Kristy L. Jackson

A Novel Interaction Between Sympathetic Overactivity and Aberrant Regulation of Renin by miR-181a in BPH/2J Genetically Hypertensive Mice

Genetically hypertensive mice (BPH/2J) are hypertensive because of an exaggerated contribution of the sympathetic nervous system to blood pressure. We hypothesize that an additional contribution to elevated blood pressure is via sympathetically mediated activation of the intrarenal renin–angiotensin system. Our aim was to determine the contribution of the renin–angiotensin system and sympathetic nervous system to hypertension in BPH/2J mice. BPH/2J and normotensive BPN/3J mice were preimplanted with radiotelemetry devices to measure blood pressure. Depressor responses to ganglion blocker pentolinium (5 mg/kg IP) in mice pretreated with the angiotensin-converting enzyme inhibitor enalaprilat (1.5 mg/kg IP) revealed a 2-fold greater sympathetic contribution to blood pressure in BPH/2J mice during the active and inactive period. However, the depressor response to enalaprilat was 4-fold greater in BPH/2J compared with BPN/3J mice, but only during the active period (P=0.01). This was associated with 1.6-fold higher renal renin messenger RNA (mRNA; P=0.02) and 0.8-fold lower abundance of micro-RNA-181a (P=0.03), identified previously as regulating human renin mRNA. Renin mRNA levels correlated positively with depressor responses to pentolinium (r=0.99; P=0.001), and BPH/2J mice had greater renal sympathetic innervation density as identified by tyrosine hydroxylase staining of cortical tubules. Although there is a major sympathetic contribution to hypertension in BPH/2J mice, the renin–angiotensin system also contributes, doing so to a greater extent during the active period and less during the inactive period. This is the opposite of the normal renin–angiotensin system circadian pattern. We suggest that renal hyperinnervation and enhanced sympathetically induced renin synthesis mediated by lower micro-RNA-181a contributes to hypertension in BPH/2J mice.

Renin-angiotensin and sympathetic nervous system contribution to high blood pressure in Schlager mice.

Objective Schlager hypertensive (BPH/2J) mice have been suggested to have high blood pressure (BP) due to an overactive sympathetic nervous system (SNS), but the contribution of the renin–angiotensin system (RAS) is unclear. In the present study, we examined the cardiovascular effects of chronically blocking the RAS in BPH/2J mice. Methods Schlager normotensive (BPN/3J, n = 6) and BPH/2J mice (n = 8) received the angiotensin AT1A-receptor antagonist losartan (150 mg/kg per day) in drinking water for 2 weeks. Pre-implanted telemetry devices were used to record mean arterial pressure (MAP), heart rate (HR) and locomotor activity. Results MAP was reduced by losartan treatment in BPN/3J (−23 mmHg, P < 0.01) and in BPH/2J mice (−25 mmHg, P < 0.001), whereas HR was increased. Losartan had little effect on initial pressor responses to feeding or to stress, but did attenuate the sustained pressor response to cage-switch stress. During the active period, the hypotension to sympathetic blockade with pentolinium was greater in BPH/2J than BPN/3J (suggesting neurogenic hypertension), but was not affected by losartan. During the inactive period, a greater depressor response to pentolinium was observed in losartan-treated animals. Conclusion The hypotensive actions of losartan suggest that although the RAS provides an important contribution to BP, it contributes little, if at all, to the hypertension-induced or the greater stress-induced pressor responses in Schlager mice. The effects of pentolinium suggest that the SNS is mainly responsible for hypertension in BPH/2J mice. However, the RAS inhibits sympathetic vasomotor tone during inactivity and prolongs sympathetic activation during periods of adverse stress, indicating an important sympatho-modulatory role.

Cardiovascular reactivity and neuronal activation to stress in Schlager genetically hypertensive mice.

Schlager inbred hypertensive mice (BPH/2J) have been suggested to have high blood pressure (BP) due to an overactive sympathetic nervous system (SNS). The brain nuclei associated with the hypertension are also those involved in the integration of the cardiovascular responses to stress. Therefore, in the present study, we hypothesize that BPH/2J mice likely have a greater response to stress that is associated with greater neuronal activation in the limbic system, hypothalamus and medulla in regions known to regulate sympathetic activity. Male hypertensive BPH/2J and normotensive BPN/3J mice were implanted with telemetry devices and exposed to dirty cage-switch, an acute model of aversive stress. Stress exposure caused a 60% greater pressor response in BPH/2J compared with BPN/3J mice and an increase in activity, by contrast the level of tachycardia was less in BPH/2J mice. Stress-induced cardiovascular responses were also associated with greater neuronal activation, as detected by c-Fos expression, in BPH/2J compared with BPN/3J mice in the medial nucleus of the amygdala (MeAm), dorsomedial hypothalamus (DMH) (P<0.001) and marginally in the rostral ventrolateral medulla (RVLM; P=0.7). These findings suggest that hypertension in the BPH/2J mice is associated with greater sympathetic vasomotor responses to central pathways mediating the arousal responses to acute aversive stress in particular the amygdala, hypothalamus and rostral ventrolateral medulla.

Contribution of Orexin to the Neurogenic Hypertension in BPH/2J Mice

BPH/2J mice are a genetic model of hypertension associated with an overactive sympathetic nervous system. Orexin is a neuropeptide which influences sympathetic activity and blood pressure. Orexin precursor mRNA expression is greater in hypothalamic tissue of BPH/2J compared with normotensive BPN/3J mice. To determine whether enhanced orexinergic signaling contributes to the hypertension, BPH/2J and BPN/3J mice were preimplanted with radiotelemetry probes to compare blood pressure 1 hour before and 5 hours after administration of almorexant, an orexin receptor antagonist. Mid frequency mean arterial pressure power and the depressor response to ganglion blockade were also used as indicators of sympathetic nervous system activity. Administration of almorexant at 100 (IP) and 300 mg/kg (oral) in BPH/2J mice during the dark-active period (2 hours after lights off) markedly reduced blood pressure (−16.1±1.6 and −11.0±1.1 mm Hg, respectively; P<0.001 compared with vehicle). However, when almorexant (100 mg/kg, IP) was administered during the light-inactive period (5 hours before lights off) no reduction from baseline was observed (P=0.64). The same dose of almorexant in BPN/3J mice had no effect on blood pressure during the dark (P=0.79) or light periods (P=0.24). Almorexant attenuated the depressor response to ganglion blockade (P=0.018) and reduced the mid frequency mean arterial pressure power in BPH/2J mice (P<0.001), but not BPN/3J mice (P=0.70). Immunohistochemical labeling revealed that BPH/2J mice have 29% more orexin neurons than BPN/3J mice which are preferentially located in the lateral hypothalamus. The results suggest that enhanced orexinergic signaling contributes to sympathetic overactivity and hypertension during the dark period in BPH/2J mice.

GABAA receptor dysfunction contributes to high blood pressure and exaggerated response to stress in Schlager genetically hypertensive mice.

Objective: Schlager BPH/2J hypertensive mice have high blood pressure (BP) likely due to overactivity of the sympathetic nervous system regulated by neurons in amygdala-hypothalamic pathways. These areas are normally under tonic inhibition by GABA containing neurons that may be deficient in Schlager hypertensive mice as suggested by microarray analysis. In the present study, cardiovascular effects of chronic activation of GABAA receptors were examined in BPH/2J mice. Methods: Male normotensive BPN/3J and hypertensive BPH/2J mice were administered diazepam in drinking water for 7 days. BP, heart rate and locomotor activity were recorded by telemetry. Results: Diazepam (2.5 mg/kg) reduced BP of BPN/3J mice during the night-time by −7.1 ± 2.0 mmHg (P = 0.001) but had no effect in BPH/2J mice (+2 ± 2 mmHg) and no effect on heart rate or locomotor activity in either strain. Diazepam reduced the responses to restraint stress in BPN/3J mice by 20% (P = 0.01) and there was no association between Fos-immunoreactive neurons and neurons expressing GABAA receptors or neuropeptide Y in the medial amygdala and paraventricular nucleus of the hypothalamus. By contrast diazepam had no effect on the pressor response to stress in BPH/2J mice and ∼50% of stress-activated neurons in these regions also expressed GABAA receptors and ∼45% were neuropeptide Y-containing. Conclusion: These findings show that BPH/2J mice are resistant to the effects of diazepam and suggest that GABAA receptor dysfunction in BPH/2J mice may be contributing to the neurogenic hypertension by not suppressing arousal-induced sympathetic activation within amygdala and hypothalamic nuclei.

Major Contribution of the Medial Amygdala to Hypertension in BPH/2J Genetically Hypertensive Mice

BPH/2J mice are recognized as a neurogenic model of hypertension primarily based on overactivity of the sympathetic nervous system and greater neuronal activity in key autonomic cardiovascular regulatory brain regions. The medial amygdala (MeAm) is a forebrain region that integrates the autonomic response to stress and is the only region found to have greater Fos during the night and daytime in BPH/2J compared with BPN/3J mice. To determine the contribution of the MeAm to hypertension, the effect of neuronal ablation on blood pressure (BP) was assessed in BPH/2J (n=7) and normotensive BPN/3J mice (n=7). Mice were preimplanted with radiotelemetry devices to measure 24-hour BP and cardiovascular responses to stress, before and 1 to 3 weeks after bilateral lesions of the MeAm. Baseline BP was 121±4 mm Hg in BPH/2J and 101±2 mm Hg in BPN/3J mice (Pstrain<0.001). MeAm lesions reduced BP by 11±2 mm Hg in BPH/2J mice (Plesion<0.001) but had no effect in BPN/3J mice. The hypotensive effect of lesions in BPH/2J mice was similar during both day and night, suggesting that the MeAm has tonic effects on BP, but the pressor response to stress was maintained in both strains. Midfrequency BP power was attenuated in both strains (Plesion<0.05) and the depressor responses to pentolinium after enalaprilat pretreatment was attenuated after lesions in BPH/2J mice (Plesion<0.001; n=3). These findings indicate that the MeAm provides a tonic contribution to hypertension in BPH/2J mice, which is independent of its role in stress reactivity or circadian BP influences.

6B.01: EFFECT OF RENAL DENERVATION ON BLOOD PRESSURE AND MICRORNA 181A IN HYPERTENSIVE SCHLAGER MICE.

Objective: Hypertensive Schlager mice (BPH) are hypertensive due to an exaggerated contribution of the sympathetic nervous system (SNS) and renin angiotensin system (RAS). The latter was associated with reduced expression of the renin regulatory micro RNA-181a. We therefore determined the effect of bilateral renal denervation (Rx) on blood pressure in BPH compared to normotensive BPN. Design and method: Blood pressure was measured by in 16 week old conscious mice by radiotelemetry and Rx was performed by surgery and 10% phenol application. Results: After 3 weeks recovery, mean 24 hour blood pressure in BPH was 110 ± 1 mmHg in BPN and 128 ± 2 mmHg in BPH. Blood pressure was 8 ± 2 mmHg lower than sham in Rx BPH mice but Rx had no effect in BPN. Following Rx, blocking the renin angiotensin system with enalapril decreased blood pressure more in BPH mice compared to sham group but had no effect in BPN. Micro RNA-181a levels in the kidney were lower and renin mRNA higher in BPH compared to BPN. The depressor response to the ganglionic blocker pentolinium (SNS contribution) was greater in BPN mice following Rx compared with the sham group but the response was unaffected by Rx in BPH. Rx reduced renal norepinephrine levels in both strains but more so in BPH. Rx normalised both miR-181a (0.72 ± 0.02 BPH vs 0.73 ± 0.03 BPN, NS) and renin mRNA (1.9 ± 0.1 BPH vs 1.61 ± 0.2 BPN, NS) in BPH/2J to levels comparable to the control strain. Conclusions: We suggest that renal sympathetic activity is essential in maintaining hypertension in BPH mice partly by overexpression of renal renin as a result of inhibiting micro RNA-181a. Importantly we demonstrate for the first time that sympathetic activity directly regulates renin expression through inhibition of micro RNA-181a. These findings may explain the positive effectiveness of Rx in neurogenic hypertension.

Cardiovascular role of angiotensin type1A receptors in the nucleus of the solitary tract of mice

AIMS The nucleus of the solitary tract (NTS) is important for cardiovascular regulation and contains angiotensin type 1A (AT1A) receptors. To assess its function, we examined the effect of expressing in AT1A receptors in the NTS of mice lacking these receptors. METHODS AND RESULTS Bilateral microinjections of lentivirus expressing AT1A receptors (AT1Av mice, n = 6) or green fluorescent protein (GFPv, n = 8, control) under the control of the PRSx8 promotor were made into the NTS of AT1A receptors null mice (AT1A(-/-)). Telemetry devices recorded blood pressure (BP), heart rate (HR), and locomotor activity. Expression of AT1A receptors in the NTS increased BP by 11.2 ± 4 mmHg (P < 0.05) at 2 and 3 weeks, whereas GFPv mice remained at pre-injection BP. Ganglion blockade reduced BP to similar levels pre- and post-transfection in GFPv and AT1Av mice. Greater pressor responses to cage-switch stress were observed following AT1A receptors expression (+18 ± 2 mmHg pre- to +24 ± 2 mmHg post-virus, P < 0.05) with similar stress-induced pressor responses pre- and post-virus in GFPv mice. Pressor responses to restraint stress pre- and post-virus were similar in AT1Av but were 20% less post-GFPv (P < 0.001). The lack of attenuation in BP to restraint was associated with four-fold greater Fos-expression in AT1A receptors mice. AT1A receptors expression in the NTS did not alter baroreflex gain differently between groups. CONCLUSION The results suggest that transfection of AT1A receptors on neurons in the NTS elevates BP independent of the SNS and pressor responses to aversive stimuli are associated with greater Fos-expression in forebrain regions. This study suggests a novel mechanism by which the NTS may modulate MAP in the long-term via AT1A receptors.

Factors Responsible for Obesity-Related Hypertension

Purpose of ReviewThe major health issue of being overweight or obese relates to the development of hypertension, insulin resistance and diabetic complications. One of the major underlying factors influencing the elevated blood pressure in obesity is increased activity of the sympathetic nerves to particular organs such as the kidney.Recent FindingsThere is now convincing evidence from animal studies that major signals such as leptin and insulin have a sympathoexcitatory action in the hypothalamus to cause hypertension. Recent studies suggest that this may involve ‘neural plasticity’ within hypothalamic signalling driven by central actions of leptin mediated via activation of melanocortin receptor signalling and activation of brain neurotrophic factors.SummaryThis review describes the evidence to support the contribution of the SNS to obesity related hypertension and the major metabolic and adipokine signals.

Actions of rilmenidine on neurogenic hypertension in BPH/2J genetically hypertensive mice

Objective: BPH/2J hypertensive mice have an exaggerated sympathetic contribution to blood pressure (BP). Premotor sympathetic neurons within the rostroventrolateral medulla (RVLM) are a major source of sympathetic vasomotor tone and major site of action of the centrally acting sympatholytic agent, rilmenidine. The relative cardiovascular effect of rilmenidine in BPH/2J versus normotensive BPN/3J mice was used as an indicator of the involvement of the RVLM in the sympathetic contribution to hypertension in BPH/2J mice. Methods: BPH/2J and BPN/3J mice were pre-implanted with telemetry devices to measure BP in conscious unrestrained mice. Rilmenidine was administered acutely (n = 7–9/group), orally for 14 days, at a wide range of doses (n = 5/group), and also infused intracerebroventricularly for 7 days (n = 6/group). Results: Acute intraperitoneal rilmenidine induced greater depressor and bradycardic responses in BPH/2J than BPN/3J mice (Pstrain < 0.01). Both responses were reduced by atropine pre-treatment, with the remaining hypotensive effect being small and comparable between strains (Pstrain = 1.0). This suggests that vagally induced reductions in cardiac output were responsible for the hypotension. Chronic intracerebroventricularly infused rilmenidine reduced BP from baseline marginally in BPH/2J mice during the dark (active) period (−6.5 ± 2 mmHg; P = 0.006). Chronic orally administered rilmenidine (1–12 mg/kg per day) also had minimal effect on 24-h BP in both strains (P > 0.16). Conclusion: The sympathetic vasomotor inhibitory effect of rilmenidine is minimal in both strains and similar in hypertensive BPH/2J and BPN/3J mice. Thus, hypertension in BPH/2J mice is not likely mediated by greater neuronal activity in the RVLM, and agents such as rilmenidine would be an ineffective treatment for this form of neurogenic hypertension.