AJP - Regu Information on EB 2010
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Regul Integr Comp Physiol 288: R1014-R1020, 2005. First published November 18, 2004; doi:10.1152/ajpregu.00479.2003
0363-6119/05 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
288/4/R1014    most recent
00479.2003v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Web of Science (1)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by El-Haddad, M. A.
Right arrow Articles by Ross, M. G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by El-Haddad, M. A.
Right arrow Articles by Ross, M. G.

DEVELOPMENTAL PHYSIOLOGY AND PREGNANCY

Central angiotensin II AT1 receptors mediate fetal swallowing and pressor responses in the near-term ovine fetus

Mostafa A. El-Haddad, Yaser Ismail, Dave Gayle, and Michael G. Ross

Perinatal Research Center, David Geffen UCLA Medical School, Research and Educational Institute, Harbor-UCLA Medical Center, Torrance, California

Submitted 22 August 2003 ; accepted in final form 11 November 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Swallowed volumes in the fetus are greater than adult values (per body weight) and serve to regulate amniotic fluid volume. Central ANG II stimulates swallowing, and nonspecific ANG II receptor antagonists inhibit both spontaneous and ANG II-stimulated swallowing. In the adult rat, AT1 receptors mediate both stimulated drinking and pressor activities, while the role of AT2 receptors is controversial. As fetal brain contains increased ANG II receptors compared with the adult brain, we sought to investigate the role of both AT1 and AT2 receptors in mediating fetal swallowing and pressor activities. Five pregnant ewes with singleton fetuses (130 ± 1 days) were prepared with fetal vascular and lateral ventricle (LV) catheters and electrocorticogram and esophageal electromyogram electrodes and received three studies over 5 days. On day 1 (ANG II), following a 2-h basal period, 1 ml artificial cerebrospinal fluid (aCSF) was injected in the LV. At time 4 h, ANG II (6.4 µg) was injected in the LV, and the fetus was monitored for a final 2 h. On day 3, AT1 receptor blocker (losartan 0.5 mg) was administered at 2 h, and ANG II plus losartan was administered at 4 h. On day 5, AT2 receptor blocker (PD-123319; 0.8mg) was administered at 2 h and ANG II plus PD-123319 at 4 h. In the ANG II study, LV injection of ANG II significantly increased fetal swallowing (0.9 ± 0.1 to 1.4 ± 0.1 swallows/min; P < 0.05). In the losartan study, basal fetal swallowing significantly decreased in response to blockade of AT1 receptors (0.9 ± 0.1 to 0.4 ± 0.1 swallows/min; P < 0.05), while central injection of ANG II in the presence of AT1 receptor antagonism did not increase fetal swallowing (0.6 ± 0.1 swallows/min). In the PD-123319 study, basal fetal swallowing did not change in response to blockade of AT2 receptor (0.9 ± 0.1 swallows/min), while central injection of ANG II in the presence of AT2 blockade significantly increased fetal swallowing (1.5 ± 0.1 swallows/min; P < 0.05). ANG II caused significant pressor responses in the control and PD-123319 studies but no pressor response in the presence of AT1 blockade. These data demonstrate that in the near-term ovine fetus, AT1 receptor but not AT2 receptors accessible via CSF contribute to dipsogenic and pressor responses.

ovine fetus; angiotensin receptors; swallowing

IN THE NEAR-TERM OVINE FETUS, fetal swallowing contributes importantly to amniotic fluid volume regulation and gastrointestinal tract development (20, 27, 47). The near-term ovine fetus demonstrates an intact swallowing response to central dipsogenic stimuli, including hypertonicity (39) and ANG II (40). Furthermore, tonic central ANG II activity contributes to spontaneous swallowing activity, as nonselective blockade of central ANG II receptors attenuated both basal as well as ANG II-mediated fetal swallowing activities (16).

In adult animals, brain-derived ANG II is a potent stimulant of water and sodium intake and an important regulator of blood pressure (21, 37, 51). The recent development of specific, highly selective, nonpeptide ANG II-receptor antagonists have identified two major ANG II-receptor subtypes, AT1 and AT2. In adult animals of several species, neuronal AT1 receptors found in the lamina terminalis, hypothalamic paraventricular nucleus, and the nucleus tractus solitarius mediate the dipsogenic, pressor, and arginine vasopressin (AVP)-releasing actions of ANG II (13, 19, 30). The AT2 receptor is expressed differently from AT1 receptor, as it is found mainly in the cerebellum and thalamus (1–3). AT2 receptors may contribute importantly to fetal development, they are highly expressed during fetal development, and expression declines rapidly after birth (29, 48). Although little is known of the function of AT2 receptors, several (22, 23) although not all studies (8, 49) have suggested that AT2 and AT1 receptors act synergistically to augment water intake. Notably, the AT2 receptor also has been demonstrated to oppose the pressor effect of AT1 receptor stimulation (31, 44).

The role of ANG II receptor subtypes in mediating fetal swallowing and cardiovascular regulation has not been previously explored. In view of the fundamental differences in expression and localization of AT2 receptors in the fetal compared with adult brain, and the evidence that AT2 receptors may synergistically augment AT1-mediated adult drinking behavior, we sought to determine the specific roles of central AT1 and AT2 receptors in mediating spontaneous and ANG II-mediated fetal swallowing as well as cardiovascular regulation.


   MATERIAL AND METHODS
TOP
 ABSTRACT
 MATERIAL AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Five time-dated pregnant ewes with singleton fetuses (130 ± 1 days' gestation on the first study day) were studied. Western mixed-breed sheep were obtained from a local farm (Nebeker Farm, Palmdale, CA). Animals were housed indoors in individual steel study cages and acclimated to a 12:12-h light-dark cycle. Surgical procedures and studies were approved by the Harbor-University of California Los Angeles Animal Use Committee. Food (alfalfa pellets) and water were provided ad libitum, except that food was withheld for 24 h before surgery (water was available to the animal until the time of surgery).

Surgical preparation. Anesthesia was induced with ketamine hydrochloride (15–20 mg/kg im), and general anesthesia was maintained with 1% to 2% isoflurane and 1 l/min of oxygen. The uterus was exposed by a midline abdominal incision, and a small hysterotomy was performed to expose the fetal hindlimb. Polyethylene catheters with inner diameters of 0.040 in. and outer diameters of 0.070 in. were placed in the fetal femoral vein and artery and threaded to the inferior vena cava and abdominal aorta, respectively. Surgical placement of bipolar electromyography (EMG) electrodes (thyrohyoid muscle and upper and lower nuchal esophagus) for determination of swallowing activity was performed. Electrodes were also implanted on the parietal dura through two drilled burr holes (5 mm above the bregma and 10 mm from each side of the sagittal suture) for the determination of fetal electrocortical (ECoG) activity. An 18-gauge needle connected to polyethylene catheter (inner diameter, 0.02 in.; outer diameter, 0.04 in.) was inserted into the lateral ventricle that was identified 20 mm above the lambdoid suture and 5 mm lateral to the sagittal suture. The lateral ventricle needle and the dural electrodes were immobilized with dental cement and two stainless steel screws fixed in the skull. An intrauterine catheter (inner diameter, 0.125 in.; outer diameter, 0.25 in.; Corometrics Medical Systems, Wallingford, CT) was inserted for measurement of amniotic fluid pressure. Uterine and maternal incisions were repaired, and all catheters and electrodes were externalized to the maternal flank and placed in a cloth pouch. Catheters also were placed in the maternal femoral vein and artery. Animals were allowed at least 5 days for postoperative recovery, which included catheter maintenance and antibiotic administration (20).

Study protocol. The study protocol was conducted over a 5-day period. The effects of central artificial cerebrospinal fluid (aCSF) and ANG II on fetal swallowing were examined on the first day of the study. On days 3 and 5, central effects of the selective ANG II AT1 (losartan, 0.5 mg, 1 ml) and ANG II AT2 (PD-123319, 0.8 mg, 1 ml) receptor blockers, respectively, were examined. Each experiment was separated by a minimum of 36 h of rest. Studies were initiated with the ewes standing in the same individual cages in which they were housed before the study. An equilibration period included preparation of the animals for the experiments and confirmation that fetal arterial pH was >7.30. All doses were injected slowly over 5 min.

aCSF-ANG II study. Following a 2-h baseline period (time 0 to 2 h), aCSF (1 ml; 145 meq/l sodium; 3.3 meq/l potassium; 121 meq/l calcium; 2.2 meq/l magnesium; 121 meq/l chloride, 25 meq/l bicarbonate; osmolality, 297 mosmol/kgH2O) was injected into the lateral ventricle [intracerebroventricularly (ICV)], and the fetuses were monitored for 2 h (from 2 to 4 h). At time 4 h, ANG II (1 ml, 6.4 µg/ml) was injected ICV, and fetuses were monitored for a final 2 h.

Losartan study. After a 2-h baseline period, losartan dissolved aCSF (0.5 mg, 1 ml; Merck, NJ) was injected ICV. At time 4 h the dose of losartan was repeated together with ANG II (1 ml, 6.4 µg), and fetuses were monitored for a final 2 h.

PD-123319 study. After a 2-h baseline period, PD-123319 dissolved in normal saline (0.8 mg, 1 ml; Sigma, St Louis, MO) was injected ICV. At time 4 h the dose of PD-123319 was repeated together with ANG II (1 ml, 6.4 µg), and fetuses were monitored for a final 2 h.

Throughout the experiments, fetal swallowing and ECoG activities, fetal and maternal arterial blood pressures and heart rates, and amniotic fluid pressures were monitored continuously. Maternal and fetal blood samples (5 ml) were collected at hourly intervals for measurement of hematocrit, blood gases, and plasma osmolality, electrolytes, and AVP. Fetal blood samples were replaced with an equivalent volume of heparinized maternal blood withdrawn before the study, and maternal blood samples were replaced with an equivalent volume of 0.15 mol/l saline solution. Five-minute segments of fetal and maternal blood pressure and heart rate data were analyzed at 1 and 2 h (control period) and at 5, 10, 15, 30, 60, and 120 min after each drug injection.

Analytical methods. Maternal and fetal blood samples were collected into chilled iced tubes that contained 10 U/ml of lithium heparin. Blood aliquots were assessed for hematocrit, pH, PO2, and PCO2. The remaining blood was centrifuged, and plasma osmolality, sodium, chloride, and potassium concentrations were measured. Maternal and fetal plasma aliquots were stored at –20°C until assayed for arginine vasopressin (AVP).

Blood PO2, PCO2, and pH were measured at 39°C with a Nova Stat 3 blood gas analyzer system (Nova Biomedical, Waltham, Mass). Plasma osmolality was measured by freezing point depression on an Advanced Digimatic osmometer (model 3 MO, Advanced Instruments, Needham Heights, MA). Plasma sodium, potassium, and chloride concentrations were determined by use of a Nova 5 electrolyte analyzer (Nova Biomedical). Fetal and maternal blood pressures and amniotic pressures were measured by means of World Precision Instruments (Sarasota, FL) signal conditioners and transducers. Fetal blood pressure was corrected for amniotic cavity pressure.

For the analysis of plasma AVP, the samples were collected into an ice-chilled glass tube that contained 10 IU heparin and 500 IU aprotinin per milliliter of blood. Samples were extracted by use of a modification of the procedure of LaRochelle et al. (28), and AVP levels were determined with radioimmunoassay. Plasma AVP recoveries in our laboratory average 70%.

Data analysis. Digitization of all signals was performed at a rate of 75 Hz. EMG and ECoG activity signals were directed to a Grass physiological recorder and to a Windaq analog digital system (Dataq Instruments, Akron, OH). An EMG-propagated swallow, representing a coordinated laryngeal-esophageal contraction, was defined by a time sequence of integrated EMG signals from the thyrohyoid muscle to the upper and lower nuchal esophagus. The time at the onset of each swallow was stored for later analysis, and swallowing activity was expressed as swallows per minute.

Fetal ECoG activity was assessed by visual analysis and was divided into periods of low voltage and high voltage. Periods of ECoG activity that did not clearly belong to either low- or high-voltage activities were considered to be intermediate ECoG activity. Intermediate ECoG activity constituted <5% of the total ECoG activity and was not considered in the analysis of the data. Total swallowing activity was calculated as defined earlier and was expressed as swallows per minute.

Statistical analysis. For each animal, swallowing rate per minute, percentage of time spent in each ECoG activity state, and percentage of swallowing in each state was calculated for the control and drug exposure periods. Data of fetal swallowing and all other fetal and maternal parameters were analyzed using one-way repeated measures ANOVA with the Dunnett's test for post hoc analysis. All data are presented as mean values ± SE, and P < 0.05 was considered to be statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIAL AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Control aCSF-ANG II study. ICV injection of aCSF did not change total fetal swallowing (0.9 ± 0.2 to 1.0 ± 0.2 swallow/min), whereas ICV injection of ANG II significantly increased swallowing activity (1.4 ± 0.2 swallow/min; P < 0.05; Fig 1A). Fetal swallowing occurred primarily during low-voltage ECoG activity (control 1.2 ± 0.2; aCSF 1.2 ± 0.1; ANG II 1.9 ± 0.4 swallow/min during low-voltage ECoG activity; P < 0.05). Fetal swallowing during high-voltage ECoG activities were as follows: control 0.6 ± 0.1, aCSF 0.6 ± 0.1, and ANG II 0.8 ± 0.1 swallow/min (P < 0.01).



View larger version (14K):
[in this window]
[in a new window]
  		Fig. 1. A: total fetal total swallows per minute during the baseline period (control), after artificial cerebrospinal fluid (aCSF) injection at 2 h, and after ANG II injection at 4 h into the intracerebroventricular space (ICV) (*P < 0.05 vs. control). B: total fetal total swallows per minute during the baseline period (control), after selective ANG II AT1 receptor antagonist (losartan), and after combined injection of losartan and ANG II into the ICV space (*P < 0.05 vs. control). C: total fetal total swallows per minute during the baseline period (control), after AT2 receptor antagonist (PD-123319), and after combined injection of PD-123319 and ANG II and into the ICV space (*P < 0.05 vs. control).

 
The percentage of time spent in low-voltage ECoG activity was stable during the control period (52 ± 5%), after injection of aCSF (52 ± 1%), and after injection of ANG II (56 ± 5%; P = NS). Fetal mean blood pressure did not change after injection of aCSF, but it significantly increased at 15 min after ICV injection of ANG II (53 ± 4 to 58 ± 3 mmHg; P < 0.003; Fig. 2A). Fetal heart rate increased at 5 min after ICV injection of ANG II; however, it achieved significance only at 30 min (170 ± 4 to 208 ± 18 beats/min; P < 0.01). Fetal plasma AVP concentration increased significantly after ICV injection of ANG II (0.9 ± 0.2 to 5.6 ± 2.3 pg/ml; P < 0.03).



View larger version (18K):
[in this window]
[in a new window]
  		Fig. 2. A: fetal mean blood pressure (BP) in response to aCSF and ANG II injection into the ICV space. Mean values ± SE are shown; *P < 0.05; see text for details. B: fetal mean BP in response to artificial ICV injection of losartan and losartan plus ANG II injection into the ICV space. Mean values ± SE are shown; *P < 0.05; see text for details. C: fetal mean BP in response to PD-123319 and after combined injection of PD-123319 and ANG II injections into the ICV space. Mean values ± SE are shown; *P < 0.05; see text for detail.

 
Losartan study. ICV injection of the AT1 receptor antagonist (losartan) significantly reduced total mean spontaneous fetal swallowing (0.9 ± 0.1 to 0.4 ± 0.1 swallow/min; P < 0.001; Fig. 1B). ICV ANG II in the presence of losartan did not increase swallowing activity (0.6 ± 0.1 swallow/min; Fig. 1B). Fetal swallowing occurred primarily during low-voltage ECoG activity (control 1.4 ± 0.2; losartan 0.5 ± 0.1; losartan plus ANG II 0.8 ± 0.1 swallow/min low-voltage ECoG activity; P < 0.003). Fetal swallowing during high-voltage ECoG activities were as follows: control 0.7 ± 0.1, losartan 0.4 ± 0.1, and losartan + ANG II 0.5 ± 0.1 swallow/min (P < 0.001).

The percentage of time spent in low-voltage ECoG activity was stable during the control period (51 ± 5%), after injection of losartan (49 ± 4%), and after injection of losartan and ANG II (53 ± 6%; P = NS). Neither fetal blood pressure (49 ± 2 mmHg; Fig. 2B), heart rate (166 ± 3 beats/min), nor plasma AVP (2 ± 1 pg/ml) changed significantly during the study.

PD-123319 study. ICV injection of AT2 antagonist (PD-123319) did not change total mean spontaneous fetal swallowing (0.9 ± 0.1 swallow/min; Fig. 1C). ICV ANG II in the presence of PD-123319 significantly increased swallowing activity (1.5 ± 0.1 swallow/min; P < 0.001; Fig. 1C) Fetal swallowing occurred primarily during low-voltage ECoG activity (control 1.1 ± 0.2; PD-123319 1.1 ± 0.2; PD-123319 plus ANG II 1.8 ± 01 swallow/min low-voltage ECoG activity; P < 0.003). Fetal swallowing during high-voltage ECoG activities were as follows: control 0.7 ± 0.1, PD-123319 0.7 ± 0.1, and PD-123319 + ANG II 1.2 ± 0.1 swallow/min (P < 0.001).

The percentage of time spent in low-voltage ECoG activity was stable during the control period (56 ± 1%), after injection of PD-123319 (52 ± 2%), and after injection of PD-123319 and ANG II (57 ± 2%; P = NS). Fetal blood pressure did not change in response to PD-123319 but significantly increased at 5 min after ICV injection of ANG II (control 51 ± 3 mmHg; ANG II 58 ± 3 mmHg; P < 0.02; Fig. 2C). Fetal heart rate did not change in response to P-123319 or ICV ANG II. Fetal plasma AVP concentration increased significantly after ICV ANG II (1.1 ± 0.5 to 5.5 ± 2.8 pg/ml; P < 0.03).

In all study protocols, fetal hematocrit, pH, PO2, PCO2, plasma osmolality, and sodium, chloride, and potassium concentrations values drawn at the end of the 1st hour of the baseline period did not change (Table 1). Similarly, maternal arterial parameters did not change from control values.


View this table:
[in this window]
[in a new window]
  		Table 1. Fetal arterial blood gases, osmolality, and chemistry during the control aCSF/ANG II, losartan, and PD-123319 studies

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
This study examined the roles of central ANG II receptor subtypes in the regulation of fetal swallowing and systemic blood pressure with the use of selective ANG II receptor antagonists. ICV injection of ANG II increased both fetal swallowing activities and systemic blood pressure, consistent with previous reports (17). Selective blockade of the central AT1 but not AT2 receptor resulted in a significant reduction in basal fetal swallowing and eliminated the dipsogenic effect of central ANG II. These data demonstrate that the high rate of basal as well as ANG II-mediated fetal swallowing is regulated via central AT1 receptors. Our results also demonstrated that central injection of AT2 receptor antagonist (PD-123319) which blocked CSF accessible AT2 receptor affected neither dipsogenic nor pressor responses of central ANG II.

The ANG II dose used in our experiment was 6.4 µg diluted in 1 ml of aCSF. The CSF volume in the near-term ovine fetus is ~7 ml (18); this resulted in a final concentration of ANG II in the CSF of 0.8 µg/ml. Higher concentration of ANG II is expected at the site of injection (lateral ventricle). Although there are no previous studies that measured the basal level of ANG II in the ovine fetus CSF, the basal ANG II concentration in the CSF of adult rats is 0.6 µg/ml (7, 38). Thus the dose administered is an appropriate physiological stimulant dose. Although we have previously used lower doses of central ANG II (40), we have found that such a low dose of ANG II (0.5 µg/kg) does not consistently produce dipsogenic and pressor responses. Therefore, we have gradually increased the dose of central ANG II to 2.0 µg/kg, which totals 6.4 µg in an approximately 2.5- to 3-kg ovine fetus. This central ANG II dose consistently and reliably produces dipsogenic and pressor responses.

Losartan is highly selective for AT1 receptors with 30,000 times more selectivity to AT1 than AT2 receptors (10, 11), while PD-123319 is characterized by its high affinity for AT2 receptors with very low affinity for AT1 receptors (50). The dose of the selective AT1 and AT2 blockers were calculated based on rat studies (41, 50), after adjustments for the differences in CSF volume between adult rat (~0.3 to 0.4 ml) (34) and near-term fetal sheep (~7 ml) (18). The chemical purity of both losartan and PD-123319 was checked by the manufacturers (Merck and Sigma, respectively) utilizing high-performance liquid chromatography.

We have used ICV PD-123319 in a dose of 0.8 mg. Converting this dose to a nanomolar concentration, the concentration of PD-123319 in the CSF would be 1,551 nM. In a study by McMullen et al. (33) in pregnant ewes, PD-123319 competition curve demonstrated that PD-123319 achieved half-maximal displacement (IC50) of ANG II from AT2 receptor in the uterine arteries at a concentration of 14.5 nM. In the brain tissue the IC50 of PD-123319 is 210 nM (9). Therefore our dose of PD-123319 used centrally was sufficient to block >50% of periventricular AT2 receptors. In agreement with our study, Weisinger et al. utilizing water-deprived adult sheep found that PD-123319 ICV infusion (5,000 pg/h) did not block the dipsogenic effect of ICV infusion of ANG II (3.8 pg/h) (49). As PD-123319 was dissolved in normal saline and we utilized aCSF in the control study (aCSF/ANG II), we have utilized three additional control normal saline studies in which 1 ml of normal saline was injected ICV after 2-h basal periods. ICV normal saline caused no hemodynamic or biochemical changes as fetal blood pressure, heart rate, blood gases, osmolality, and chemistry did not change from basal values. Similarly, fetal swallowing did not change from basal values (0.8 swallow/min).

In the adult rat (21) central AT1 blockade completely prevented drinking and AVP responses to central ANG II, while central AT2 blockade potentiated the ANG II-induced drinking and AVP release (24), suggesting an inhibitory effect of AT2 receptors on AT1-mediated responses. In the adult sheep, ICV injection of AT1 receptor antagonism blocked the water intake caused by ICV hypertonic NaCl in sodium-depleted animals, while ICV PD-123319 did not alter ANG II-induced water intake (49, 51). Similarly, central AT1 antagonism blocked postprandial and systemic ANG II-induced drinking in adult sheep (32). These reports indicate that in the adult rat and sheep, AT1 but not AT2 receptors mediate stimulated drinking behavior.

In contrast to the above data, several studies in the adult rat have indicated that central blockade of AT2 receptors significantly reduced drinking activities induced by central ANG II and hypertonic saline (23, 42). Furthermore, AT2 knockout mice have an impaired drinking response to water deprivation (22, 31), indicating a role for AT2 receptors in mediating osmotic drinking. Other studies have demonstrated that both AT1 and AT2 blockers inhibit central ANG II-induced water intake (6) and AT2 blockers (PD-123319) inhibit ANG II mediated drinking only in rats, which are fed high-salt diets (10). Thus AT2 receptors may synergistically enhance AT1-mediated water intake only under special circumstances.

In accord with the high density of AT1 receptors in various brain regions involved in cardiovascular regulation in several species (2, 4, 5, 35), the AT1 receptor contributes to adult blood pressure control. The AT1 receptor maintains resting blood pressure in anaesthetized rats on a low-salt diet, suggesting a role in maintaining resting blood pressure under conditions of increased central ANG II levels (15). In the conscious adult rat, chronic lateral ventricle infusion of AT1 antagonist significantly decreased basal blood pressure (53), suggesting that AT1 receptor in the brain plays a significant physiological role in the regulation of basal blood pressure. In conscious newborn lambs, both systemic and lateral ventricle administration of AT1 antagonist (losartan) but not AT2 antagonist (PD-123319) significantly decreased resting mean arterial blood pressure (43). Similarly, there is a marked attenuation of pressor and AVP-releasing effects of central ANG II in AT1a knockout and in wild-type mice treated with central AT1 receptor antagonist (31). Furthermore, AT1a knockout mice demonstrate reduced basal blood pressures compared with wild-type mice (31, 46).

The AT2 receptor has been repeatedly shown to oppose the pressor and AVP-releasing effects of AT1 receptors in the adult animals. AT2 knockout mice as well as mice treated with central AT2 blocker (PD-123319) demonstrate exaggerated pressor and AVP-releasing responses to central ANG II (31). Similarly, AT2 knockout mice had increased sensitivity to the pressor effect of peripheral injection of ANG II (22). Our results in the ovine fetus demonstrated that AT1 but not AT2 receptors mediate both the pressor and AVP-releasing effects of central ANG II. Although AT2 blockade resulted in small rise in basal blood pressure, the variance was large, and to detect a significant increase in basal blood pressure in response to AT2 blockade with 80% confidence, a sample size of 13 would be required.

ANG II receptors situated within the dipsogenic neurons are easily accessible from the CSF compartment. In fact, dipsogenic neurons from the subfornical organ (SFO) and organ vasculosum lamina terminalis (OVLT) are in direct contact with CSF. As detected with electron microscopy of neuronal composition of the SFO, axonal terminals and dendrites making direct contact with ependymal cells and CSF (14). Further studies revealed that direct ICV injection of ANG II was as effective as direct tissue injection into dipsogenic sensitive sites close to the ventricles (26). Conversely deep sites away from the ventricles were insensitive to direct injection of ANG II not crossing the ventricles, supporting the hypothesis of periventricular receptor site for angiotensin (26).

Few studies have been published describing the ontogeny of ANG II receptors (AT) in the fetal rat brain. Utilizing in situ hybridization technique, AT1 receptor mRNA subtype appears in late gestation at embryonic day 19 (E19) in forebrain areas involved in fluid homeostasis and cardiovascular regulation (35). AT2 receptor mRNA appears earlier at E13 and is strongly but transiently expressed in certain structures involved mainly in motor functions and sensory integration (36).

Although the precise signaling pathways and the functional roles are unclear, AT2 receptors may antagonize, under physiological conditions, several AT1-mediated actions (1, 11, 22, 25, 36). The mechanism by which AT2 receptors inhibit AT1 function is not clear. However, stimulation of the AT1 receptor increases neuronal depolarization and firing rate (12, 45), while AT2 stimulation results in neuronal hyperpolarization and reduced neuronal excitability (52). Although AT2 receptors are highly expressed in fetal brain (48), they do not appear to exert antagonistic effects to either AT1-mediated drinking or pressor effects. Teleologically, the high expression of AT2 receptors in early fetal life may facilitate rapid cell turnover and differentiation by opposing the growth-promoting effect of AT1 receptor (29).

In conclusion, we report an important role for AT1 receptors in maintaining ANG II-stimulated ingestive behavior in the near-term ovine fetus. We have also demonstrated that ANG II-mediated AVP-releasing effects are mediated via AT1 receptors. Despite the relative overexpression of AT2 receptors in the fetal compared with the adult brain, the role for AT2 receptors remains uncertain.

Perspectives

Near-term ovine fetus has functional central dipsogenic mechanism as demonstrated by increased drinking activities in response to central ANG II. Similar to the adult, ovine fetus utilizes AT1 receptors to mediate dipsogenic response. Our data demonstrate that AT2 receptors, in regions accessible to the CSF, do not contribute to fetal drinking, pressor, or AVP-releasing effects of central ANG II in ovine fetus. In light of the controversial role of AT2 receptor in mediating drinking activities in the adult rat, we must interpret with caution our findings regarding the role of AT2 receptor in the fetus. We emphasize that our studies were physiological in nature and utilized ICV route to access periventricular ANG II receptors, and to rule out with certainty any role of AT2 receptors in drinking and cardiovascular regulation it would be necessary to carry out other studies at hypothalamic tissue level including binding, molecular and electrophysiological studies.


   GRANTS
TOP
 ABSTRACT
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This research was supported by grants RO1-DK-43311 and RO3-HD-39671–01 (M. G. Ross) and K08-HD-40251–02 (M. A. El-Haddad) from the National Institutes of Health.


   ACKNOWLEDGMENTS
 
We thank both C. Guerra and L. Day for technical assistance.

This work was presented at the Society for Gynecological Investigation, March 2003.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. A. El-Haddad, Harbor/UCLA Medical Center, 1124 W. Carson St., RB-1, Torrance, CA 90502 (E-mail: el-haddad{at}gcrc.rei.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Akishita M, Ito M, Lehtonen JY, Daviet L, Dzau VJ, and Horiuchi M. Expression of the AT2 receptor developmentally programs extracellular signal-regulated kinase activity and influences fetal vascular growth. J Clin Invest 103: 63–71, 1999.[Web of Science][Medline]
  2. Aldred GP, Chai SY, Song K, Zhuo J, MacGregor DP, and Mendelsohn FA. Distribution of angiotensin II receptor subtypes in the rabbit brain. Regul Pept 44: 119–130, 1993.[CrossRef][Web of Science][Medline]
  3. Allen AM, Zhuo J, and Mendelsohn FA. Localization and function of angiotensin AT1 receptors. Am J Hypertens: 31S–38S, 2000.
  4. Allen AM, Dampney RA, and Mendelsohn FA. Angiotensin receptor binding and pressor effects in cat subretrofacial nucleus. Am J Physiol Heart Circ Physiol 255: H1011–H1017, 1988.[Abstract/Free Full Text]
  5. Allen AM, Chai SY, Clevers J, McKinley MJ, Paxinos G, and Mendelsohn FA. Localization and characterization of angiotensin II receptor binding and angiotensin converting enzyme in the human medulla oblongata. J Comp Neurol 269: 249–264, 1988.[CrossRef][Web of Science][Medline]
  6. Alova LG, Stancheva SL, Matsoukas J, and Georgiev VP. Effects of peptide and non-peptide antagonists of angiotensin II receptors on drinking behavior in rats. J Physiol Paris 93: 219–224, 1999.[CrossRef][Web of Science][Medline]
  7. Batt CM, Jensen LL, Hanesworth JM, Harding JW, and Wright JW. Intracerebroventricularly applied peptidase inhibitors increase endogenous angiotensin levels. Brain Res 529: 126–130, 1990.[CrossRef][Web of Science][Medline]
  8. Beresford MJ and Fitzsimons JT. Intracerebroventricular angiotensin II-induced thirst and sodium appetite in rat are blocked by the AT1 receptor antagonist, Losartan (DuP 753), but not by the AT2 antagonist, CGP 42112B. Exp Physiol 77: 761–764, 1992.[Abstract]
  9. Blankley CJ, Hodges JC, Klutchko SR, Himmelsbach RJ, Chucholowski A, Connolly CJ, Neergaard SJ, Van Nieuwenhze MS, Sebastian A, and Quin 3rd J. Synthesis and structure-activity relationships of a novel series of non-peptide angiotensin II receptor binding inhibitors specific for the AT2 subtype. J Med Chem 34: 3248–3260, 1991.[CrossRef][Web of Science][Medline]
  10. Camara AK and Osborn J. Central AT1 and AT2 receptors mediate chronic intracerebroventricular angiotensin II-induced drinking in rats fed high sodium chloride diet from weaning. Acta Physiol Scand 171: 195–201, 2001.[CrossRef][Web of Science][Medline]
  11. Chiu AT, McCall DE, Ardecky RJ, Duncia JV, Nguyen TT, and Timmermans PB. Angiotensin II receptor subtypes and their selective nonpeptide ligands. Receptor 1: 33–40, 1990–91.[Medline]
  12. Ciuffo GM, Alvarez SE, and Fuentes LB. Angiotensin II receptors induce tyrosine dephosphorylation in rat fetal membranes. Regul Pept 74: 129–135, 1998.[CrossRef][Web of Science][Medline]
  13. Culman J, Höhle S, Qadri F, Edling O, Blume A, Lebrun C, and Unger T. Angiotensin as neuromodulator/neurotransmitter in central control of body fluid and electrolyte homeostasis. Clin Exp Hypertens 17: 281–293, 1995.[Web of Science][Medline]
  14. Dellmann HD. Fine structural organization of the subfornical organ. A concise review. Brain Res Bull 15: 71–78, 1985.[CrossRef][Web of Science][Medline]
  15. DiBona GF and Jones SY. Effect of dietary sodium intake on the responses to bicuculline in the paraventricular nucleus of rats. Hypertension 38: 192–197, 2001.[Abstract/Free Full Text]
  16. El-Haddad MA, Chao CR, Abdel Sayed A, El-Haddad H, and Ross MG. Effects of central angiotensin-II (Ang II) receptor antagonism on fetal swallowing and cardiovascular activity. Am J Obstet Gynecol 185: 828–833, 2001.[CrossRef][Web of Science][Medline]
  17. El-Haddad MA, Chao CR, Ma SX, and Ross MG. Neuronal NO modulates spontaneous and ANG II-stimulated fetal swallowing behavior in the near-term ovine fetus. Am J Physiol Regul Integr Comp Physiol 282: R1521–R1527, 2002.[Abstract/Free Full Text]
  18. Evans CAN, Reynolds JM, Reynolds ML, Saunders NR, and Segal MB. The development of a blood-brain barrier mechanism in foetal sheep. J Physiol 238: 371–378, 1974.[Abstract/Free Full Text]
  19. Ferguson AV and Washburn DLS. Angiotensin II: a peptidergic neurotransmitter in central autonomic pathways. Prog Neurobiol 54:169–192, 1998.[CrossRef][Web of Science][Medline]
  20. Fujino Y, Agnew CL, Schreyer P, Ervin MG, Sherman DJ, Ross MG. Amniotic fluid volume response to esophageal occlusion in fetal sheep. Am J Obstet Gynecol 165: 1620–1626, 1991.[Web of Science][Medline]
  21. Gronan DH and York RJ. Effects of chronic intraventricular administration of angiotensin II on drinking behavior and blood pressure. Pharmacol Biochem Behav 10: 121–126, 1979.[CrossRef][Web of Science][Medline]
  22. Hein L, Barsh GS, Pratt RE, Dzau VJ, Kobilka BK. Behavioural and cardiovascular effects of disrupting the angiotensin II type-2 receptor in mice. Nature 377(6551): 744–747, 1995 [Corrigendum Nature 380(6572): 366, 1996].[CrossRef][Medline]
  23. Hogarty DC, Speakman EA, Puig V, and Phillips MI. The role of angiotensin, AT1 and AT2 receptors in the pressor, drinking and vasopressin responses to central angiotensin. Brain Res 586: 289–294, 1992.[CrossRef][Web of Science][Medline]
  24. Hohle S, Spitznagel H, Rascher W, Culman J, Unger T. Angiotensin AT1 receptor-mediated vasopressin release and drinking is potentiated by an AT2 receptor antagonist. Eur J Pharmacol 275: 277–282, 1995.[CrossRef][Web of Science][Medline]
  25. Ichiki T, Labosky PA, Shiota C, Okuyama S, Imagawa Y, Fogo A, Niimura F, Ichikawa I, Hogan BL, and Inagami T. Effects on blood pressure and exploratory behaviour of mice lacking angiotensin II type-2 receptor. Nature 377(6551): 748–750, 1995.[CrossRef][Medline]
  26. Johnson AK and Epstein AN. The cerebral ventricles as the avenue for the dipsogenic action of intracranial angiotensin. Brain Res 86: 399–418, 1975.[CrossRef][Web of Science][Medline]
  27. Kimble RM, Harding JE, and Kolbe A. Does gut atresia cause polyhydramnios? Pediatr Surg Int 13(2–3): 115–117, 1998.[CrossRef][Web of Science][Medline]
  28. La Rochelle FT, North WG, and Stern P. A new extraction of arginine vasopressin from blood. The use of octadecasilylsilica. Pflügers Arch 387: 79–81, 1980.[CrossRef][Web of Science][Medline]
  29. Lazard D, Briend-Sutren MM, Villageois P, Mattei MG, Strosberg AD, and Nahmias C. Molecular characterization and chromosome localization of a human angiotensin II AT2 receptor gene highly expressed in fetal tissues. Receptors Channels 2: 271–280, 1994.[Web of Science][Medline]
  30. Lenkei Z, Palkovits M, Corvol P, and Llorens-Cortes C. Expression of angiotensin type-1 (AT1) and type-2 (AT2) receptor mRNAs in the adult rat brain: a functional neuroanatomical review. Front Neuroendocrinol 18: 383–439, 1997.[CrossRef][Web of Science][Medline]
  31. Li Z, Iwai M, Wu L, Shiuchi T, Jinno T, Cui TX, and Horiuchi M. Role of AT2 receptor in the brain in regulation of blood pressure and water intake. Am J Physiol Heart Circ Physiol 284: H116–H121, 2003.[Abstract/Free Full Text]
  32. Mathai M, Evered MD, and McKinley MJ. Intracerebroventricular losartan inhibits postprandial drinking in sheep. Am J Physiol Regul Integr Comp Physiol 272: R1055–R1059, 1997.[Abstract/Free Full Text]
  33. McMullen JR, Gibson KJ, Lumbers ER, Burrell JH. Selective down-regulation of AT2 receptors in uterine arteries from pregnant ewes given 24-h intravenous infusions of angiotensin II. Regul Pept 99: 119–129, 2001.[CrossRef][Web of Science][Medline]
  34. Meek JL and Neff NH. Is cerebrospinal fluid the major avenue for the removal of 5-hydroxyindoleacetic acid from the brain? Neuropharmacology 12: 401–499, 1973.[CrossRef][Web of Science][Medline]
  35. Nuyt AM, Lenkei Z, Corvol P, Palkovits M, and Llorens-Cortes C. Ontogeny of angiotensin II type 1 receptor mRNAs in fetal and neonatal rat brain. J Comp Neurol 440: 192–203, 2001.[CrossRef][Web of Science][Medline]
  36. Nuyt AM, Lenkei Z, Palkovits M, Corvol P, Llorens-Cortes C. Ontogeny of angiotensin II type 2 receptor mRNA expression in fetal and neonatal rat brain. J Comp Neurol 407:193–206, 1999.[CrossRef][Web of Science][Medline]
  37. Richardson GJ and Mogenson DB. Water intake elicited by injection of angiotensin II into preoptic area of rats. Am J Physiol Regul Integr Comp Physiol 240: R70–R74, 1981.[Abstract/Free Full Text]
  38. Rodriguez EM. The cerebrospinal fluid as a pathway in neuroendocrine integration. J Endocrinol 71: 407–443, 1976.[Abstract/Free Full Text]
  39. Ross MG, Kullama LK, Ogundipe OA, Chan K, and Ervin MG. Ovine fetal swallowing response to intracerebroventricular hypertonic saline. J Appl Physiol 78: 2267–2271, 1995.[Abstract/Free Full Text]
  40. Ross MG, Kullama LK, Ogundipe OA, Chan K, and Ervin MG. Central angiotensin II stimulation of ovine fetal swallowing. J Appl Physiol 76: 1340–1345, 1994.[Abstract/Free Full Text]
  41. Rowland NE, Rozelle A, Riley PJ, and Fregly MJ. Effect of nonpeptide angiotensin receptor antagonists on water intake and salt appetite in rats. Brain Res Bull 3–4: 389–393, 1992.
  42. Rowland NE, Fregly MJ. Brain angiotensin AT-2 receptor antagonism and water intake. Brain Res Bull 32: 391–394, 1993.[CrossRef][Web of Science][Medline]
  43. Segar JL, Minnick A, Nuyt AM, and Robillard JE. Role of endogenous ANG II and AT1 receptors in regulating arterial baroreflex responses in newborn lambs. Am J Physiol Regul Integr Comp Physiol 272: R1862–R1873, 1997.[Abstract/Free Full Text]
  44. Sugaya T, Nishimatsu S, Tanimoto K, Takimoto E, Yamagishi T, Imamura K, Goto S, Imaizumi K, Hisada Y, Otsuka A, Uchida H, Sugiura M, Fukuta K, Fukamizu A, and Murakami K. Angiotensin II type 1a receptor-deficient mice with hypotension and hyperreninemia. J Biol Chem 270: 18719–18722, 1995.[Abstract/Free Full Text]
  45. Sumners C, Zhu M, Gelband CH, and Posner P. Angiotensin II type 1 receptor modulation of neuronal K+ and Ca2+ currents: intracellular mechanisms. Am J Physiol Cell Physiol 271: C154–C163, 1996.[Abstract/Free Full Text]
  46. Timmermans PB, Wong PC, Chiu AT, Herblin WF, Benfield P, Carini DJ, Lee RJ, Wexler RR, Saye JA, and Smith RD. Angiotensin II receptors and angiotensin II receptor antagonists. Pharmacol Rev 45: 205–251, 1993.[Web of Science][Medline]
  47. Trahair JF and Harding R. Restitution of swallowing in the fetal sheep restores intestinal growth after midgestation esophageal obstruction. J Pediatr Gastroenterol Nutr 20: 156–161, 1995.[Web of Science][Medline]
  48. Tsutsumi K, Viswanathan M, Stromberg C, Saavedra JM. Type-1 and type-2 angiotensin II receptors in fetal rat brain. Eur J Pharmacol 198: 89–92, 1991.[CrossRef][Web of Science][Medline]
  49. Weisinger RS, Blair-West JR, Denton DA, Tarjan E. Role of brain angiotensin-II in thirst and sodium appetite of sheep. Am J Physiol Regul Integr Comp Physiol 273: R187–R196, 1997.[Abstract/Free Full Text]
  50. Widdop RE, Gardiner SM, Kemp PA, and Bennett T. Differential blockade of central effects of angiotensin II by AT2-receptor antagonists. Am J Physiol Heart Circ Physiol 265: H226–H231, 1993.[Abstract/Free Full Text]
  51. Wright JW, Harding JW. Brain angiotensin receptor subtypes in the control of physiological and behavioral responses. Neurosci Biobehav Rev 18: 21–53,1994.[CrossRef][Web of Science][Medline]
  52. Xiong H and Marshall KC. Angiotensin II depresses glutamate depolarization and excitatory postsynaptic potentials in locus coeruleus through angiotensin II subtype 2 receptors. Neuroscience 62: 163–175, 1994.[CrossRef][Web of Science][Medline]
  53. Yang EK, Lee WJ, Park YY, Ahn DK, Park JS, and Kim HJ. Effects of chronic central administration of losartan on the cardiovascular and hormonal responses to hemorrhage in conscious rats. Regul Pept 67: 107–113, 1996.[CrossRef][Web of Science][Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
288/4/R1014    most recent
00479.2003v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Web of Science (1)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by El-Haddad, M. A.
Right arrow Articles by Ross, M. G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by El-Haddad, M. A.
Right arrow Articles by Ross, M. G.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2005 by the American Physiological Society.