Hypotensive effect of ANG II and ANG-(1-7) at the caudal ventrolateral medulla involves different mechanisms

The objective of the present study was to determine the contribution of the autonomic nervous system and nitric oxide to the depressor effect produced by unilateral microinjection of ANG-(1-7) and ANG II into the caudal ventrolateral medulla (CVLM). Unilateral microinjection of ANG-(1-7), ANG II (40 pmol), or saline (100 nl) was made into the CVLM of male Wistar rats anesthetized with urethane before and after intravenous injection of 1) methyl-atropine, 2.5 mg/kg; 2) prazosin, 25 microg/kg; 3) the nitric oxide synthase (NOS) inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME), 5 mg/kg; or 4) the specific inhibitor of neuronal NOS, 7-nitroindazole (7-NI), 45 mg/kg. Arterial pressure and heart rate (HR) were continuously monitored. Microinjection of ANG-(1-7) or ANG II into the CVLM produced a significant decrease in mean arterial pressure (MAP; -11 +/- 1 mmHg, n = 12 and -10 +/- 1 mmHg, n = 10, respectively) that was not accompanied by consistent changes in HR or in cardiac output. The effect of ANG-(1-7) was abolished after treatment with methyl-atropine (-3 +/- 0.6 mmHg, n = 9) or L-NAME (-2.3 +/- 0.5 mmHg, n = 8) or 7-NI (-2.8 +/- 0.6 mmHg, n = 5). In contrast, these treatments did not significantly interfere with the ANG II effect (-10 +/- 2.6 mmHg, n = 8; -8 +/- 1.5 mmHg, n = 8; and -12 +/- 3.6 mmHg, n = 6; respectively). Peripheral treatment with prazosin abolished the hypotensive effect of ANG-(1-7) and ANG II. Microinjection of saline did not produce any significant change in MAP or in HR. These results suggest that the hypotensive effect produced by ANG II at the CVLM depends on changes in adrenergic vascular tonus and, more importantly, the hypotensive effect produced by ANG-(1-7) also involves a nitric oxide-related mechanism.     

Nitric oxide at the CVLM is involved in the attenuation of the reflex bradycardia in renovascular hypertensive rats

Hypertension is associated to an increase in central oxidative stress and an attenuation of the baroreflex control of arterial pressure. The present study evaluated the effect of alterations in the levels of nitric oxide (NO) and superoxide anion in the caudal ventrolateral medulla (CVLM), a key area of the brainstem for the baroreflex control of arterial pressure, in renovascular hypertensive rats (2K1C). Baseline mean arterial pressure (MAP), heart rate (HR), and reflex bradycardia were evaluated 30 days after renal artery occlusion in anesthetized (urethane, 1.2 g/kg, i.p.) 2K1C or normotensive (SHAM) rats. The MAP, HR, and baroreflex control of HR were evaluated before and after CVLM microinjections of the non-selective NOS inhibitor L-NAME (10 nmol), the NO precursor L-ARG (50 nmol), or the antioxidant ascorbic acid, Vit C (10 nmol). In both 2K1C and SHAM animals, CVLM microinjection of L-NAME produced a decrease in MAP, whereas L-ARG induced a significant increase in MAP. However, microinjection of Vit C into the CVLM produced a decrease in MAP and HR only in 2K1C and not in SHAM rats. Cardiovascular effects produced by microinjection of l-ARG into the CVLM were abolished by prior microinjection of L-NAME in the CVLM of 2K1C and SHAM rats. Microinjection of L-NAME into the CVLM increased the sensitivity of reflex bradycardia in 2K1C animals. In contrast, the CVLM microinjection of L-ARG reduced reflex bradycardia only in SHAM rats. Vit C in the CVLM did not change reflex bradycardia in either 2K1C or in SHAM rats. These results suggest that increased oxidative stress in the CVLM during hypertension contributes to the reduced baroreflex sensitivity and to maintain hypertension in the 2K1C model.     

Sex differences in the development of angiotensin II-induced hypertension in conscious mice

Sex has an important influence on blood pressure (BP) regulation. There is increasing evidence that sex hormones interfere with the renin-angiotensin system. Thus the purpose of this study was to determine whether there are sex differences in the development of ANG II-induced hypertension in conscious male and female mice. We used telemetry implants to measure aortic BP and heart rate (HR) in conscious, freely moving animals. ANG II (800 ng.kg(-1).min(-1)) was delivered via an osmotic pump implanted subcutaneously. Our results showed baseline BP in male and female mice to be similar. Chronic systemic infusion of ANG II induced a greater increase in BP in male (35.1 +/- 5.7 mmHg) than in female mice (7.2 +/- 2.0 mmHg). Gonadectomy attenuated ANG II-induced hypertension in male mice (15.2 +/- 2.4 mmHg) and augmented it in female mice (23.1 +/- 1.0 mmHg). Baseline HR was significantly higher in females relative to males (630.1 +/- 7.9 vs. 544.8 +/- 16.2 beats/min). In females, ANG II infusion significantly decreased HR. However, the increase in BP with ANG II did not result in the expected decrease in HR in either intact male or gonadectomized mice. Moreover, the slope of the baroreflex bradycardia to phenylephrine was blunted in males (-5.6 +/- 0.3 to -2.9 +/- 0.5) but not in females (-6.5 +/- 0.5 to -5.6 +/- 0.3) during infusion of ANG II, suggesting that, in male mice, infusion of ANG II results in a resetting of the baroreflex control of HR. Ganglionic blockade resulted in greater reduction in BP on day 7 after ANG II infusion in males compared with females (-61.0 +/- 8.9 vs. -36.6 +/- 6.6 mmHg), suggesting an increased contribution of sympathetic nerve activity in arterial BP maintenance in male mice. Together, these data indicate that there are sex differences in the development of chronic ANG II-induced hypertension in conscious mice and that females may be protected from the increases in BP induced by ANG II.     

Baroreceptor reflex modulation by circulating angiotensin II is mediated by AT1 receptors in the nucleus tractus solitarius

Circulating ANG II modulates the baroreceptor reflex control of heart rate (HR), at least partly via activation of ANG II type 1 (AT1) receptors on neurons in the area postrema. In this study, we tested the hypothesis that the effects of circulating ANG II on the baroreflex also depend on AT1 receptors within the nucleus tractus solitarius (NTS). In confirmation of previous studies in other species, increases in arterial pressure induced by intravenous infusion of ANG II had little effect on HR in urethane-anesthetized rats, in contrast to the marked bradycardia evoked by equipressor infusion of phenylephrine. In the presence of a continuous background infusion of ANG II, the baroreflex control of HR was shifted to higher levels of HR but had little effect on the baroreflex control of renal sympathetic activity. The modulatory effects of circulating ANG II on the cardiac baroreflex were significantly reduced by microinjection of candesartan, an AT1 receptor antagonist, into the area postrema and virtually abolished by microinjections of candesartan into the medial NTS. After acute ablation of the area postrema, a background infusion of ANG II still caused an upward shift of the cardiac baroreflex curve, which was reversed by subsequent microinjection of candesartan into the medial NTS. The results indicate that AT1 receptors in the medial NTS play a critical role in modulation of the cardiac baroreflex by circulating ANG II via mechanisms that are at least partly independent of AT1 receptors in the area postrema.     

Effects of angiotensins on day-night fluctuations and stress-induced changes in blood pressure

In this study we evaluated by telemetry the effects of ANG II and ANG-(1-7) infusion on the circadian rhythms of blood pressure (BP) and heart rate (HR) and on the cardiovascular adjustment resulting from restraint stress in rats. ANG II or ANG-(1-7) or vehicle were infused subcutaneously for 7 days. Restraint stress was carried out before, during, and after infusion at 7-day intervals. Parallel with an increase in MAP, ANG II infusion produced an inversion of MAP circadian rhythm with a significant MAP acrophase inversion. It also produced bradycardia during the first 3 days of infusion. Thereafter, HR progressively increased, reaching values similar to or above those of the control period at the end of the infusion period. HR circadian variation was not changed by ANG II infusion. Strikingly, ANG II significantly attenuated the increase in MAP induced by restraint stress without altering the HR response. ANG-(1-7) infusion produced a slight but significant decrease in MAP restricted to the daytime period. No significant changes in the MAP acrophase were observed. In addition, ANG-(1-7) infusion produced a small but significant sustained bradycardia. ANG-(1-7) did not change cardiovascular responses to restraint stress. These data indicate that ANG II can influence the activity of brain areas involved in the determination of stress-induced or circadian-dependent variations of blood pressure without changing HR fluctuations. A significant modulatory influence of ANG-(1-7) on basal MAP and HR is also suggested.     

Evidence for a role of AT(2) receptors at the CVLM in the cardiovascular changes induced by low-intensity physical activity in renovascular hypertensive rats

In the present study, we evaluated the involvement of the rennin-angiotensin system (RAS) in the control of the blood pressure (BP), baroreceptor-mediated bradycardia and the reactivity of caudal ventrolateral medulla (CVLM) neurons to Ang II and to AT(2) receptor antagonist in sedentary or trained renovascular hypertensive rats. Physical activity did not significantly change the baseline mean arterial pressure (MAP), heart rate (HR) or the sensitivity of the baroreflex bradycardia in normotensive Sham rats. However, in 2K1C hypertensive rats, physical activity induced a significant fall in baseline MAP and HR and produced an improvement of the baroreflex function (bradycardic component). The microinjections of Ang II into the CVLM produced similar decreases in MAP in all groups, Sham and 2K1C, sedentary and trained rats. The hypotensive effect of Ang II at the CVLM was blocked by previous microinjection of the AT(2) receptors antagonist, PD123319, in all groups of rats. Unexpectedly, microinjection of PD123319 at the CVLM produced a depressor effect in 2K1C sedentary that was attenuated in 2K1C trained rats. No significant changes in MAP were observed after PD123319 in Sham rats, sedentary or trained. These data showed that low-intensity physical activity is effective in lowering blood pressure and restoring the sensitivity of the baroreflex bradycardia, however these cardiovascular effects are not accompanied by changes in the responsiveness to Ang II at CVLM in normotensive or hypertensive, 2K1C rats. In addition, the blood pressure changes observed after AT(2) blockade in 2K1C rats suggest that hypertension may trigger an imbalance of AT(1)/AT(2) receptors at the CVLM that may be restored, at least in part, by low-intensity physical activity.     

The hypothalamic arcuate nucleus: a new site of cardiovascular action of angiotensin-(1-12) and angiotensin II

The hypothalamic arcuate nucleus (ARCN) has been reported to play a significant role in cardiovascular regulation. It has been hypothesized that the ARCN may be one of the sites of cardiovascular actions of angiotensins (ANGs). Experiments were carried out in urethane-anesthetized, artificially ventilated, adult male Wistar rats. The ARCN was identified by microinjections of N-methyl-d-aspartic acid (NMDA; 10 mM). Microinjections (50 nl) of ANG-(1-12) (1 mM) into the ARCN elicited increases in mean arterial pressure (MAP), heart rate (HR), and greater splanchnic nerve activity (GSNA). The tachycardic responses to ANG-(1-12) were attenuated by bilateral vagotomy. The cardiovascular responses elicited by ANG-(1-12) were attenuated by microinjections of ANG II type 1 receptor (AT(1)R) antagonists but not ANG type 2 receptor (AT(2)R) antagonist. Combined inhibition of ANG-converting enzyme (ACE) and chymase in the ARCN abolished ANG-(1-12)-induced responses. Microinjections of ANG II (1 mM) into the ARCN also increased MAP and HR. Inhibition of ARCN by microinjections of muscimol (1 mM) attenuated the pressor and tachycardic responses to intravenously administered ANG-(1-12) and ANG II (300 pmol/kg each). These results indicated that 1) microinjections of ANG-(1-12) into the ARCN elicited increases in MAP, HR, and GSNA; 2) HR responses were mediated via both sympathetic and vagus nerves; 3) AT(1)Rs, but not AT(2)Rs, in the ARCN mediated ANG-(1-12)-induced responses; 4) both ACE and chymase were needed to convert ANG-(1-12) to ANG II in the ARCN; and 5) ARCN plays a role in mediating the cardiovascular responses to circulating ANGs.     

Altered regulation of the rostral ventrolateral medulla in hypertensive obese Zucker rats

Cardiovascular effects of angiotensin-(1-12) [ANG-(1-12)] were studied in the medial nucleus of the tractus solitarius (mNTS) in anesthetized, artificially ventilated, adult male Wistar rats. Microinjections (100 nl) of ANG-(1-12) (0.06 mM) into the mNTS elicited maximum decreases in mean arterial pressure (MAP; 34 ± 5.8 mmHg) and heart rate (HR; 39 ± 3.7 beats/min). Bilateral vagotomy abolished ANG-(1-12)-induced bradycardia. Efferent greater splanchnic nerve activity was decreased by microinjections of ANG-(1-12) into the mNTS. Blockade of ANG type 1 receptors (AT(1)Rs; using ZD-7155 or L-158,809), but not ANG type 2 receptors (AT(2)Rs; using PD-123319), significantly attenuated ANG-(1-12)-induced cardiovascular responses. Simultaneous inhibition of both angiotensin-converting enzyme (ACE; using captopril) and chymase (using chymostatin) completely blocked the effects of ANG-(1-12). Microinjections of A-779 [ANG-(1-7) antagonist] did not attenuate ANG-(1-12)-induced responses. Pressure ejection of ANG-(1-12) (0.06 mM, 2 nl) caused excitation of barosensitive mNTS neurons, which was blocked by prior application of the AT(1)R antagonist. ANG-(1-12)-induced excitation of mNTS neurons was also blocked by prior sequential applications of captopril and chymostatin. These results indicate that 1) microinjections of ANG-(1-12) into the mNTS elicited depressor and bradycardic responses by exciting barosensitive mNTS neurons; 2) the decreases in MAP and HR were mediated via sympathetic and vagus nerves, respectively; 3) AT(1)Rs, but not AT(2)Rs, mediated these actions of ANG-(1-12); 4) the responses were mediated via the conversion of ANG-(1-12) to ANG II and both ACE and chymase were involved in this conversion; and 5) ANG-(1-7) was not one of the metabolites of ANG-(1-12) in the mNTS.     

nNOS gene transfer to RVLM improves baroreflex function in rats with chronic heart failure

We hypothesized that gene transfer of neuronal nitric oxide synthase (nNOS) into the rostral ventrolateral medulla (RVLM) improves baroreflex function in rats with chronic heart failure (CHF). Six to eight weeks after coronary artery ligation, rats showed hemodynamic signs of CHF. A recombinant adenovirus, either Ad.nNOS or Ad.beta-Gal, was transfected into the RVLM. nNOS expression in the RVLM was confirmed by Western blot analysis, NADPH-diaphorase, and immunohistochemical staining. We studied baroreflex control of the heart rate (HR) and renal sympathetic nerve activity (RSNA) in the anesthetized state 3 days after gene transfer by intravenous injections of phenylephrine and nitroprusside. Baroreflex sensitivity was depressed for HR and RSNA regulation in CHF rats (2.0 +/- 0.3 vs. 0.8 +/- 0.2 beats.min-1.mmHg-1, P < 0.01 and 3.8 +/- 0.3 vs. 1.2 +/- 0.1% max/mmHg, P < 0.01, respectively). Ad.nNOS transfer into RVLM significantly increased the HR and RSNA ranges (152 +/- 19 vs. 94 +/- 12 beats/min, P < 0.05 and 130 +/- 16 vs. 106 +/- 5% max/mmHg, P < 0.05) compared with the Ad.beta-Gal in CHF rats. Ad.nNOS also improved the baroreflex gain for the control of HR and RSNA (1.8 +/- 0.2 vs. 0.8 +/- 0.2 beats.min-1.mmHg-1, P < 0.01 and 2.6 +/- 0.2 vs. 1.2 +/- 0.1% max/mmHg, P < 0.01). In sham-operated rats, we found that Ad.nNOS transfer enhanced the HR range compared with Ad.beta-Gal gene transfer (188 +/- 15 vs. 127 +/- 14 beats/min, P < 0.05) but did not alter any other parameter. This study represents the first demonstration of altered baroreflex function following increases in central nNOS in the CHF state. We conclude that delivery of Ad.nNOS into the RVLM improves baroreflex function in rats with CHF.     

Angiotensin receptors contribute to blood pressure homeostasis in salt-depleted SHR

This study evaluated the contribution of angiotensin peptides acting at various receptor subtypes to the arterial pressure and heart rate of adult 9-wk-old male conscious salt-depleted spontaneously hypertensive rats (SHR). Plasma ANG II and ANG I in salt-depleted SHR were elevated sevenfold compared with peptide levels measured in sodium-replete SHR, whereas plasma ANG-(1-7) was twofold greater in salt-depleted SHR compared with salt-replete SHR. Losartan (32.5 micromol/kg), PD-123319 (0.12 micromol. kg(-1). min(-1)), [d-Ala(7)]ANG-(1-7) (10 and 100 pmol/min), and a polyclonal ANG II antibody (0.08 mg/min) were infused intravenously alone or in combination. Combined blockade of AT(2) and AT((1-7)) receptors significantly increased the blood pressure of losartan-treated SHR (+15 +/- 1 mmHg; P < 0.01); this change did not differ from the blood pressure elevation produced by the sole blockade of AT((1-7)) receptors (15 +/- 4 mmHg). On the other hand, sole blockade of AT(2) receptors in losartan-treated SHR increased mean arterial pressure by 8 +/- 1 mmHg (P < 0.05 vs. 5% dextrose in water as vehicle), and this increase was less than the pressor response produced by blockade of AT((1-7)) receptors alone or combined blockade of AT((1-7)) and AT(2) receptors. The ANG II antibody increased blood pressure to the greatest extent in salt-depleted SHR pretreated with only losartan (+11 +/- 2 mmHg) and to the least extent in salt-depleted SHR previously treated with the combination of losartan, PD-123319, and [d-Ala(7)]ANG-(1-7) (+7 +/- 1 mmHg; P < 0.01). Losartan significantly increased heart rate, whereas other combinations of receptor antagonists or the ANG II antibody did not alter heart rate. Our results demonstrate that ANG II and ANG-(1-7) act through non-AT(1) receptors to oppose the vasoconstrictor actions of ANG II in salt-depleted SHR. Combined blockade of AT(2) and AT((1-7)) receptors and ANG II neutralization by the ANG II antibody reversed as much as 67% of the blood pressure-lowering effect of losartan.     

AT(1) and glutamatergic receptors in paraventricular nucleus support blood pressure during water deprivation

Water deprivation activates sympathoexcitatory neurons in the paraventricular nucleus (PVN); however, the neurotransmitters that mediate this activation are unknown. To test the hypothesis that ANG II and glutamate are involved, effects on blood pressure (BP) of bilateral PVN microinjections of ANG II type 1 receptor (AT1R) antagonists, candesartan and valsartan, or the ionotropic glutamate receptor antagonist, kynurenate, were determined in urethane-anesthetized water-deprived and water-replete male rats. Because PVN may activate sympathetic neurons via the rostral ventrolateral medulla (RVLM) and because PVN disinhibition increases sympathetic activity in part via increased drive of AT1R in the RVLM, candesartan was also bilaterally microinjected into the RVLM. Total blockade of the PVN with bilateral microinjections of muscimol, a GABA(A) agonist, decreased BP more (P < 0.05) in water-deprived (-29 +/- 8 mmHg) than in water-replete (-7 +/- 2 mmHg) rats, verifying that the PVN is required for BP maintenance during water deprivation. PVN candesartan slowly lowered BP by 7 +/- 1 mmHg (P < 0.05). In water-replete rats, however, candesartan did not alter BP (1 +/- 1 mmHg). Valsartan also produced a slowly developing decrease in arterial pressure (-6 +/- 1 mmHg; P < 0.05) in water-deprived but not in water-replete (-1 +/- 1 mmHg) rats. In water-deprived rats, PVN kynurenate rapidly decreased BP (-19 +/- 3 mmHg), and the response was greater (P < 0.05) than in water-replete rats (-4 +/- 1 mmHg). Finally, as in PVN, candesartan in RVLM slowly decreased BP in water-deprived (-8 +/- 1 mmHg; P < 0.05) but not in water-replete (-3 +/- 1 mmHg) rats. These data suggest that activation of AT(1) and glutamate receptors in PVN, as well as of AT1R in RVLM, contributes to BP maintenance during water deprivation.     

Influence of rostral ventrolateral medulla on renal sympathetic baroreflex in conscious rabbits

Previous studies with anesthetized animals have shown that the pressor region of the rostral ventrolateral medulla (RVLM) is a critical site in vasomotor control. The aim of this study was to develop, in conscious rabbits, a technique for microinjecting into the RVLM and to determine the influence of this area on renal sympathetic nerve activity (RSNA) and arterial pressure (AP) using local injections of glutamate, rilmenidine, ANG II and sarile. Rabbits were implanted with guide cannulas for bilateral microinjections into the RVLM (n = 7) or into the intermediate ventrolateral medulla (IVLM, n = 6) and an electrode for measuring RSNA. After 7 days of recovery, injections of glutamate (10 and 20 nmol) into the RVLM increased RSNA by 81 and 88% and AP by 17 and 25 mmHg, respectively. Infusion of glutamate (2 nmol/min) into the RVLM increased AP by 15 mmHg and the RSNA baroreflex range by 38%. By contrast, injection of the imidazoline receptor agonist rilmenidine (4 nmol) into the RVLM decreased AP by 8 mmHg and the RSNA baroreflex range by 37%. Injections of rilmenidine into the IVLM did not alter AP or RSNA. Surprisingly, treatments with ANG II (4 pmol/min) or the ANG II receptor antagonist sarile (500 pmol) into the RVLM did not affect the resting or baroreflex parameters. Infusion of ANG II (4 pmol/min) into the fourth ventricle increased AP and facilitated the RSNA baroreflex. Our results show that agents administered via a novel microinjecting system for conscious rabbits can selectively modulate neuronal activity in circumscribed regions of the ventrolateral medulla. We conclude that the RVLM plays a key role in circulatory control in conscious rabbits. However, we find no evidence for the role of ANG II receptors in the RVLM in the moment-to-moment regulation of AP and RSNA.     

Aging and baroreflex control of RSNA and heart rate in rats

Aging is associated with altered autonomic control of cardiovascular function, but baroreflex function in animal models of aging remains controversial. In this study, pressor and depressor agent-induced reflex bradycardia and tachycardia were attenuated in conscious old (24 mo) rats [57 and 59% of responses in young (10 wk) Wistar rats, respectively]. The intrinsic heart rate (HR, 339 +/- 5 vs. 410 +/- 10 beats/min) was reduced in aged animals, but no intergroup differences in resting mean arterial blood pressure (MAP, 112 +/- 3 vs. 113 +/- 5 mmHg) or HR (344 +/- 9 vs. 347 +/- 9 beats/min) existed between old and young rats, respectively. The aged group also exhibited a depressed (49%) parasympathetic contribution to the resting HR value (vagal effect) but preserved sympathetic function after intravenous methylatropine and propranolol. An implantable electrode revealed tonic renal sympathetic nerve activity (RSNA) was similar between groups. However, old rats showed impaired baroreflex control of HR and RSNA after intravenous nitroprusside (-0.63 +/- 0. 18 vs. -1.84 +/- 0.4 bars x cycle(-1) x mmHg(-1) x s(-1)). Therefore, aging in rats is associated with 1) preserved baseline MAP, HR, and RSNA, 2) impaired baroreflex control of HR and RSNA, and 3) altered autonomic control of resting HR.     

Sympathoinhibitory pathway from caudal midline medulla to RVLM is independent of baroreceptor reflex pathway

Glutamate stimulation of the caudal midline medulla (CMM) causes profound sympathoinhibition due to GABAergic inhibition of presympathetic neurons in the rostral ventrolateral medulla (RVLM). We investigated whether the sympathoinhibitory pathway from CMM to RVLM, like the central baroreceptor reflex pathway, includes a glutamatergic synapse in the caudal ventrolateral medulla (CVLM). In pentobarbital sodium-anesthetized rats, the RVLM on one side was inhibited by a muscimol microinjection. Then the response evoked by glutamate microinjections into the CMM or by baroreceptor stimulation was determined before and after 1) microinjection of the GABA receptor antagonist bicuculline into the RVLM on the other side or 2) microinjections of the glutamate receptor antagonist kynurenate bilaterally into the CVLM. Bicuculline in the RVLM greatly reduced both CMM- and baroreceptor-evoked sympathoinhibition. Compared with the effect of vehicle solution, kynurenate in the CVLM greatly reduced baroreceptor-evoked sympathoinhibition, whereas its effect on CMM-evoked sympathoinhibition was not different from that of the vehicle solution. These findings indicate that the output pathway from CMM sympathoinhibitory neurons, unlike the baroreceptor and other reflex sympathoinhibitory pathways, does not include a glutamatergic synapse in the CVLM.     

Angiotensin-(1--7) in the ovine fetus

In the adult animal, ANG-(1-7) may counterbalance some effects of ANG II. Its effects in the fetus are unknown. Basal ANG-(1-7), ANG I, ANG II, and renin concentrations were measured in plasma from ovine fetuses and their mothers (n = 10) at 111 days of gestation. In the fetus, concentrations of ANG I, ANG-(1-7), and ANG II were 86 +/- 21, 13 +/- 2, and 14 +/- 2 fmol/ml, respectively. In the ewe, concentrations of ANG I were significantly lower (20 +/- 4 fmol/ml, P < 0.05) as were concentrations of ANG-(1-7) (2.9 +/- 0.6 fmol/ml), whereas ANG II concentrations were not different (10 +/- 1 fmol/ml). Plasma renin concentrations were higher in the fetus (4.8 +/- 1.1 pmol ANG I x ml(-1) x h(-1)) than in the ewe (0.9 +/- 0.2 pmol x ml(-1) x h(-1), P < 0.05). Infusion of ANG-(1-7) (approximately 9 microg/h) for a 3-day period caused a significant increase in plasma concentrations of ANG-(1-7) reaching a maximum of 448 +/- 146 fmol/ml on day 3 of infusion. Plasma levels of ANG I and II as well as renin were unchanged by the infusion. Urine flow rate, glomerular filtration rate, and fetal arterial blood pressure did not change and were not different than values in fetuses receiving a saline infusion for 3 days (n = 5). However, the osmolality of amniotic and allantoic fluid was significantly higher in fetuses that received ANG-(1-7). Also, compared with the saline-infused animals, mRNA expression levels of renin, the AT(1) receptor, and AT(2) receptor were elevated in kidneys of fetuses that received infusions of ANG-(1-7). Infusion of an ANG-(1-7) antagonist ([D-Ala(7)]-ANG-(1-7), 20 microg/h) for 3 days had no effect on fetal blood pressure or renal function. In conclusion, although infusion of ANG-(1-7) did not affect fetal urine flow rate, glomerular filtration rate, or blood pressure, changes in fetal fluids and gene expression indicate that ANG-(1-7) may play a role in the fetal kidney.     

Attenuation of aortic baroreflex responses by microinjections of endomorphin-2 into the rostral ventrolateral medullary pressor area of the rat

The presence of mu-opioid receptors and endomorphins has been demonstrated in the general area encompassing the rostral ventrolateral medullary pressor area (RVLM). This investigation was carried out to test the hypothesis that endomorphins in the RVLM may have a modulatory role in regulating cardiovascular function. Blood pressure and heart rate (HR) were recorded in urethane-anesthetized male Wistar rats. Unilateral microinjections of endomorphin-2 (0.0125-0.5 mmol/l) into the RVLM elicited decreases in mean arterial pressure (16-30 mmHg) and HR (12-36 beats/min), which lasted for 2-4 min. Bradycardia was not vagally mediated. The effects of endomorphin-2 were mediated via mu-opioid receptors because prior microinjections of naloxonazine (1 mmol/l) abolished these responses; the blocking effect of naloxonazine lasted for 15-20 min. Unilateral stimulations of aortic nerve for 30 s (at frequencies of 5, 10, and 25 pulses/s; each pulse 0.5 V and 1-ms duration) elicited depressor and bradycardic responses. These responses were significantly attenuated by microinjections of endomorphin-2 (0.2 and 0.4 mmol/l). The inhibitory effect of endomorphin-2 on baroreflex responses was prevented by prior microinjections of naloxonazine. Microinjections of naloxonazine alone did not affect either baseline blood pressure and HR or baroreflex responses. These results indicate that endomorphin-2 elicits depressor and bradycardic responses and inhibits baroreflex function when injected into the RVLM. These effects are consistent with the known hyperpolarizing effect of opioid peptides on RVLM neurons.     

ACE2 and ANG-(1-7) in the rat uterus during early and late gestation

The present study was designed to determine ANG peptide content [ANG I, ANG II, ANG-(1-7)], ACE2 mRNA, and the immunocytochemical distribution of ANG-(1-7) and ACE2 in the uteroembryonic unit during early and late gestation in Sprague-Dawley rats and in a rat model of pregnancy-induced hypertension, the reduced uterine perfusion pressure (RUPP) model. At early pregnancy ANG-(1-7) and ACE2 staining were localized in the primary and secondary decidual zone and luminal and glandular epithelial cells. During late gestation, ANG-(1-7) and ACE2 staining was visualized in the labyrinth placenta and amniotic and yolk sac epithelium. Uterine ANG II concentration at early pregnancy was significantly decreased by 21-55% in the implantation and interimplantation sites compared with virgin rats, whereas ANG-(1-7) levels were maintained at prepregnancy levels. At late gestation, uterine concentrations of ANG I and ANG II were significantly increased (30% and 25%, respectively). In RUPP animals, ANG-(1-7) concentration is significantly reduced in the uterus (181 +/- 16 vs. 372 +/- 74 fmol/g of tissue) and placenta (143 +/- 26 vs. 197 +/- 20 fmol/g of tissue). ACE2 mRNA increased in the uterus of early pregnant compared with virgin rats, yet within the implantation site it was downregulated. At late pregnancy, ACE2 mRNA is elevated by 58% in the uterus and decreased by 59% in RUPP animals. The regulation of ANG-(1-7) and ACE2 in early and late pregnancy supports the hypothesis that ANG-(1-7) and ACE2 may act as a local autocrine/paracrine regulator throughout pregnancy, participating in the early (angiogenesis, apoptosis, and growth) and late (uteroplacental blood flow) events of pregnancy.     

Differential cardiorespiratory control elicited by activation of ventral medullary sites in mice.

We studied the respiratory and blood pressure responses to chemical stimulation of two regions of the ventral brainstem in mice: the rostral and caudal ventrolateral medulla (RVLM and CVLM, respectively). Stimulation of the RVLM by microinjections of the excitatory amino acid L-glutamate induced increases in diaphragm activity and breathing frequency, elevation of blood pressure (BP), and a slight increase in heart rate (HR). However, activation of the CVLM induced a decrease in breathing frequency, mainly due to prolongation of expiratory time (TE), and hypotension associated with a slight slowing of HR. Because adrenergic mechanisms are known to participate in the control of respiratory timing, we examined the role of alpha(2)-adrenergic receptors in the RVLM region in mediating these inhibitory effects. The findings demonstrated that blockade of the alpha(2)-adrenergic receptors within the RVLM by prior microinjection of SKF-86466 (an alpha(2)-adrenergic receptor blocker) significantly reduced changes in TE induced by CVLM stimulation but had little effect on BP responses. These results indicate that, in mice, activation of the RVLM increases respiratory drive associated with an elevation of BP, but stimulation of CVLM induces prolongation of TE via an alpha(2)-adrenergic signal transduction pathway.     

Chronic infusion of angiotensin-(1-7) into the lateral ventricle of the brain attenuates hypertension in DOCA-salt rats

Angiotensin-(ANG)-(1-7) is known by its central and peripheral actions, which mainly oppose the deleterious effects induced by accumulation of ANG II during pathophysiological conditions. In the present study we evaluated whether a chronic increase in ANG-(1-7) levels in the brain would modify the progression of hypertension. After DOCA-salt hypertension was induced for seven days, Sprague-Dawley rats were subjected to 14 days of intracerebroventricular (ICV) infusion of ANG-(1-7) (200 ng/h, DOCA-A7) or 0.9% sterile saline. As expected, on the 21st day, DOCA rats presented increased mean arterial pressure (MAP) (≈40%), and impaired baroreflex control of heart rate (HR) and baroreflex renal sympathetic nerve activity (RSNA) in comparison with that in normotensive control rats (CTL). These changes were followed by an overactivity of the cardiac sympathetic tone and reduction of the cardiac parasympathetic tone, and exaggerated mRNA expression of collagen type I (≈9-fold) in the left ventricle. In contrast, DOCA rats treated with ANG-(1-7) ICV had an improvement of baroreflex control of HR, which was even higher than that in CTL, and a restoration of the baroreflex control of RSNA, the balance of cardiac autonomic tone, and normalized mRNA expression of collagen type I in the left ventricle. Furthermore, DOCA-A7 had MAP lowered significantly. These effects were not accompanied by significant circulating or cardiac changes in angiotensin levels. Taken together, our data show that chronic increase in ANG-(1-7) in the brain attenuates the development of DOCA-salt hypertension, highlighting the importance of this peptide in the brain for the treatment of cardiovascular diseases.     

Does angiotensin II have a significant tonic action on cardiovascular neurons in the rostral and caudal VLM?

The peptidic ANG II receptor antagonists [Sar(1),Ile(8)]ANG II (sarile) or [Sar(1),Thr(8)]ANG II (sarthran) are known to decrease arterial pressure and sympathetic activity when injected into the rostral part of the ventrolateral medulla (VLM). In anesthetized rabbits and rats, the profound depressor and sympathoinhibitory response after bilateral microinjections of sarile or sarthran into the rostral VLM was unchanged after prior selective blockade of angiotensin type 1 (AT(1)) and ANG-(1---7) receptors, although this abolished the effects of exogenous ANG II. Unlike the neuroinhibitory compounds muscimol or lignocaine, microinjections of sarile in the rostral VLM did not affect respiratory activity. Sarile or sarthran in the caudal VLM resulted in a large pressor and sympathoexcitatory response, which was also unaffected by prior blockade of AT(1) and ANG-(1---7) receptors. The results indicate that the peptidic ANG receptor antagonists profoundly inhibit the tonic activity of cardiovascular but not respiratory neurons in the VLM and that these effects are independent of ANG II or ANG-(1---7) receptors.