Dmitry N. Mayorov

AT1 receptors in the RVLM mediate pressor responses to emotional stress in rabbits

Abstract—In this study, we examined the role of angiotensin type 1 (AT1) receptors in the rostral ventrolateral medulla (RVLM) in mediating the pressor action of emotional stress in conscious rabbits. Rabbits were chronically instrumented with guide cannulas for bilateral microinjections into the RVLM and an electrode for measuring renal sympathetic nerve activity (RSNA). Airjet stress evoked increases in arterial pressure, heart rate, and RSNA, which reached a maximum (+9±1 mm Hg, +20±5 beats/min, and +93±17%, respectively) in the first 2 minutes of stress exposure. Then RSNA rapidly returned to prestress values, while arterial pressure and heart rate remained close to the maximal level until the conclusion of the 7-minute airjet exposure. Microinjections of the nonselective angiotensin receptor antagonist sarile (0.5 nmol, n=8) or AT1 receptor antagonists losartan (2 nmol, n=6) or candesartan (0.2 nmol, n=6) into the RVLM did not alter resting cardiovascular parameters. By contrast, the antagonists attenuated the sustained phase (4 to 7 minutes) of the pressor stress response by 55% to 89%. However, only sarile decreased the onset of this response. The antagonists affected neither the stress-induced tachycardia nor the pressor response to glutamate microinjections. Microinfusion of angiotensin II (4 pmol/min, n=8) into the RVLM did not change the pressor response to airjet stress but attenuated tachycardic response by 47%. Microinjections of vehicle did not alter the cardiovascular stress response. Sarile, losartan, and angiotensin II did not affect the sympathoexcitatory response to baroreceptor unloading. These results suggest that AT1 receptors in the RVLM are important in mediating the pressor effects of emotional stress in conscious rabbits.

Renin Enhancer Is Critical for Control of Renin Gene Expression and Cardiovascular Function

The important cardiovascular regulator renin contains a strong in vitro enhancer 2.7 kb upstream of its gene. Here we tested the in vivo role of the mouse Ren-1c enhancer. In renin-expressing As4.1 cells stably transfected with Ren-1c promoter with or without enhancer, expression of linked β-geo reporter, stable expression, and colony formation were dependent on the presence of the enhancer. We then generated mice carrying a targeted deletion of the enhancer (REKO mice) and found marked depletion of renin in renal juxtaglomerular and submandibular ductal cells, as well as hyperplasia of macula densa cells. Plasma creatinine was increased, but electrolytes were normal. Male REKO mice implanted with telemetry devices had 9 ± 1 mm Hg lower mean arterial pressure (p < 0.001), which was partly normalized by a high NaCl diet. Locomotor activity was lower, and baroreflex sensitivity was normal. Markedly reduced mean arterial pressure variability in the midfrequency band indicated a contribution of reduced sympathetic vasomotor tone to the hypotension. In conclusion, the renin enhancer is critical for renin gene expression and physiological sequelae, including response to alteration in salt intake. The REKO mouse may be useful as a low renin expression model.

Central Angiotensin and Baroreceptor Control of Circulation

Abstract: Angiotensin (Ang) receptors are located in many important central nuclei involved in the regulation of the cardiovascular system. While most interest has focused on forebrain circumventricular actions, areas of the brainstem such as the nucleus of the solitary tract and the ventrolateral medulla contain high concentrations of AT1 receptors. The present review encompasses the physiological role of Ang II in the hindbrain, particularly in relation to its influence on baroreflex control mechanisms. In rabbits there are sympatho‐excitatory AT1 receptors in the rostral ventrolateral medulla (RVLM), accessible to Ang II from the cerebrospinal fluid. Activation of these receptors acutely increases renal sympathetic nerve activity (RSNA) and RSNA baroreflex responses. However, blockade of endogenous Ang receptors in the brainstem also shows sympathoexcitation, suggesting there is greater endogenous activity of a sympathoinhibitory Ang II action. Microinjections of angiotensin antagonists into the RVLM showed relatively little tonic activity of endogenous Ang II influencing sympathetic activity in conscious rabbits. However, Ang II receptors in the RVLM mediate sympathetic responses to airjet stress in conscious rabbits. Similarly with respect to heart rate baroreflexes, there appears to be little tonic effect of angiotensin in the brainstem in normal conscious animals. Chronic infusion of Ang II for two weeks into the fourth ventricle of conscious rabbits inhibits the cardiac baroreflex while infusion of losartan increases the gain of the reflex. These actions suggest that Ang II in the brainstem modulates sympathetic responses depending on specific afferent and synaptic inputs in both the short term but importantly also in the long term, thus forming an important mechanism for increasing the range of adaptive response patterns.

Role of Angiotensin II Type 1A Receptors in Cardiovascular Reactivity and Neuronal Activation After Aversive Stress in Mice

We determined whether genetic deficiency of angiotensin II Type 1A (AT1A) receptors in mice results in altered neuronal responsiveness and reduced cardiovascular reactivity to stress. Telemetry devices were used to measure mean arterial pressure, heart rate, and activity. Before stress, lower resting mean arterial pressure was recorded in AT1A−/− (85±2 mm Hg) than in AT1A+/+ (112±2 mm Hg) mice; heart rate was not different between groups. Cage-switch stress for 90 minutes elevated blood pressure by +24±2 mm Hg in AT1A+/+ and +17±2 mm Hg in AT1A−/− mice (P<0.01), and heart rate increased by +203±9 bpm in AT1A+/+ and +121±9 bpm in AT1A−/− mice (P<0.001). Locomotor activation was less in AT1A−/− (3.0±0.4 U) than in AT1A+/+ animals (6.0±0.4 U), but differences in blood pressure and heart rate persisted during nonactive periods. In contrast to wild-type mice, spontaneous baroreflex sensitivity was not inhibited by stress in AT1A−/− mice. After cage-switch stress, c-Fos immunoreactivity was less in the paraventricular (P<0.001) and dorsomedial (P=0.001) nuclei of the hypothalamus and rostral ventrolateral medulla (P<0.001) in AT1A−/− compared with AT1A+/+ mice. Conversely, greater c-Fos immunoreactivity was observed in the medial nucleus of the amygdala, caudal ventrolateral medulla, and nucleus of the solitary tract (P<0.001) of AT1A−/− compared with AT1A+/+ mice. Greater activation of the amygdala suggests that AT1A receptors normally inhibit the degree of stress-induced anxiety, whereas the lesser activation of the hypothalamus and rostral ventrolateral medulla suggests that AT1A receptors play a key role in autonomic cardiovascular reactions to acute aversive stress, as well as for stress-induced inhibition of the baroreflex.

Selective Sensitization by Nitric Oxide of Sympathetic Baroreflex in Rostral Ventrolateral Medulla of Conscious Rabbits

Nitric oxide (NO) deficiency in the rostral ventrolateral medulla (RVLM) has been implicated in impaired baroreflex control in hypertensive and heart failure animals. However, the role of local NO in normal baroreflex regulation remains unclear. This study aimed to examine the role of NO in tonic and baroreflex control of blood pressure (BP) in the RVLM of conscious rabbits. Microinjections of NO donors, S-nitroso-N-acetylpenicillamine and sodium nitroprusside (5 to 20 nmol), or NO itself (20 to 200 pmol) into the RVLM dose-dependently increased BP. Bilateral microinjections of an NO synthase (NOS) inhibitor NG-nitro-l-arginine methyl ester (l-NAME; 10 nmol), its inactive enantiomer d-NAME, or soluble guanylate cyclase (sGC) inhibitors, 1-H-[1,2,4]oxadiaolo[4,3-a]quinoxalin-1-one (ODQ, 250 pmol) and methylene blue (10 nmol), into the RVLM did not affect resting BP, heart rate, or renal sympathetic nerve activity (RSNA). However, l-NAME, methylene blue, and ODQ decreased RSNA baroreflex gain by 42% to 55%, whereas d-NAME did not affect this reflex. Co-microinjections of l-NAME and superoxide scavenger tempol (20 nmol) decreased RSNA baroreflex gain by 37±8%. Microinjections of a neuronal NOS (nNOS) inhibitor, 7-nitroindazole (500 pmol), into the RVLM decreased RSNA baroreflex gain by 42±12%, without altering resting BP, heart rate, or RSNA. Local administration of inducible NOS (iNOS) inhibitors, S-methylisothiourea (0.25 nmol) and aminoguanidine (0.25 and 2.5 nmol), affected neither resting nor baroreflex parameters. These results suggest that nNOS-derived NO facilitates sympathetic baroreflex transmission in the RVLM at least in part via a sGC-dependent, superoxide-independent mechanism. However, local nNOS and iNOS play little role in the tonic support of BP in conscious rabbits.

Blood pressure reactivity to emotional stress is reduced in AT1A-receptor knockout mice on normal, but not high salt intake

Pharmacological evidence suggests that angiotensin II type 1 (AT1) receptors are involved in the regulation of cardiovascular response to emotional stress and reinforcing effect of dietary salt on this response. In this study, we examined the effect of genetic deletion of AT1A receptors on the cardiovascular effects of stress and salt in mice. AT1A receptor knockout (AT1A−/−) and wild-type (AT1A+/+) mice were implanted with telemetry devices and placed on a normal (0.4%) or high (3.1%) salt diet (HSD). Resting blood pressure (BP) in AT1A−/− mice (84±3 mm Hg) was lower than in AT1A+/+ mice (107±2 mm Hg). Negative emotional (restraint) stress increased BP by 33±3 mm Hg in AT1A+/+ mice. This response was attenuated by 40% in AT1A−/− mice (18±3 mm Hg). Conversely, the BP increase caused by food presentation and feeding was similar in AT1A−/− (25±3 mm Hg) and AT1A+/+ mice (26±3 mm Hg). HSD increased resting BP by 14±4 mm Hg in AT1A−/− mice without affecting it significantly in AT1A+/+ mice. Under these conditions, the pressor response to restraint stress in AT1A−/− mice (30±3 mm Hg) was no longer different from that in wild-type animals (28±3 mm Hg). The BP response to feeding was not altered by HSD in either AT1A−/− or AT1A+/+ mice (25±2 and 27±3 mm Hg, respectively). These results indicate that AT1A receptor deficiency leads to a reduction in BP reactivity to negative emotional stress, but not feeding. HSD can selectively reinforce the cardiovascular response to negative stress in AT1A−/− mice. However, there is little interaction between AT1A receptors, excess dietary sodium and feeding-induced cardiovascular arousal.

Ionotropic glutamate receptors in the rostral ventrolateral medulla mediate sympathetic responses to acute stress in conscious rabbits.

In conscious, chronically instrumented rabbits (n = 7), airjet stress evoked increases in arterial pressure (AP) and renal sympathetic nerve activity (RSNA), which were greatest in the first 2 min (+ 10 mm Hg and + 127%, respectively), but then rapidly reached a stable level (+ 7 mm Hg and + 37%, respectively). Bilateral microinjection into the rostral ventrolateral medulla (RVLM) of an ionotropic excitatory amino acid (EAA) receptor antagonist kynurenate (10 nmol/200 nl) did not affect resting AP and RSNA, but reduced their initial peak responses to airjet by 80% and 52%, respectively, without altering the stable levels of these responses. By contrast, bilateral microinfusion of glutamate (2 nmol/20 nl/min) into the RVLM increased resting AP by 13 mm Hg, but did not alter the RSNA and AP responses to airjet stress. These results suggest that the RVLM is an essential site for conveying excitatory environmental influences to the sympathetic nervous system in conscious rabbits. The EAA receptors are critically important in initiating the pressor and sympathoexcitatory responses to acute emotional stress, but play relatively little role in the maintenance of these responses.

Relative importance of rostral ventrolateral medulla in sympathoinhibitory action of rilmenidine in conscious and anesthetized rabbits

The pressor region of the rostral ventrolateral medulla (RVLM) is a critical site in the sympathoinhibitory action of imidazoline receptor agonists as shown by studies in anesthetized animals. The aim of this study was to compare the importance of the RVLM in mediating the inhibitory action of rilmenidine on renal sympathetic nerve activity (RSNA) and arterial pressure in urethane-anesthetized rabbits (n = 11) and in conscious, chronically instrumented rabbits (n = 6). Bilateral microinjection of rilmenidine (4 nmol in 100 nl) into the RVLM caused a greater decrease in resting arterial pressure in anesthetized animals (−19 mm Hg) than in conscious animals (−8 mm Hg). By contrast, the decrease in resting RSNA evoked by rilmenidine was similar in conscious (−27%) and anesthetized (−36%) rabbits. Furthermore, rilmenidine microinjection into the RVLM was equally effective in inhibiting the RSNA baroreflex in both groups of animals. The upper plateau of the RSNA baroreflex decreased by 37% and 42%, and gain decreased by 41% and 44% after rilmenidine treatments in conscious and anesthetized rabbits, respectively. We conclude that the RVLM plays an equally important role in the inhibitory action of rilmenidine on RSNA in conscious and anesthetized rabbits either at rest or during baroreflex responses. A relatively moderate effect of rilmenidine on arterial pressure in conscious, chronically instrumented rabbits may relate to a lower level of sympathetic drive compared with anesthetized animals.

Brain angiotensin AT1 receptors as specific regulators of cardiovascular reactivity to acute psychoemotional stress.

1. Cardiovascular reactivity, an abrupt rise in blood pressure (BP) and heart rate in response to psychoemotional stress, is a risk factor for heart disease. Pharmacological and molecular genetic studies suggest that brain angiotensin (Ang) II and AT1 receptors are required for the normal expression of sympathetic cardiovascular responses to various psychological stressors. Moreover, overactivity of the brain AngII system may contribute to enhanced cardiovascular reactivity in hypertension.

The Day–Night Difference of Blood Pressure Is Increased in AT1A-Receptor Knockout Mice on a High-Sodium Diet

BACKGROUND Abnormal circadian variation of blood pressure (BP) increases cardiovascular risk. In this study, we examined the influence of angiotensin AT(1A) receptors on circadian BP variation, and specifically on its behavioral activity-related and -unrelated components. METHODS BP and locomotor activity were recorded by radiotelemetry in AT(1A)-receptor knockout mice (AT(1A)(-/-)) and their wild-type controls (AT(1A)(+/+)) placed on a normal-salt diet (NSD) or high-salt diet (HSD, 3.1% Na). RESULTS The 24-h BP was lower in AT(1A)(-/-) than AT(1A)(+/+) mice on a NSD (92 +/- 2 and 118 +/- 2 mm Hg, respectively), whereas the day-night BP difference (DeltaDNBP) was similar between groups (11 +/- 2 and 12 +/- 1 mm Hg, respectively). HSD increased BP by 20 +/- 2 mm Hg and DeltaDNBP by 7 +/- 1 mm Hg in AT(1A)(-/-) mice, without affecting these parameters much in AT(1A)(+/+) mice. The DeltaDNBP increase in AT(1A)(-/-) mice was caused by nondipping BP during the inactive late-dark period. Conversely, BP rise associated with circadian behavioral activation during the early dark period was not altered by HSD in AT(1A)(-/-) mice. The BP change associated with spontaneous ultradian activity-inactivity bouts was also similar between strains on HSD as was the BP rise associated with induced (cage-switch) behavioral activity. Ganglionic or alpha(1)-adrenergic blockade decreased BP in both strains; HSD did not affect this response in AT(1A)(-/-), but abolished it in AT(1A)(+/+) mice. CONCLUSIONS AT(1A)-receptor deficiency, when combined with HSD, can increase circadian BP difference in mice. This increase is mediated principally by activity-unrelated factors, such as the nonsuppressibility of basal resting sympathetic tone by HSD, thus suggesting a form of salt-/volume-dependent hypertension.