F 14
102 F 10 61 F 7 82 F 8 99 F 11
5 min
15 min
30 min
60 min
90 min
66 F 9* 52 F 8* 59 F 7* 52 F 2* 140 F 17
66 F 3* 53 F 8* 60 F 5* 53 F 3* 142 F 24
66 F 7* 53 F 8* 59 F 6* 53 F 1* 132 F 46
62 F 5 48 F 6 57 F 4 51 F 41 140 F 29
58 F 5 45 F 5 51 F 3 49 F 1 142 F 22
Z. Xu et al. / Developmental Brain Research 145 (2003) 169–176
171
Fig. 1. The effect of icv injection of vehicle or ANG II on fetal MAP (mm Hg). 0 min: time for icv injection. *p < 0.01. icv: intracerebroventricular. MAP: mean arterial pressure.
lality was measured on an Advanced Digimatic osmometer (model 3 MO, Advanced Instruments, Needham Heights, MA). Plasma sodium and potassium concentrations were determined by a Nova 5 electrolyte analyzer (Nova Biomedical, Waltham, MA). 2.4. Immunohistochemistry experiments The animals were anesthetized and perfused, and the fetal brain was collected at the end of the cardiovascular testing. Twenty-micrometer coronal sections of fetal forebrain were cut on a cryostat. The sections of the hypothalamus were used for FOS immunoreactivity (FOS-ir) staining using the avidin – biotin-peroxidase technique. The tissue sections were first incubated in the primary antibody (1:20,000, Santa Cruz Biotech, CA) overnight, then incubated in goat antirabbit serum (1:500) and processed using the ABC kit (Vector Labs, Burlingame). The tissue sections were treated with 1 mg/ml diaminobenzidine tetrahydrochloride. 2.5. Data analysis All signals in cardiovascular studies were digitized at 500 Hz and recorded on a computer with Win-DAQ acquisition software (Data Q instruments, Akron). Heart rate, systolic and diastolic pressure, and MAP were calculated from the pressure waveforms by means of Advanced CODAS software. The number of FOS-ir positive cells in the supraoptic and paraventricular nuclei (SON and PVN) was evaluated in a qualitative and blinded manner. Statistical analysis was preformed with repeated-measures ANOVA. Comparisons before and after treatments were determined with one-way ANOVA followed by Tukey test or t-test. p < 0.05 or 0.01 was the probability level used to define statistical significance. All data were expressed as mean F S.E.M.
3. Results 3.1. Cardiovascular responses Histological analysis confirmed that all icv cannulae were inserted into the fetal lateral ventricle. There was no significant difference between the control and experimental groups for maternal systolic pressure ( F8,1 = 0.05), diastolic pressure ( F8,1 = 0.24), and MAP ( F8,1 = 0.01, all p: no significance) (Table 1). However, icv injection of ANG II into the fetus significantly increased fetal BP. Fetal systolic, diastolic pressure, and MAP were increased in the experimental group as compared to the control fetuses
Table 2 Maternal (upper panel) and fetal (lower panel) arterial values before and after icv injection of ANG II (1.5 Ag/kg) Baseline
15 min
30 min
60 min
pH 7.46 F 1.15 7.48 F 2.70 7.45 F 3.63 7.47 F 1.94 PCO2 (Torr) 34.7 F 1.1 35.2 F 1.6 37.4 F 3.9 35.8 F 2.6 PO2 (Torr) 113.1 F 6.2 112.3 F 9.0 108.6 F 9.8 111.4 F 10.3 Osmolality 301.2 F 2.3 301.4 F 4.1 300.4 F 4.5 300.6 F 3.5 (mOsm/kg) Na+ (meq/l) 145.6 F 3.5 147.4 F 1.5 146.6 F 2.8 146.3 F 1.8 K+ (meq/l) 4.0 F 0.2 4.1 F 0.2 4.0 F 0.2 4.0 F 0.2 Hb (g/dl) 8.1 F 1.1 8.3 F 0.4 7.9 F 0.8 7.9 F 1.2 Baseline
15 min
30 min
60 min
pH 7.37 F 0.21 7.38 F 0.24 7.37 F 0.27 7.37 F 0.20 PCO2 (Torr) 51.1 F 4.5 48.8 F 3.9 50.3 F 4.6 50.2 F 3.6 PO2 (Torr) 20.3 F 2.6 20.8 F 1.3 20.4 F 1.3 22.0 F 2.4 Osmolality 296.2 F 5.4 297.1 F 3.3 297.4 F 2.3 295.3 F 4.1 (mOsm/kg) Na+ (meq/l) 140.2 F 2.4 141.3 F 2.6 140.2 F 1.7 141.3 F 3.3 K+ (meq/l) 4.1 F 0.6 4.0 F 0.6 4.1 F 0.6 3.9 F 4.2 Hb (g/dl) 8.4 F 1.7 8.0 F 1.2 8.3 F 1.8 8.1 F 1.5
172
Z. Xu et al. / Developmental Brain Research 145 (2003) 169–176
( F8,1 = 5.70, 7.13, and 12.26, respectively, p < 0.01). There was significant difference for adjusted fetal MAP between the animals treated with icv ANG II and icv vehicle ( F8,1 = 11.09, p < 0.01). In the experimental animals, fetal MAP was increased from the baseline level, 49 F 4 mm Hg, to 60 F 5 mm Hg 15 min after icv injection of ANG II ( p < 0.01). This was a 20% increase from the baseline MAP to the peak level MAP. The increased MAP lasted for at least 30 min (Fig. 1). The fetal MAP, adjusted fetal MAP, systolic and diastolic pressure still remained at higher levels above the baseline ( F = 7.62, 8.94, and 3.76, respectively, p < 0.01 for both MAP and adjusted MAP, p < 0.05 for diastolic pressure) for 30 min after administration of ANG II (Table 1). In the control animals, icv injection of the vehicle had no effect on fetal systolic, diastolic pressure, MAP, and adjusted MAP in the nearterm fetus (Tukey test, all p: no significance). Maternal
heart rate was not changed by the icv injection of ANG II into the fetus ( F8,1 = 1.69, p: no significance). Fetal heart rate was not significantly changed in the control and the experimental animals (Tukey test, p: no significance). 3.2. Blood values For both the control and the experimental animals, icv injection of ANG II or vehicle had no effect on plasma osmolality, Cl , Na+, and K+ concentrations in either maternal or fetal animals. There was no significant difference for arterial blood pH, PO2, PCO2, Na+, K+, osmolality, hemoglobin, and hematocrit before and after icv injection of ANG II (all p: no significance). All arterial values were within normal ranges and did not vary significantly between the control and the experimental groups (all p: no significance) (Table 2).
Fig. 2. The effect of icv injection of vehicle or ANG II on fetal and maternal plasma AVP levels. 0 min: time for icv injection. *p < 0.01. icv: intracerebroventricular. AVP: vasopressin.
Z. Xu et al. / Developmental Brain Research 145 (2003) 169–176
173
Fig. 3. FOS-ir induced by icv ANG II in the fetal SON (upper panel) and PVN (bottom panel). A and a: the animal injected with icv ANG II; B and b: the animal treated with icv CSF. OC: Optic chiasma. 3V: Third ventricle. SON: supraoptic nuclei, PVN: paraventricular nuclei. 40 .
3.3. Plasma AVP assay There was no change in plasma AVP levels between the control and the experimental ewes ( F7,1 = 1.76, p: no significance). However, the fetal AVP concentrations were significantly higher in the icv ANG II injected fetuses than in the icv vehicle-treated animals ( F7,1 = 7.71, p < 0.05). In the control group, icv injection of vehicle did not change fetal plasma AVP ( F19,3 = 0.34, p: no significance, the
baseline period vs. the period after icv injection). The icv ANG II significantly increased fetal plasma AVP levels ( F19,3 = 4.01 p < 0.05, the baseline period vs. the period after icv injection) (Fig. 2). Fetal plasma AVP increased about sixfold at 15 min after icv ANG II, and the peak level of plasma AVP was observed at 30 min (ninefold) after icv injection of ANG II. Plasma fetal AVP concentrations dropped at 60 min, although the level was still higher than that at the baseline.
Fig. 4. The effect of icv injection of vehicle or ANG II on FOS-ir in the SON and PVN in the near-term ovine fetus. *p < 0.01. icv: intracerebroventricular. SON: supraoptic nuclei, PVN: paraventricular nuclei.
174
Z. Xu et al. / Developmental Brain Research 145 (2003) 169–176
3.4. FOS-immunoreactivity In the control fetuses following injection with icv vehicle, there was little FOS-ir in the fetal hypothalamic structures, including the putative pressor nuclei PVN and the SON. However, icv injection of ANG II produced FOS-ir in the hypothalamus of the fetus. Intense FOS-ir was observed in the SON and PVN (Fig. 3). There were significant differences of FOS-ir between icv vehicle and icv ANG II-injected fetuses (t = 8.2; p < 0.05) (Fig. 4). ANG II-induced FOS-ir in the PVN was detected in both magnocellular and parvicellular parts.
4. Discussion The present study demonstrated that icv application of ANG II could produce pressor responses in the near-term ovine fetus. Intracerebroventricular ANG II not only increased fetal MAP in utero, but also stimulated the neural activity in the central cardiovascular centers in utero. The finding that c-fos expression was induced by icv ANG II in the fetal cardiovascular centers and AVP secretion nuclei indicates that the central RAS is functional before birth. Thus, the central ANG systems for cardiovascular homeostasis are likely to be intact before birth. Previous studies have shown that central ANG-produced pressor responses occur reliably within the first 5 – 8 min after administration of ANG II in adult models [24]. Our study demonstrated that fetal systolic pressure, diastolic pressure, and MAP significantly increased within 5 min after icv injection of ANG II. Therefore, the latency for icv ANG II-stimulated pressor responses in the near-term ovine fetus is similar to that observed in the adult. ANG II is a potent vasoconstrictive hormone in the fetus and adult. Earlier work has shown that an active RAS is present in the ovine fetus before birth [5,12]. Its major function appears to be maintenance of fetal arterial pressure under conditions of fetal stress, including acute hemorrhage or hypoxia [16]. However, previous fetal studies on ANGregulated cardiovascular responses focused exclusively on the action of this peptide at the peripheral side. This study demonstrated that the brain RAS in the near-term fetus is functional and important in the control of pressor responses. In the present study, increased fetal systolic, diastolic, and mean arterial pressures were maintained for at least 30 min before they returned to the baseline. Biological effects of ANG II are mediated via the ANG receptors. ANG receptors have been demonstrated in both fetal and adult brains [9,19,21]. This study provides evidence that the fetal ANG receptors are functional in the mediation of cardiovascular regulation at near-term. Fetal physiological status remained stable during the testing periods, as did arterial values, including PCO2, PO2, pH, plasma electrolytes, hematocrit, and hemoglobin, following icv injections. The lack of change of plasma electro-
lytes, particularly sodium and osmolality, indicated that systemic sodium/osmolality did not affect cardiovascular responses in the present study. Maternal systolic and diastolic pressure and MAP were not influenced by the administration of icv ANG II into the fetus. Maternal plasma AVP levels were not affected by application of ANG II in the fetal animals either. These data suggest that icv injection of ANG II into the fetus has no effect on the maternal animals. A number of studies support the concept that there are two interacting systems in the brain responsible for ANG-increased blood pressure: autonomic and hormonal mechanisms. Several hormonal factors, including AVP, may contribute to ANG-mediated pressor responses. Intracerebroventricular ANG increases plasma AVP, a potent vasoconstrictor, in both rat and sheep [9]. Infusion of V1-AVP receptor antagonist, AVP antibodies, and hypophysectomy, have shown to reduce, but not eliminate, icv ANG II-induced pressor responses [5,10]. Moreover, rats with hereditary hypothalamic diabetes insipidus fail to show a blood pressure response to icv application of ANG II [11]. Thus, AVP mechanisms are partially involved in central ANG-induced pressor responses. In the present study, fetal plasma AVP levels were significantly increased following icv injection of ANG II, and increased fetal AVP concentrations lasted for about 30 min before returning to the baseline. Moreover, intense c-fos was expressed in the SON and PVN following icv ANG II in the fetal brain. Since the SON and PVN are the main source for the plasma AVP [6], activation of a large number of cells in the hypothalamus associated with a significant increase of fetal plasma AVP levels, in the present study, strongly indicates that AVP-containing neurons in these nuclei may be activated after icv injection of ANG II in the near-term fetus. As mentioned above, autonomic mechanisms are another major contribution to the increased fetal blood pressure by icv injection of ANG II [5]. Many brain nuclei and pathways that process signals for the control of cardiovascular responses have been identified by employing functional analysis and anatomical tract tracing techniques. Presumably, every region and pathway in this network can potentially influence signal integration in the pressor net. However, certain nuclei have demonstrated to be particularly critical to the maintenance of arterial pressure. Most notable among these are the nuclei in the hypothalamusPVN. The present study focused specifically on the action sites of central ANG in the fetal hypothalamus. The results showed that icv ANG II induced FOS-ir in the PVN and SON, as evidence that the areas in the hypothalamus were activated, and consistent with the previous adult studies [25,26]. Associated with the increased blood pressure and plasma AVP levels in the same fetal animals, the c-fos expression suggests that the critical areas in the CNS like the PVN for ANG-mediated cardiovascular mechanisms are functional during the last third of the gestation. Exogenous ANG II in the PVN augments the cardiac sympathetic
Z. Xu et al. / Developmental Brain Research 145 (2003) 169–176
afferent reflex evoked by stimulation of cardiac sympathetic afferent nerves [28]. In addition, the PVN is replete with ANG receptor, especially AT1 subtype receptors, which may account for the biological actions of the octapeptide [25]. In the present study, the neural activity marked with cfos in the PVN, in both magnocellular and parvicellular parts, suggests that the ANG receptors in the fetal hypothalamus have matured enough in response to ANG II stimulation. The present study is the first to demonstrate icv ANG IIstimulated c-fos expression in the fetal hypothalamus. These results provide evidence that the near-term fetal hypothalamus is functional in the brain RAS in the face of ANG stimulation. Although there is a possibility that the increased fetal blood pressure and baroreflex may induce c-fos expression in the brain under certain conditions [23], c-fos expression induced by increased blood pressure and/or baroreflex mainly locates in the hindbrain. Particularly, in the tractus solitarius nuclei (NTS), the lateral parabrachial nucleus (LPBN), and the rostral ventrolateral medulla (RVLM) [4,15,23]. The patterns of FOS-ir in the fetal PVN and SON following icv injection of ANG II were similar to that in previous observations in the adult hypothalamus under the same condition [25]. In addition, both maternal and fetal heart rates were not significantly changed following injection of ANG II. This suggests that it was unlikely that c-fos expression in the present study was due to baroreflex. There are two important results in the present study: ANG II in the fetal brain can reliably increase fetal blood pressure, and c-fos expression is observed in the fetal hypothalamus in utero. These results provide evidence that the putative cardiovascular centers, especially the critical areas like the PVN (i.e., the part of ‘‘sympathetic’’ neural network), and the SON (i.e., the main source of plasma AVP), are functional in the last third of gestation. It is apparent that brain ANG-mediated cardiovascular homeostasis is intact before birth. Considering that a major function of ANG II in utero appears to be maintenance of fetal BP under condition of fetal stress [13,14], and that recent progress in demonstrating that the development of regulatory mechanisms for cardiovascular homeostasis in utero may also be important to adult BP, the data gained in the present study provide useful information to the development of the central regulatory system related to ANGstimulated pressor responses starting during fetal life.
Acknowledgements These studies were conducted at the biomedical research facilities of the Research and Education Institute at HarborUCLA Medical Center. Research described in this article is supported by March of Dimes Research Grant, External Research Grant from Philip Morris, USA, Inc. and UCLA FGP Award to Z. Xu.
175
References [1] K. Allikmets, T. Parik, M. Viigimaah, The renin – angiotensin system in essential hypertension: associations with cardiovascular risk, Blood Press. 8 (1991) 70 – 78. [2] E. Badoer, Hypothalamic paraventricular nucleus and cardiovascular regulation, Clin. Exp. Pharmacol. Physiol. 28 (2001) 95 – 99. [3] J.R.G. Challis, B.F. Mitchell, Endocrinology of pregnancy and parturition, in: J.B. Warshaw (Ed.), The Biological Basis of Reproductive and Developmental Medicine, Elsevier, New York, 1983, p. 106. [4] R.K. Chan, P.E. Sawchenko, Organization and transmitter specificity of medullary neurons activated by sustained hypertension: implications for understanding baroreceptor reflex circuitry, J. Neurosci. 18 (1998) 371 – 387. [5] K.E. Clark, G.L. Irion, C.E. Mack, Differential responses of uterine and umbilical vasculatures to angiotensin II and norepinephrine, Am. J. Physiol. 259 (1990) H197 – H203. [6] K. Dierickx, F. Vandesande, Immunocytochemical demonstration of separate vasopressin – neurophysin and oxytocin – neurophysin neurons in the human hypothalamus, Cell Tissue Res. 19 (1979) 203 – 212. [7] L.J. Edwards, G. Simonetta, J.A. Owens, J.S. Robinson, I.C. McMillen, Restriction of placental and fetal growth in sheep alters fetal blood pressure responses to angiotensin II and captopril, J. Physiol. (London) 515 (1999) 897 – 904. [8] J.J. Faber, D.F. Anderson, Hydrops fetalis in nephrectomized fetal lambs infused with angiotensin I, Am. J. Physiol. 267 (1994) R1522 – R1527. [9] J.T. Fitzsimons, Angiotensin, thirst, and sodium appetite, Physiol. Rev. 78 (1998) 583 – 686. [10] D. Haack, J. Mohring, Vasopressin-mediated blood pressure response to intraventricular injection of angiotensin II in the rat, Pflugers Arch. 373 (1978) 167 – 173. [11] J.S. Hutchinson, P. Schelling, J. Mohring, D. Ganten, Pressor action of centrally perfused angiotensin II in rats with hereditary hypothalamic diabetes insipidus, Endocrinology 99 (1976) 819 – 823. [12] H.S. Iwamoto, A.M. Rudolph, Effects of endogenous angiotensin II on the fetal circulation, J. Dev. Physiol. 1 (1979) 283 – 293. [13] H.S. Iwamoto, A.M. Rudolph, Role of renin – angiotensin system in response to hemorrhage in fetal sheep, Am. J. Physiol. 240 (1981) H848 – H854. [14] H.S. Iwamoto, A.M. Rudolph, L.C. Keil, M.A. Heymann, Hemodynamic responses of the sheep fetus to vasopressin infusion, Circ. Res. 44 (1979) 430 – 436. [15] J.H. Jhamandas, K.H. Harris, T. Petrov, H.Y. Yang, K.H. Jhamandas, Activation of neuropeptide FF neurons in the brainstem nucleus tractus solitarius following cardiovascular challenge and opiate withdrawal, J. Comp. Neurol. 14 (1998) 210 – 221. [16] O.W. Jones, C.Y. Cheung, R.A. Brace, Dose-dependent effects of angiotensin II on the ovine fetal cardiovascular system, Am. J. Obstet. Gynecol. 165 (1991) 1524 – 1533. [17] S.C. Langley-Evans, R.C. Sherman, S.J. Welham, M.O. Nwagwu, D.S. Gardner, A.A. Jackson, Intrauterine programming of hypertension: the role of the renin – angiotensin system, Biochem. Soc. Trans. 27 (1999) 88 – 93. [18] J.M. Neutel, D.H. Smith, Hypertension control: multifactorial contributions, Am. J. Hypertens. 12 (1999) 164S – 169S. [19] N. Rowland, A. Rozelle, P. Riley, M. Fregly, Effect of nonpeptide angiotensin receptor antagonists on water intake and salt appetite in rats, Brain Res. Bull. 29 (1992) 389 – 393. [20] S.R. Siegel, G. Oaks, S. Palmer, Effects of angiotensin II on blood pressure, plasma renin activity, and aldosterone in the fetal lamb, Dev. Pharmacol. Ther. 3 (1981) 144 – 149. [21] K.M. Stevenson, K.J. Gibson, E.R. Lumbers, Effects of losartan on the cardiovascular system, renal haemodynamics and function and lung liquid flow in fetal sheep, Clin. Exp. Pharmacol. Physiol. 23 (1996) 125 – 130.
176
Z. Xu et al. / Developmental Brain Research 145 (2003) 169–176
[22] E. Stricker, The renin – angiotensin system and thirst: a reevaluation: II. Drinking elicited in rats by caval ligation or isoproterenol, J. Comp. Physiol. Psychol. 91 (1977) 1220 – 1231. [23] C.A. Williams, S.D. Loyd, T.A. Hampton, D.B. Hoover, Expression of c-fos-like immunoreactivity in the feline brainstem in response to isometric muscle contraction and baroreceptor reflex changes in arterial pressure, Brain Res. 852 (2000) 424 – 435. [24] J.W. Wright, J.W. Harding, Regulatory role of brain angiotensin in the control of physiological and behavioral responses, Brain Res Rev. 17 (1992) 227 – 262. [25] Z. Xu, J. Herbert, Regional suppression by water intake of c-fos
expression induced by intraventricular infusions of angiotensin II, Brain Res. 659 (1994) 157 – 168. [26] Z. Xu, A.K. Johnson, Intracerebroventricular renin-produced endogenous angiotensin induces drinking, FOS and egr-1 in the forebrain and brainstem of rat, Brain Res. 782 (1998) 24 – 35. [27] Z. Xu, G. Calvario, J. Yao, L. Day, M.G. Ross, Central angiotensin induction of fetal brain c-fos expression and swallowing activity, Am. J. Physiol. 280 (2001) R1837 – R1843. [28] G.Q. Zhu, K.P. Patel, I.H. Zucker, W. Wang, Microinjection of ANG II into paraventricular nucleus enhances cardiac sympathetic afferent reflex in rats, Am. J. Physiol. 282 (2002) H2039 – H2045.
Research report
In utero development of central ANG-stimulated pressor response and hypothalamic fos expression Zhice Xu *, Lijun Shi, Fang Hu, Rodney White, Lauren Stewart, Jiaming Yao Department of Obstetrics and Gynecology and Division of Vascular Surgery, Harbor-UCLA Medical Center and Research and Education Institute, 1124 West Carson Street, RB-1, Torrance, CA 90502, USA Accepted 24 July 2003
Abstract Central renin – angiotensin system (RAS) is as important as the peripheral RAS in the control of the cardiovascular homeostasis in the adult. However, previous fetal studies on angiotensin II (ANG II)-induced cardiovascular responses focused exclusively on the peripheral side. Thus, few data exist characterizing the in utero development of central angiotensin-mediated pressor responses. The present study determined cardiovascular responses to central application of ANG II in the chronically prepared near-term ovine fetus, and determined the action sites marked by c-fos expression in the fetal hypothalamus following intracerebroventricular (icv) injection of ANG II in utero. ANG II significantly increased fetal systolic, diastolic, and mean arterial pressure (MAP) within 5 min after injection of this peptide into the brain. Adjusted fetal MAP against amniotic pressure was also increased by icv ANG II, associated with increased c-fos in the central putative cardiovascular area—the paraventricular nuclei (PVN). Application of ANG II also induced intense c-fos expression in the supraoptic nuclei (SON), accompanied by a significant increase of fetal plasma vasopressin (AVP) levels, while maternal blood pressure (BP) and plasma AVP concentration were not changed. These results indicate that the central ANG II-mediated pressor response is functional at the last third of gestation, acting at the sites consistent with the cardiovascular neural network in the hypothalamus. D 2003 Elsevier B.V. All rights reserved. Theme: Endocrine and autonomic regulation Topic: Cardiovascular regulation Keywords: Angiotensin II; Pressor response; c-fos; Fetal brain development; AVP
1. Introduction Recent progress has been made in demonstrating that the development of regulatory mechanisms for cardiovascular homeostasis may start during fetal life and that the in utero development of cardiovascular mechanisms is important for fetal and ultimately adult blood pressure (BP). There are numerous studies that have demonstrated the importance of renin – angiotensin system (RAS) in regulation of BP [1,17,18]. A major function of angiotensin (ANG) in the healthy fetus appears to be maintenance of fetal arterial pressure under conditions of fetal stress, such as hemorrhage or hypoxia [13,14]. However, investigation of the development of the brain RAS and its role in the control of BP in
* Correspondence author. Tel.: +1-310-222-8179; fax: +1-310-2224131. E-mail address: [email protected] (Z. Xu). 0165-3806/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0165-3806(03)00226-8
utero has been limited. Although it is well known that the RAS plays an important role in the central nervous systems mediated cardiovascular responses, it is noted that previous work performed in the fetus focused exclusively on actions of ANG at the peripheral side in the control of BP at the developmental stages [7,8,12,16,20]. However, in the course of conducting experiments in our laboratory on the capacity of ANG II to stimulate fetal swallowing activity, we frequently saw cardiovascular responses after injection of this peptide into the lateral ventricle of the near-term ovine brain in utero [27] This observation prompted these initial experiments investigating the cardiovascular action of fetal intracerebroventricular (icv) injection of octapeptide ANG II. Administration of either ANG II or its precursors into the brain produces a pressor response, induces drinking behavior, and causes the release of adrenocorticotropic hormone and vasopressin (AVP) [2,9,22]. In the adult, there is significant information regarding the effects of central ANG [9]. Intracerebroventricular ANG II has been repeat-
170
Z. Xu et al. / Developmental Brain Research 145 (2003) 169–176
fied with histological analysis after sacrificing the animal. An intrauterine catheter was inserted for measuring amniotic fluid pressure. The fetus was then returned into the uterus, and the hysterotomy was closed in two layers. All catheters were externalized to the maternal flank and placed in a cloth pouch. Catheters were filled with heparinized saline, and the animals were treated with antibiotics [27], and given 5– 6 days for postoperative recovery.
edly shown to produce a reliable pressor response in adult animals. Furthermore, administration of ANG II icv also induces neuroendocrinological responses (e.g., vasopressin release) [3,20]. However, as mentioned above, few data exist regarding effects of central action of ANG on the cardiovascular system in utero. As a result, knowledge concerning the development of central mechanisms for ANG-regulated cardiovascular homeostasis is very limited. The present study determined the effects of central ANG II on fetal pressor responses in utero and determined whether central ANG can activate the brain pathways in the near-term fetus. Information gained increases our understanding of the ontogeny of central ANG mechanisms for cardiovascular homeostasis and provides information to the development of central RAS-related cardiovascular neural network in the early stages of life.
2.2. Cardiovascular experiments Studies began with a baseline ( 120 – 0 min) followed by study period (0 – 100 min). There were two groups (control: n = 5; experimental: n = 5). Beginning at time 0, ANG II (1.5 Ag/kg, 1 ml) (Sigma) in isotonic saline was injected icv into the fetus over 5 min. For the control animals, isotonic saline was injected. Throughout the study, maternal and fetal systolic and diastolic pressure, amniotic pressure, and heart rate were monitored continuously. The fetal mean arterial pressure (MAP) was corrected for amniotic cavity pressure. Fetal and maternal BP was measured by means of a Beckman R612 (Beckman Instruments, Fullerton, CA) physiological recorder with Statham (Garret, Oxnard, CA) P23 transducers. BP and heart rate was determined by computer analysis of waveforms utilizing a customized pattern recognition algorithm.
2. Material and methods 2.1. Surgical preparation Time-dated pregnant ewes (Nebeker) with singleton fetuses (130 F 3 days gestation at test) were used. Experimental protocols have been approved by the Institute Animal Care Committee. Ketamine hydrochloride (20 mg/ kg, i.m.) was used for anesthesia and general anesthesia was maintained with 5% isoflurane and 1 l/min oxygen. The uterus was exposed by a midline abdominal incision, and a small hysterotomy was performed to provide access to a fetal hindlimb and head. Polyethylene catheters were placed in the maternal and fetal femoral vein and artery and threaded to the inferior vena cava and abdominal aorta, respectively. An intracranial cannulae was placed in the fetal lateral ventricle and held in place with dental cement. The coordinates were anterior –posterior: + 0.1 cm in front of the bregma; medial – lateral: 0.8 cm from the middle line; and ventral: 1.8 cm below the dura. Patency of the catheter at insertion was assessed by free flow of cerebrospinal fluid via gravity drainage. The placement of cannulae was veri-
2.3. Endocrine experiments Maternal and fetal blood samples were collected and assessed for hematocrit, pH, PO2, and PCO2, remaining blood was centrifuged and plasma osmolality, sodium, chloride, and potassium concentrations were measured as reported [27]. Plasma samples were used for AVP radioimmunoassay. The sensitivity of the AVP antiserum is 0.8 pg/tube with intra-assay and interassay coefficients of variation of 6% and 9%, respectively. Plasma AVP recoveries average 70%. Blood PO2, PCO2, pH, hemoglobin (Hb) were measured with a Radiometer BM 33 MK2-PHM 72 MKS acid – base analyzer system (Radiometer, Copenhagan). Plasma osmo-
Table 1 Effects of icv ANG II (1.5 Ag/kg) on maternal (upper panel) and fetal (lower panel) cardiovascular responses 60 min Systolic pressure (mm Hg) Diastolic pressure (mm Hg) MAP (mm Hg) Heart rate (beats/min)
100 F 14 62 F 8 80 F 11 106 F 7
Systolic pressure (mm Hg) Diastolic pressure (mm Hg) MAP (mm Hg) Adjusted MAP (mm Hg) Heart rate (beats/min)
56 F 7 43 F 6 49 F 6 42 F 1 153 F 5
60 min
* p < 0.01.
30 min 98 F 14 63 F 5 81 F 11 108 F 10 30 min 55 F 5 44 F 5 49 F 4 43 F 1 149 F 13
5 min
15 min
30 min
60 min
96 F 14 63 F 8 79 F 11 103 F 6
103 F 8 64 F 6 83 F 6 102 F 9
103 F 11 64 F 6 83 F 8 104 F 14
102 F 10 61 F 7 82 F 8 99 F 11
5 min
15 min
30 min
60 min
90 min
66 F 9* 52 F 8* 59 F 7* 52 F 2* 140 F 17
66 F 3* 53 F 8* 60 F 5* 53 F 3* 142 F 24
66 F 7* 53 F 8* 59 F 6* 53 F 1* 132 F 46
62 F 5 48 F 6 57 F 4 51 F 41 140 F 29
58 F 5 45 F 5 51 F 3 49 F 1 142 F 22
Z. Xu et al. / Developmental Brain Research 145 (2003) 169–176
171
Fig. 1. The effect of icv injection of vehicle or ANG II on fetal MAP (mm Hg). 0 min: time for icv injection. *p < 0.01. icv: intracerebroventricular. MAP: mean arterial pressure.
lality was measured on an Advanced Digimatic osmometer (model 3 MO, Advanced Instruments, Needham Heights, MA). Plasma sodium and potassium concentrations were determined by a Nova 5 electrolyte analyzer (Nova Biomedical, Waltham, MA). 2.4. Immunohistochemistry experiments The animals were anesthetized and perfused, and the fetal brain was collected at the end of the cardiovascular testing. Twenty-micrometer coronal sections of fetal forebrain were cut on a cryostat. The sections of the hypothalamus were used for FOS immunoreactivity (FOS-ir) staining using the avidin – biotin-peroxidase technique. The tissue sections were first incubated in the primary antibody (1:20,000, Santa Cruz Biotech, CA) overnight, then incubated in goat antirabbit serum (1:500) and processed using the ABC kit (Vector Labs, Burlingame). The tissue sections were treated with 1 mg/ml diaminobenzidine tetrahydrochloride. 2.5. Data analysis All signals in cardiovascular studies were digitized at 500 Hz and recorded on a computer with Win-DAQ acquisition software (Data Q instruments, Akron). Heart rate, systolic and diastolic pressure, and MAP were calculated from the pressure waveforms by means of Advanced CODAS software. The number of FOS-ir positive cells in the supraoptic and paraventricular nuclei (SON and PVN) was evaluated in a qualitative and blinded manner. Statistical analysis was preformed with repeated-measures ANOVA. Comparisons before and after treatments were determined with one-way ANOVA followed by Tukey test or t-test. p < 0.05 or 0.01 was the probability level used to define statistical significance. All data were expressed as mean F S.E.M.
3. Results 3.1. Cardiovascular responses Histological analysis confirmed that all icv cannulae were inserted into the fetal lateral ventricle. There was no significant difference between the control and experimental groups for maternal systolic pressure ( F8,1 = 0.05), diastolic pressure ( F8,1 = 0.24), and MAP ( F8,1 = 0.01, all p: no significance) (Table 1). However, icv injection of ANG II into the fetus significantly increased fetal BP. Fetal systolic, diastolic pressure, and MAP were increased in the experimental group as compared to the control fetuses
Table 2 Maternal (upper panel) and fetal (lower panel) arterial values before and after icv injection of ANG II (1.5 Ag/kg) Baseline
15 min
30 min
60 min
pH 7.46 F 1.15 7.48 F 2.70 7.45 F 3.63 7.47 F 1.94 PCO2 (Torr) 34.7 F 1.1 35.2 F 1.6 37.4 F 3.9 35.8 F 2.6 PO2 (Torr) 113.1 F 6.2 112.3 F 9.0 108.6 F 9.8 111.4 F 10.3 Osmolality 301.2 F 2.3 301.4 F 4.1 300.4 F 4.5 300.6 F 3.5 (mOsm/kg) Na+ (meq/l) 145.6 F 3.5 147.4 F 1.5 146.6 F 2.8 146.3 F 1.8 K+ (meq/l) 4.0 F 0.2 4.1 F 0.2 4.0 F 0.2 4.0 F 0.2 Hb (g/dl) 8.1 F 1.1 8.3 F 0.4 7.9 F 0.8 7.9 F 1.2 Baseline
15 min
30 min
60 min
pH 7.37 F 0.21 7.38 F 0.24 7.37 F 0.27 7.37 F 0.20 PCO2 (Torr) 51.1 F 4.5 48.8 F 3.9 50.3 F 4.6 50.2 F 3.6 PO2 (Torr) 20.3 F 2.6 20.8 F 1.3 20.4 F 1.3 22.0 F 2.4 Osmolality 296.2 F 5.4 297.1 F 3.3 297.4 F 2.3 295.3 F 4.1 (mOsm/kg) Na+ (meq/l) 140.2 F 2.4 141.3 F 2.6 140.2 F 1.7 141.3 F 3.3 K+ (meq/l) 4.1 F 0.6 4.0 F 0.6 4.1 F 0.6 3.9 F 4.2 Hb (g/dl) 8.4 F 1.7 8.0 F 1.2 8.3 F 1.8 8.1 F 1.5
172
Z. Xu et al. / Developmental Brain Research 145 (2003) 169–176
( F8,1 = 5.70, 7.13, and 12.26, respectively, p < 0.01). There was significant difference for adjusted fetal MAP between the animals treated with icv ANG II and icv vehicle ( F8,1 = 11.09, p < 0.01). In the experimental animals, fetal MAP was increased from the baseline level, 49 F 4 mm Hg, to 60 F 5 mm Hg 15 min after icv injection of ANG II ( p < 0.01). This was a 20% increase from the baseline MAP to the peak level MAP. The increased MAP lasted for at least 30 min (Fig. 1). The fetal MAP, adjusted fetal MAP, systolic and diastolic pressure still remained at higher levels above the baseline ( F = 7.62, 8.94, and 3.76, respectively, p < 0.01 for both MAP and adjusted MAP, p < 0.05 for diastolic pressure) for 30 min after administration of ANG II (Table 1). In the control animals, icv injection of the vehicle had no effect on fetal systolic, diastolic pressure, MAP, and adjusted MAP in the nearterm fetus (Tukey test, all p: no significance). Maternal
heart rate was not changed by the icv injection of ANG II into the fetus ( F8,1 = 1.69, p: no significance). Fetal heart rate was not significantly changed in the control and the experimental animals (Tukey test, p: no significance). 3.2. Blood values For both the control and the experimental animals, icv injection of ANG II or vehicle had no effect on plasma osmolality, Cl , Na+, and K+ concentrations in either maternal or fetal animals. There was no significant difference for arterial blood pH, PO2, PCO2, Na+, K+, osmolality, hemoglobin, and hematocrit before and after icv injection of ANG II (all p: no significance). All arterial values were within normal ranges and did not vary significantly between the control and the experimental groups (all p: no significance) (Table 2).
Fig. 2. The effect of icv injection of vehicle or ANG II on fetal and maternal plasma AVP levels. 0 min: time for icv injection. *p < 0.01. icv: intracerebroventricular. AVP: vasopressin.
Z. Xu et al. / Developmental Brain Research 145 (2003) 169–176
173
Fig. 3. FOS-ir induced by icv ANG II in the fetal SON (upper panel) and PVN (bottom panel). A and a: the animal injected with icv ANG II; B and b: the animal treated with icv CSF. OC: Optic chiasma. 3V: Third ventricle. SON: supraoptic nuclei, PVN: paraventricular nuclei. 40 .
3.3. Plasma AVP assay There was no change in plasma AVP levels between the control and the experimental ewes ( F7,1 = 1.76, p: no significance). However, the fetal AVP concentrations were significantly higher in the icv ANG II injected fetuses than in the icv vehicle-treated animals ( F7,1 = 7.71, p < 0.05). In the control group, icv injection of vehicle did not change fetal plasma AVP ( F19,3 = 0.34, p: no significance, the
baseline period vs. the period after icv injection). The icv ANG II significantly increased fetal plasma AVP levels ( F19,3 = 4.01 p < 0.05, the baseline period vs. the period after icv injection) (Fig. 2). Fetal plasma AVP increased about sixfold at 15 min after icv ANG II, and the peak level of plasma AVP was observed at 30 min (ninefold) after icv injection of ANG II. Plasma fetal AVP concentrations dropped at 60 min, although the level was still higher than that at the baseline.
Fig. 4. The effect of icv injection of vehicle or ANG II on FOS-ir in the SON and PVN in the near-term ovine fetus. *p < 0.01. icv: intracerebroventricular. SON: supraoptic nuclei, PVN: paraventricular nuclei.
174
Z. Xu et al. / Developmental Brain Research 145 (2003) 169–176
3.4. FOS-immunoreactivity In the control fetuses following injection with icv vehicle, there was little FOS-ir in the fetal hypothalamic structures, including the putative pressor nuclei PVN and the SON. However, icv injection of ANG II produced FOS-ir in the hypothalamus of the fetus. Intense FOS-ir was observed in the SON and PVN (Fig. 3). There were significant differences of FOS-ir between icv vehicle and icv ANG II-injected fetuses (t = 8.2; p < 0.05) (Fig. 4). ANG II-induced FOS-ir in the PVN was detected in both magnocellular and parvicellular parts.
4. Discussion The present study demonstrated that icv application of ANG II could produce pressor responses in the near-term ovine fetus. Intracerebroventricular ANG II not only increased fetal MAP in utero, but also stimulated the neural activity in the central cardiovascular centers in utero. The finding that c-fos expression was induced by icv ANG II in the fetal cardiovascular centers and AVP secretion nuclei indicates that the central RAS is functional before birth. Thus, the central ANG systems for cardiovascular homeostasis are likely to be intact before birth. Previous studies have shown that central ANG-produced pressor responses occur reliably within the first 5 – 8 min after administration of ANG II in adult models [24]. Our study demonstrated that fetal systolic pressure, diastolic pressure, and MAP significantly increased within 5 min after icv injection of ANG II. Therefore, the latency for icv ANG II-stimulated pressor responses in the near-term ovine fetus is similar to that observed in the adult. ANG II is a potent vasoconstrictive hormone in the fetus and adult. Earlier work has shown that an active RAS is present in the ovine fetus before birth [5,12]. Its major function appears to be maintenance of fetal arterial pressure under conditions of fetal stress, including acute hemorrhage or hypoxia [16]. However, previous fetal studies on ANGregulated cardiovascular responses focused exclusively on the action of this peptide at the peripheral side. This study demonstrated that the brain RAS in the near-term fetus is functional and important in the control of pressor responses. In the present study, increased fetal systolic, diastolic, and mean arterial pressures were maintained for at least 30 min before they returned to the baseline. Biological effects of ANG II are mediated via the ANG receptors. ANG receptors have been demonstrated in both fetal and adult brains [9,19,21]. This study provides evidence that the fetal ANG receptors are functional in the mediation of cardiovascular regulation at near-term. Fetal physiological status remained stable during the testing periods, as did arterial values, including PCO2, PO2, pH, plasma electrolytes, hematocrit, and hemoglobin, following icv injections. The lack of change of plasma electro-
lytes, particularly sodium and osmolality, indicated that systemic sodium/osmolality did not affect cardiovascular responses in the present study. Maternal systolic and diastolic pressure and MAP were not influenced by the administration of icv ANG II into the fetus. Maternal plasma AVP levels were not affected by application of ANG II in the fetal animals either. These data suggest that icv injection of ANG II into the fetus has no effect on the maternal animals. A number of studies support the concept that there are two interacting systems in the brain responsible for ANG-increased blood pressure: autonomic and hormonal mechanisms. Several hormonal factors, including AVP, may contribute to ANG-mediated pressor responses. Intracerebroventricular ANG increases plasma AVP, a potent vasoconstrictor, in both rat and sheep [9]. Infusion of V1-AVP receptor antagonist, AVP antibodies, and hypophysectomy, have shown to reduce, but not eliminate, icv ANG II-induced pressor responses [5,10]. Moreover, rats with hereditary hypothalamic diabetes insipidus fail to show a blood pressure response to icv application of ANG II [11]. Thus, AVP mechanisms are partially involved in central ANG-induced pressor responses. In the present study, fetal plasma AVP levels were significantly increased following icv injection of ANG II, and increased fetal AVP concentrations lasted for about 30 min before returning to the baseline. Moreover, intense c-fos was expressed in the SON and PVN following icv ANG II in the fetal brain. Since the SON and PVN are the main source for the plasma AVP [6], activation of a large number of cells in the hypothalamus associated with a significant increase of fetal plasma AVP levels, in the present study, strongly indicates that AVP-containing neurons in these nuclei may be activated after icv injection of ANG II in the near-term fetus. As mentioned above, autonomic mechanisms are another major contribution to the increased fetal blood pressure by icv injection of ANG II [5]. Many brain nuclei and pathways that process signals for the control of cardiovascular responses have been identified by employing functional analysis and anatomical tract tracing techniques. Presumably, every region and pathway in this network can potentially influence signal integration in the pressor net. However, certain nuclei have demonstrated to be particularly critical to the maintenance of arterial pressure. Most notable among these are the nuclei in the hypothalamusPVN. The present study focused specifically on the action sites of central ANG in the fetal hypothalamus. The results showed that icv ANG II induced FOS-ir in the PVN and SON, as evidence that the areas in the hypothalamus were activated, and consistent with the previous adult studies [25,26]. Associated with the increased blood pressure and plasma AVP levels in the same fetal animals, the c-fos expression suggests that the critical areas in the CNS like the PVN for ANG-mediated cardiovascular mechanisms are functional during the last third of the gestation. Exogenous ANG II in the PVN augments the cardiac sympathetic
Z. Xu et al. / Developmental Brain Research 145 (2003) 169–176
afferent reflex evoked by stimulation of cardiac sympathetic afferent nerves [28]. In addition, the PVN is replete with ANG receptor, especially AT1 subtype receptors, which may account for the biological actions of the octapeptide [25]. In the present study, the neural activity marked with cfos in the PVN, in both magnocellular and parvicellular parts, suggests that the ANG receptors in the fetal hypothalamus have matured enough in response to ANG II stimulation. The present study is the first to demonstrate icv ANG IIstimulated c-fos expression in the fetal hypothalamus. These results provide evidence that the near-term fetal hypothalamus is functional in the brain RAS in the face of ANG stimulation. Although there is a possibility that the increased fetal blood pressure and baroreflex may induce c-fos expression in the brain under certain conditions [23], c-fos expression induced by increased blood pressure and/or baroreflex mainly locates in the hindbrain. Particularly, in the tractus solitarius nuclei (NTS), the lateral parabrachial nucleus (LPBN), and the rostral ventrolateral medulla (RVLM) [4,15,23]. The patterns of FOS-ir in the fetal PVN and SON following icv injection of ANG II were similar to that in previous observations in the adult hypothalamus under the same condition [25]. In addition, both maternal and fetal heart rates were not significantly changed following injection of ANG II. This suggests that it was unlikely that c-fos expression in the present study was due to baroreflex. There are two important results in the present study: ANG II in the fetal brain can reliably increase fetal blood pressure, and c-fos expression is observed in the fetal hypothalamus in utero. These results provide evidence that the putative cardiovascular centers, especially the critical areas like the PVN (i.e., the part of ‘‘sympathetic’’ neural network), and the SON (i.e., the main source of plasma AVP), are functional in the last third of gestation. It is apparent that brain ANG-mediated cardiovascular homeostasis is intact before birth. Considering that a major function of ANG II in utero appears to be maintenance of fetal BP under condition of fetal stress [13,14], and that recent progress in demonstrating that the development of regulatory mechanisms for cardiovascular homeostasis in utero may also be important to adult BP, the data gained in the present study provide useful information to the development of the central regulatory system related to ANGstimulated pressor responses starting during fetal life.
Acknowledgements These studies were conducted at the biomedical research facilities of the Research and Education Institute at HarborUCLA Medical Center. Research described in this article is supported by March of Dimes Research Grant, External Research Grant from Philip Morris, USA, Inc. and UCLA FGP Award to Z. Xu.
175
References [1] K. Allikmets, T. Parik, M. Viigimaah, The renin – angiotensin system in essential hypertension: associations with cardiovascular risk, Blood Press. 8 (1991) 70 – 78. [2] E. Badoer, Hypothalamic paraventricular nucleus and cardiovascular regulation, Clin. Exp. Pharmacol. Physiol. 28 (2001) 95 – 99. [3] J.R.G. Challis, B.F. Mitchell, Endocrinology of pregnancy and parturition, in: J.B. Warshaw (Ed.), The Biological Basis of Reproductive and Developmental Medicine, Elsevier, New York, 1983, p. 106. [4] R.K. Chan, P.E. Sawchenko, Organization and transmitter specificity of medullary neurons activated by sustained hypertension: implications for understanding baroreceptor reflex circuitry, J. Neurosci. 18 (1998) 371 – 387. [5] K.E. Clark, G.L. Irion, C.E. Mack, Differential responses of uterine and umbilical vasculatures to angiotensin II and norepinephrine, Am. J. Physiol. 259 (1990) H197 – H203. [6] K. Dierickx, F. Vandesande, Immunocytochemical demonstration of separate vasopressin – neurophysin and oxytocin – neurophysin neurons in the human hypothalamus, Cell Tissue Res. 19 (1979) 203 – 212. [7] L.J. Edwards, G. Simonetta, J.A. Owens, J.S. Robinson, I.C. McMillen, Restriction of placental and fetal growth in sheep alters fetal blood pressure responses to angiotensin II and captopril, J. Physiol. (London) 515 (1999) 897 – 904. [8] J.J. Faber, D.F. Anderson, Hydrops fetalis in nephrectomized fetal lambs infused with angiotensin I, Am. J. Physiol. 267 (1994) R1522 – R1527. [9] J.T. Fitzsimons, Angiotensin, thirst, and sodium appetite, Physiol. Rev. 78 (1998) 583 – 686. [10] D. Haack, J. Mohring, Vasopressin-mediated blood pressure response to intraventricular injection of angiotensin II in the rat, Pflugers Arch. 373 (1978) 167 – 173. [11] J.S. Hutchinson, P. Schelling, J. Mohring, D. Ganten, Pressor action of centrally perfused angiotensin II in rats with hereditary hypothalamic diabetes insipidus, Endocrinology 99 (1976) 819 – 823. [12] H.S. Iwamoto, A.M. Rudolph, Effects of endogenous angiotensin II on the fetal circulation, J. Dev. Physiol. 1 (1979) 283 – 293. [13] H.S. Iwamoto, A.M. Rudolph, Role of renin – angiotensin system in response to hemorrhage in fetal sheep, Am. J. Physiol. 240 (1981) H848 – H854. [14] H.S. Iwamoto, A.M. Rudolph, L.C. Keil, M.A. Heymann, Hemodynamic responses of the sheep fetus to vasopressin infusion, Circ. Res. 44 (1979) 430 – 436. [15] J.H. Jhamandas, K.H. Harris, T. Petrov, H.Y. Yang, K.H. Jhamandas, Activation of neuropeptide FF neurons in the brainstem nucleus tractus solitarius following cardiovascular challenge and opiate withdrawal, J. Comp. Neurol. 14 (1998) 210 – 221. [16] O.W. Jones, C.Y. Cheung, R.A. Brace, Dose-dependent effects of angiotensin II on the ovine fetal cardiovascular system, Am. J. Obstet. Gynecol. 165 (1991) 1524 – 1533. [17] S.C. Langley-Evans, R.C. Sherman, S.J. Welham, M.O. Nwagwu, D.S. Gardner, A.A. Jackson, Intrauterine programming of hypertension: the role of the renin – angiotensin system, Biochem. Soc. Trans. 27 (1999) 88 – 93. [18] J.M. Neutel, D.H. Smith, Hypertension control: multifactorial contributions, Am. J. Hypertens. 12 (1999) 164S – 169S. [19] N. Rowland, A. Rozelle, P. Riley, M. Fregly, Effect of nonpeptide angiotensin receptor antagonists on water intake and salt appetite in rats, Brain Res. Bull. 29 (1992) 389 – 393. [20] S.R. Siegel, G. Oaks, S. Palmer, Effects of angiotensin II on blood pressure, plasma renin activity, and aldosterone in the fetal lamb, Dev. Pharmacol. Ther. 3 (1981) 144 – 149. [21] K.M. Stevenson, K.J. Gibson, E.R. Lumbers, Effects of losartan on the cardiovascular system, renal haemodynamics and function and lung liquid flow in fetal sheep, Clin. Exp. Pharmacol. Physiol. 23 (1996) 125 – 130.
176
Z. Xu et al. / Developmental Brain Research 145 (2003) 169–176
[22] E. Stricker, The renin – angiotensin system and thirst: a reevaluation: II. Drinking elicited in rats by caval ligation or isoproterenol, J. Comp. Physiol. Psychol. 91 (1977) 1220 – 1231. [23] C.A. Williams, S.D. Loyd, T.A. Hampton, D.B. Hoover, Expression of c-fos-like immunoreactivity in the feline brainstem in response to isometric muscle contraction and baroreceptor reflex changes in arterial pressure, Brain Res. 852 (2000) 424 – 435. [24] J.W. Wright, J.W. Harding, Regulatory role of brain angiotensin in the control of physiological and behavioral responses, Brain Res Rev. 17 (1992) 227 – 262. [25] Z. Xu, J. Herbert, Regional suppression by water intake of c-fos
expression induced by intraventricular infusions of angiotensin II, Brain Res. 659 (1994) 157 – 168. [26] Z. Xu, A.K. Johnson, Intracerebroventricular renin-produced endogenous angiotensin induces drinking, FOS and egr-1 in the forebrain and brainstem of rat, Brain Res. 782 (1998) 24 – 35. [27] Z. Xu, G. Calvario, J. Yao, L. Day, M.G. Ross, Central angiotensin induction of fetal brain c-fos expression and swallowing activity, Am. J. Physiol. 280 (2001) R1837 – R1843. [28] G.Q. Zhu, K.P. Patel, I.H. Zucker, W. Wang, Microinjection of ANG II into paraventricular nucleus enhances cardiac sympathetic afferent reflex in rats, Am. J. Physiol. 282 (2002) H2039 – H2045.