Role In Central Cardiovascular Regulation Research Articles

Temporal Profiling of Molecular Alterations in the Hypothalamic Paraventricular Nucleus During Angiotensin‐II Hypertension

The hypothalamic paraventricular nucleus (PVN) plays a central role in cardiovascular regulation including hypertension development in response to elevations in angiotensin-II (Ang-II). Upstream circumventricular nuclei located outside of the blood-brain-barrier sense circulating Ang-II and influence PVN endocrine and autonomic regulation via efferent synaptic projections. Despite progress in unraveling the function of circumventricular-PVN networks in neurogenic hypertension development, the underlying PVN mechanisms responsible for hypertension development remain unclear. In particular, most investigations have examined PVN alterations during established hypertension, which limits insight into molecular changes that may occur prior to hypertension development. Based on this, we performed a detailed temporal profiling of key molecular targets in the PVN during Ang-II hypertension progression. Male C57Bl/6J mice were implanted with subcutaneous osmotic minipumps for chronic delivery of low dose Ang-II (600 ng/kg/min). Brains were harvested for immunohistochemistry analysis of target molecules across rostral to caudal subregions of the PVN (n=3-4/group) at baseline (day 0), pre-hypertensive (days 3 and 7), and established-hypertension (day 14) timepoints. Previous work has suggested a role for arginine vasopressin (AVP) in Ang-II-induced hypertension. In line with this, AVP immunoreactivity in the PVN was elevated within 3 days of Ang-II infusion; this was evident only in the caudal PVN (day 3 fold change: 2.5±0.2, density/cell, p<0.05), whereas AVP expression in the rostral and medial portions was unchanged. Interestingly, caudal AVP immunoreactivity returned to basal levels by day 7 and remained low for the remainder of the protocol. In contrast to AVP, oxytocin expression was unchanged across the rostral to caudal PVN during 2-week Ang-II hypertension development. We also measured preproenkephalin (PPE) immunoreactivity, and found a progressive increase throughout the entire PVN with elevations initiated at pre-hypertensive times (day 7 fold change: 2.0±0.1, density/area, p=0.08) that were sustained during established hypertension (day 14 fold change: 2.6±0.3, density/area, p<0.05). To test possible involvement of the arcuate nucleus, which transmits circulating information from the median eminence to the PVN, in Ang-II hypertension, we measured alpha-melanocortin stimulating hormone (alpha-MSH) levels. Histological analysis for alpha-MSH immunoreactivity in the PVN, as an indicator of synaptic innervation strength from arcuate nucleus proopiomelanocortin neurons, revealed no changes during Ang-II infusion. Collectively, these findings indicate elevations in PVN AVP and PPE, but not oxytocin and alpha-MSH, in response to elevations in circulating Ang-II. Furthermore, the temporal responses suggest that different PVN molecules may be responsible for the initiation (e.g. AVP) versus maintenance (e.g. PPE) of Ang-II hypertension.

Potential Role of the Amygdala and Posterior Claustrum in Exercise Intensity-dependent Cardiovascular Regulation in Rats

Tuning of the cardiovascular response is crucial to maintain performance during high-intensity exercise. It is well known that the nucleus of the solitary tract (NTS) in the brainstem medulla plays a central role in cardiovascular regulation; however, where and how upper brain regions form circuits with NTS and coordinately control cardiovascular responses during high-intensity exercise remain unclear. Here focusing on the amygdala and claustrum, we investigated part of the mechanism for regulation of the cardiovascular system during exercise. In rats, c-Fos immunostaining was used to examine whether the amygdala and claustrum were activated during treadmill exercise. Further, we examined arterial pressure responses to electrical and chemical stimulation of the claustrum region. We also confirmed the anatomical connections between the amygdala, claustrum, and NTS by retrograde tracer injections. Finally, we performed simultaneous electrical stimulation of the claustrum and amygdala to examine their functional connectivity. c-Fos expression was observed in the amygdala and the posterior part of the claustrum (pCL), but not in the anterior part, in an exercise intensity-dependent manner. pCL stimulation induced a depressor response. Using a retrograde tracer, we confirmed direct projections from the amygdala to the pCL and NTS. Simultaneous stimulation of the central nucleus of the amygdala and pCL showed a greater pressor response compared with the stimulation of the amygdala alone. These results suggest the amygdala and pCL are involved in different phases of exercise. More speculatively, these areas might coordinately tune cardiovascular responses that help maintain performance during high-intensity exercise.

Involvement of Endothelial Nitric Oxide Synthase Activation in Midkine-Mediated Central Hypotensive Effects.

The growth factor midkine (MK) has been implicated in various biologic and pathologic events. It has been shown that the peripheral influence of MK on cardiovascular regulation is due to an influence on the renin-angiotensin system (RAS). The nucleus tractus solitarii (NTS) is the primary integrative center for cardiovascular control and other autonomic functions in the central nervous system. However, the signaling mechanisms involved in MK-mediated cardiovascular effects in the NTS remain unclear. In this study, we investigated whether the RAS and/or N-methyl-D-aspartate (NMDA) receptor-calmodulin-endothelial nitric oxide synthase (eNOS) signaling pathways were both involved in MK-mediated blood pressure (BP) regulation in the NTS of Wistar-Kyoto (WKY) rats. Intra-NTS microinjection and immunoblot analysis were used to evaluate the signal pathway. WKY rats were anesthetized with urethane. Unilateral microinjection of MK (600 fmol) into the NTS produced a dose-dependent decrease in BP and heart rate (HR). The depressor effects were observed before and after microinjection of the angiotensin-converting enzyme (ACE) inhibitor lisinopril (2.4 fmol), or the angiotensin receptor blockers (ARB) inhibitor valsartan (7.5 pmol). However, lisinopril and valsartan did not diminish the MK-mediated cardiovascular effects in the NTS. Microinjection of the NMDA receptor antagonist MK801 (1 nmol) or the NOS inhibitor N-nitro l-arginine methyl ester (L-NAME), (33 nmol), into the NTS attenuated the MK-induced hypotensive effects. Pretreatment with an eNOS inhibitor N5-iminoethyl-l-ornithine (L-NIO) (6 nmol) attenuated the MK-induced hypotensive effects. In this study, the data showed that MK might play a role in central cardiovascular regulation in the NTS. These results suggest that MK decreased BP and HR in the NTS probably acting via the NMDA receptor-calmodulin-eNOS signaling pathway.

Spectral Analysis Related to Bare-Metal and Drug-Eluting Coronary Stent Implantation

BackgroundThe autonomic nervous system plays a central role in cardiovascular regulation; sympathetic activation occurs during myocardial ischemia. ObjectiveTo assess the spectral analysis of heart rate variability during stent implantation, comparing the types of stent. MethodsThis study assessed 61 patients (mean age, 64.0 years; 35 men) with ischemic heart disease and indication for stenting. Stent implantation was performed under Holter monitoring to record the spectral analysis of heart rate variability (Fourier transform), measuring the low-frequency (LF) and high-frequency (HF) components, and the LF/HF ratio before and during the procedure. ResultsBare-metal stent was implanted in 34 patients, while the others received drug-eluting stents. The right coronary artery was approached in 21 patients, the left anterior descending, in 28, and the circumflex, in 9. As compared with the pre-stenting period, all patients showed an increase in LF and HF during stent implantation (658 versus 185 ms2, p = 0.00; 322 versus 121, p = 0.00, respectively), with no change in LF/HF. During stent implantation, LF was 864 ms2 in patients with bare-metal stents, and 398 ms2 in those with drug-eluting stents (p = 0.00). The spectral analysis of heart rate variability showed no association with diabetes mellitus, family history, clinical presentation, beta-blockers, age, and vessel or its segment. ConclusionsStent implantation resulted in concomitant sympathetic and vagal activations. Diabetes mellitus, use of beta-blockers, and the vessel approached showed no influence on the spectral analysis of heart rate variability. Sympathetic activation was lower during the implantation of drug-eluting stents.

Involvement of NMDA Receptors in Nicotine-Mediated Central Control of Hypotensive Effects

It is known that enrichment of glutamatergic transmission in the nucleus tractus solitarii (NTS) plays an important role in central cardiovascular regulation. Our previous study demonstrated that nicotine decreased blood pressure and heart rate in the NTS probably acting via the nicotinic acetylcholine receptors (nAChRs)-Ca²⁺-calmodulin-eNOS-NO signaling pathway. The possible relationship between glutamate and nicotine in the NTS for cardiovascular regulation is poorly understood. This study investigated the involvement of glutamate receptors in the cardiovascular effects of nicotine in the NTS. Nicotine (a non-selective nAChRs agonist), MK801 (a non-competitive NMDA receptor antagonist), APV (a competitive NMDA receptor antagonist), or NBQX (a selective AMPA receptor antagonist) was microinjected into the NTS of anesthetized Wistar-Kyoto rats. Microinjection of nicotine (1.5 pmol) into the NTS produced decreases in blood pressure and heart rate. The hypotensive and bradycardic effects of nicotine were abolished by prior administration of MK801 (1 nmol) and APV (10 nmol), but was completely restored after 60 min of recovery. In contrast, prior administration of NBQX (10 pmol) into the NTS did not alter the cardiovascular effects of nicotine. The nitrate (served as total NO) production in response to nicotine microinjection into the NTS was suppressed by prior administration of APV. These results suggest that the hypotensive and bradycardic effects of nicotine in the NTS might be mediated through NMDA receptors, and that the nAChRs-NMDA receptor-NO pathway could be involved.

Sympathetic nervous system activity in health and disease—advances with microneurographic recordings

Sympathetic nervous system activity in health and disease—advances with microneurographic recordings

Endoplasmic Reticulum Ca2+ Release Modulates Endothelial Nitric-oxide Synthase via Extracellular Signal-regulated Kinase (ERK) 1/2-mediated Serine 635 Phosphorylation

Endothelial nitric-oxide synthase (eNOS) plays a central role in cardiovascular regulation. eNOS function is critically modulated by Ca(2+) and protein phosphorylation, but the interrelationship between intracellular Ca(2+) mobilization and eNOS phosphorylation is poorly understood. Here we show that endoplasmic reticulum (ER) Ca(2+) release activates eNOS by selectively promoting its Ser-635/633 (bovine/human) phosphorylation. With bovine endothelial cells, thapsigargin-induced ER Ca(2+) release caused a dose-dependent increase in eNOS Ser-635 phosphorylation, leading to elevated NO production. ER Ca(2+) release also promoted eNOS Ser-633 phosphorylation in mouse vessels in vivo. This effect was independent of extracellular Ca(2+) and selective to Ser-635 because the phosphorylation status of other eNOS sites, including Ser-1179 or Thr-497, was unaffected in thapsigargin-treated cells. Blocking ERK1/2 abolished ER Ca(2+) release-induced eNOS Ser-635 phosphorylation, whereas inhibiting protein kinase A or Ca(2+)/calmodulin-dependent protein kinase II had no effect. Protein phosphorylation assay confirmed that ERK1/2 directly phosphorylated the eNOS Ser-635 residue in vitro. Further studies demonstrated that ER Ca(2+) release-induced ERK1/2 activation mediated the enhancing action of purine or bradykinin receptor stimulation on eNOS Ser-635/633 phosphorylation in bovine/human endothelial cells. Mutating the Ser-635 to nonphosphorylatable alanine prevented ATP from activating eNOS in cells. Taken together, these studies reveal that ER Ca(2+) release enhances eNOS Ser-635 phosphorylation and function via ERK1/2 activation. Because ER Ca(2+) is commonly mobilized by agonists or physicochemical stimuli, the identified ER Ca(2+)-ERK1/2-eNOS Ser-635 phosphorylation pathway may have a broad role in the regulation of endothelial function.

Structure, signaling mechanism and regulation of the natriuretic peptide receptor guanylate cyclase

Atrial natriuretic peptide (ANP) and the homologous B-type natriuretic peptide are cardiac hormones that dilate blood vessels and stimulate natriuresis and diuresis, thereby lowering blood pressure and blood volume. ANP and B-type natriuretic peptide counterbalance the actions of the renin-angiotensin-aldosterone and neurohormonal systems, and play a central role in cardiovascular regulation. These activities are mediated by natriuretic peptide receptor-A (NPRA), a single transmembrane segment, guanylyl cyclase (GC)-linked receptor that occurs as a homodimer. Here, we present an overview of the structure, possible chloride-mediated regulation and signaling mechanism of NPRA and other receptor GCs. Earlier, we determined the crystal structures of the NPRA extracellular domain with and without bound ANP. Their structural comparison has revealed a novel ANP-induced rotation mechanism occurring in the juxtamembrane region that apparently triggers transmembrane signal transduction. More recently, the crystal structures of the dimerized catalytic domain of green algae GC Cyg12 and that of cyanobacterium GC Cya2 have been reported. These structures closely resemble that of the adenylyl cyclase catalytic domain, consisting of a C1 and C2 subdomain heterodimer. Adenylyl cyclase is activated by binding of G(s)α to C2 and the ensuing 7° rotation of C1 around an axis parallel to the central cleft, thereby inducing the heterodimer to adopt a catalytically active conformation. We speculate that, in NPRA, the ANP-induced rotation of the juxtamembrane domains, transmitted across the transmembrane helices, may induce a similar rotation in each of the dimerized GC catalytic domains, leading to the stimulation of the GC catalytic activity.

Continuous hypertension and tachycardia after resection of a hemangioblastoma behind the dorsal medulla oblongata: relationship to sympathetic overactivity at the neurogenic vasomotor center

A very rare case of continuous hypertension and tachycardia after excision of a cerebellar hemangioblastoma at the dorsal medulla oblongata is presented. This 21-year-old man was admitted to the authors' hospital with a headache and dizziness. Radiological examination revealed a tumor located behind the dorsal medulla oblongata and compressing it substantially. The tumor was completely resected, but after the surgery the patient experienced prolonged hypertension and tachycardia. Postoperative MR imaging showed a small injury at the dorsocaudal medulla that was located at the caudal site of the nucleus of the tractus solitarius (NTS). Because the NTS has been reported to play a central role in cardiovascular regulation along with the rostral ventrolateral medulla, the authors considered it possible that the NTS injury was the cause of the prolonged elevation of sympathetic tone.

Cell-based methods for ex vivo evaluation of human endothelial biology

The endothelium plays a central role in cardiovascular regulation. Endothelial cells produce a variety of vasculoregulatory and vasculotropic molecules that act locally or at distant sites. Alteration of the vascular endothelium is a primary event in the pathogenesis of vascular diseases, such as atherosclerosis, as well as systemic and pulmonary hypertension. For these reasons, the study of human endothelium has become central in cardiovascular research. Unfortunately, technologies handling endothelial cells in vitro are often criticized due to the uncertain transferability of results to intact organs and, importantly, to humans. Although methods to assess endothelial function non-invasively have been available for decades, cell-based approaches for the direct ex vivo evaluation of endothelial cell biology in humans have been devised only recently. The discovery of endothelial progenitor cells opened the way for studies on vascular regeneration, while it has been recognized that mature circulating endothelial cells mainly represent a consequence of the ongoing vascular damage. Coupled with a minimally invasive way to obtain fresh human endothelial cells through an endovascular biopsy, these new methods provide a novel outlook on human endothelial cells as close as possible to their natural environment. In this review, we will summarize the current knowledge and the methodological perspective of these cell-based methods.

Cardiovascular effects of noradrenaline microinjection into the medial part of the superior colliculus of unanesthetized rats

The superior colliculus (SC) is a mesencephalic area involved in the mediation of defensive movements associated with cardiovascular changes. Noradrenaline (NA) is a neurotransmitter with an important role in central cardiovascular regulation exerted by several structures of the central nervous system. Although noradrenergic nerve terminals have been observed in the SC, there are no reports on the effects of local NA injection into this area. Taking this into consideration, we studied the cardiovascular effects of NA microinjection into the SC of unanesthetized rats. Microinjection of NA into the SC evoked a dose-dependent blood pressure increase and a heart rate decrease in unanesthetized rats. The pressor response to NA was not modified by intravenous pretreatment with the vasopressin v 1-receptor antagonist dTyr(CH 2) 5(Me)AVP, indicating a lack of vasopressin involvement in the response mediation. The effect of NA microinjection into the SC was blocked by intravenous pretreatment with the ganglionic blocker pentolinium, indicating its mediation by the sympathetic nervous system. Although the pressor response to NA was not affected by adrenal demedullation, the accompanying bradycardia was potentiated, suggesting some involvement of the sympathoadrenal system in the cardiovascular response to NA microinjection into the SC. In summary, results indicate that stimulation of noradrenergic receptors in the SC causes cardiovascular responses which are mediated by activation of both neural and adrenal sympathetic nervous system components.

Involvement of NO and KATP Channel in Adenosine A2B Receptors Induced Cardiovascular Regulation in the Posterior Hypothalamus of Rats

Previous reports have suggested that the posterior hypothalamic adenosine A2 receptors may play a role in central cardiovascular regulation. In this study, we examined the influence of posterior hypothalamic adenosine A2B receptors on the regulation of blood pressure and heart rate. Drugs were injected into the posterior hypothalamus of anesthetized, artificially ventilated, male Sprague-Dawley rats. Four nanomoles of 5'-N-ethylcarboxamidoadenosine (NECA), an adenosine A 2A receptor agonist, decreased arterial blood pressure and heart rate, whereas 5 nmol of alloxazine, an adenosine A2B receptor antagonist, blocked the depressor and bradycardiac effects of 4 nmol NECA. We examined the role of nitric oxide (NO) and K+ channels on cardiovascular regulation by adenosine A2B receptors in the posterior hypothalamus. Pretreatment with 40 nmol of NG-nitro-L-arginine methyl ester, a NO synthase inhibitor, significantly attenuated the effects of NECA, and 10 nmol of sodium nitroprusside, a NO releaser, strengthened the action of drug. In addition, posterior hypothalamic administration of 20 nmol of glipizide, an K ATP blocker, blocked the cardiovascular depression elicited by NECA. These results suggest that NO mediates cardiovascular regulation by activation of A2B receptors in the posterior hypothalamus. Additionally, ATP-sensitive K+ channels modulate the action of adenosine A2B receptors.

Modification of the cardiovascular response of posterior hypothalamic adenosine A 2A receptor stimulation by adenylate cyclase and KATP channel blockade in anesthetized rats

In this experiment, we examined the influence of the posterior hypothalamic adenosine A 2A receptors on the central cardiovascular regulation of blood pressure (BP) and heart rate (HR). Posterior hypothalamic injection of drugs was performed in anesthetized, artificially ventilated male Sprague–Dawley rats. Injection of CGS-21680HCl (CGS; 20 nmol), an adenosine A 2A receptor agonist, elicited a decrease of arterial BP and HR, while injection of 8-(3-Chlorostyryl)caffeine (CSC; 10 nmol), an adenosine A 2A receptor antagonist, blocked the depressor and bradycardiac effects of CGS (20 nmol). To examine the mechanisms of cardiovascular regulation of adenosine A 2A receptors in the posterior hypothalamus, we applied the adenylate cyclase and guanylate cyclase inhibitors, to the posterior hypothalamus. Pretreatment with MDL-12,330 (MDL; 10 nmol), an adenylate cylase inhibitor, attenuated the depressor and bradycardiac effects of CGS. However, pretreatment with, LY-83,583 (LY; 5 nmol), a soluble guanylate cyclase inhibitor, did not alter the effects of CGS. Additionally, we examined the modification of the cardiovascular effects of adenosine A 2A receptors through the ATP-sensitive K + channel in the posterior hypothalamus. Posterior hypothalamic administration of glipizide (20 nmol) significantly attenuated the cardiovascular depressor actions elicited by CGS. These results suggest that adenosine A 2A receptors in the posterior hypothalamus play an inhibitory role in central cardiovascular regulation, and that adenylate cyclase, but not guanylate cyclase, mediates the depressor and bradycardiac actions of adenosine A 2A receptors. Furthermore, ATP-sensitive K + channels mediate the posterior hypothalamic cardiovascular regulation of adenosine A 2A receptors.

Involvement of guanylate cyclase in the cardiovascular response induced by adenosine A 2B receptor stimulation in the posterior hypothalamus of the anesthetized rats

Cardiovascular inhibitory effects induced by the posterior hypothalamic adenosine A 2 receptors were suggested by our previous reports. In this experiment, we examined the influence of the posterior hypothalamic adenosine A 2B receptors on central cardiovascular regulation of blood pressure (BP) and heart rate (HR). Posterior hypothalamic injection of drugs was performed in anesthetized, artificially ventilated male Sprague–Dawley rats. Injection of 5′-( N-cyclopropyl)-carboxamidoadenosine (CPCA; 2 nmol), an adenosine A 2 receptor agonist, showed the decrease of arterial blood pressure and heart rate, and the alloxazine, an adenosine A 2B receptor antagonist, partially blocked the depressor and bradycardiac effects of CPCA (2 nmol). To examine the role of adenosine A 2B receptors among the adenosine A 2 subtypes, we applied the 5′- N-Ethylcarboxamidoadenosine (NECA), an adenosine A 2B receptor agonist, to the posterior hypothalamus. Injection of NECA (1, 4 and 8 nmol) produced a dose-dependent decrease of arterial blood pressure and HR. Pretreatment with alloxazine (5 nmol) partially blocked the depressor and bradycardiac effects of NECA (4 nmol). Also, pretreatment with LY-83,583 (5 nmol), a soluble guanylate cyclase inhibitor, attenuated the depressor and bradycardiac effects of NECA (4 nmol). However, pretreatment with MDL-12,330 (10 nmol), an adenylate cyclase inhibitor, did not affect these effects of NECA (4 nmol). These results suggest that adenosine A 2B receptor in the posterior hypothalamus plays an inhibitory role in central cardiovascular regulation, and that guanylate cyclase mediates the depressor and bradycardiac actions of adenosine A 2B receptors.

In situ Akt phosphorylation in the nucleus tractus solitarii is involved in central control of blood pressure and heart rate.

Previously, we have shown that nitric oxide (NO) plays a significant role in central cardiovascular regulation and modulates the baroreflex in the nucleus tractus solitarii (NTS) of rats. NO production is mediated by activation of NO synthase (NOS). Insulin signaling was involved in controlling peripheral blood pressure via the activation of endothelial NOS. Here, we investigated whether the insulin signal transduction pathway is involved in controlling central cardiovascular effects. Insulin was injected into NTS of urethane-anesthetized male Wistar-Kyoto (WKY) rats. Unilateral microinjection (60 nL) of insulin (100 IU/mL) into the NTS produced prominent depressor and bradycardic effects in 8- and 16-week-old WKY rats. In addition, pretreatment with the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 and the NOS inhibitor L-NAME into the NTS caused attenuation of the cardiovascular response evoked by insulin in either 8- or 16-week-old WKY rats. Western blot analysis showed a significant increase (2.6+/-0.4-fold; P<0.05) in Akt phosphorylation after insulin injection, whereas LY294002 abolished the insulin-induced effects. In situ Akt phosphorylation was found in NTS by immunohistochemistry analysis after injection of insulin. This in situ Akt phosphorylation was abolished significantly after injection of LY294002. Take together, these results suggest that the insulin-PI3K-Akt-NOS signaling pathway may play a significant role in central cardiovascular regulation.

The involvement of nitric oxide on the adenosine A 2 receptor-induced cardiovascular inhibitory responses in the posterior hypothalamus of rats

This study was performed to investigate the putative relationship between nitric oxide (NO) and adenosine A 2 receptors on central cardiovascular regulation in the posterior hypothalamus of rats. Posterior hypothalamic injection of drugs was performed in anesthetized, artificially ventilated male Sprague–Dawley rats. Injection of adenosine A 2 receptor agonist 5′-( N-cyclopropyl)-carboxamidoadenosine (CPCA; 1, 2 and 5 nmol) produced a dose-dependent decrease of blood pressure and heart rate. Pretreatment with adenosine A 2 receptor antagonist 3,7-dimethyl-1-propargylxanthine (10 nmol) blocked the depressor and bradycardiac effects of CPCA (5 nmol). Pretreatment with soluble guanylate cyclase inhibitor LY-83,583 (5 nmol) attenuated the depressor and bradycardiac effects of CPCA (5 nmol). In addition, pretreatment with NO synthase inhibitor N G -nitro- l-arginine methyl ester (40 nmol) attenuated the depressor and bradycardiac responses of CPCA (5 nmol). These results suggest that adenosine A 2 receptor in the posterior hypothalamus plays an inhibitory role in central cardiovascular regulation and that NO participates in the inhibitory response induced by adenosine A 2 receptor stimulation in the posterior hypothalamus.

Thyrotropin-Releasing Hormone Receptor (TRHR) Gene Is Associated With Essential Hypertension

In essential hypertension, a polygenic and multifactorial syndrome, several genes interact with the environment to produce high blood pressure. Thyrotropin-releasing hormone (TRH) plays an important role in central cardiovascular regulation. We have described that TRH overexpression induces hypertension in a normal rat, which was reversed by TRH antisense treatment. This treatment also reduces the central TRH hyperactivity in spontaneously hypertensive rats and normalizes blood pressure. Human TRH receptor (TRHR) belongs to the G protein-coupled seven-transmembrane domain receptor superfamily. Mutations of these receptors may result in constitutive activation. As it has been demonstrated that hypertensive patients have a blunted TSH response to TRH injection, suggesting a defect in the TRHR, we postulate that the TRHR gene is involved in human hypertension. We studied 2 independent populations from different geographic regions of our country: a sample of adult subjects from a referral clinic and a population-based sample of high school students. In search of molecular variants of TRHR, we disclosed that a polymorphic TG dinucleotide repeat (STR) at -68 bp and a novel single nucleotide polymorphism, a G-->C conversion at -221 located in the promoter of the TRHR are associated with essential hypertension. As STRs detected in gene promoters are potential Z-DNA-forming sequences and seem to affect gene expression, we studied the potentially different transcriptional activity of these TRHR promoter variants and found that the S/-221C allele has a higher affinity than does the L/G-221 allele to nuclear protein factor(s). Our findings support the hypothesis that the TRHR gene participates in the etiopathogenesis of essential hypertension.

Antisense inhibition of thyrotropin-releasing hormone reduces arterial blood pressure in spontaneously hypertensive rats.

Thyrotropin-releasing hormone (TRH) plays an important role in central cardiovascular regulation. Recently, we described that the TRH precursor gene overexpression induces hypertension in the normal rat. In addition, we published that spontaneously hypertensive rats (SHR) have central extrahypothalamic TRH hyperactivity with increased TRH synthesis and release and an elevated TRH receptor number. In the present study, we report that intracerebroventricular antisense (AS) treatment with a phosphorothioate oligonucleotide against the TRH precursor gene significantly diminished up to 72 hours and in a dose-dependent manner the increased diencephalic TRH content, whereas normalized systolic blood pressure (SABP) was present in the SHR compared with Wistar-Kyoto (WKY) rats. Although basal thyrotropin was higher in SHR compared with WKY rats and this difference disappeared after antisense treatment, no differences were observed in plasma T4 or T3 between strains with or without AS treatment, indicating that the effect of the AS on SABP was independent of the thyroid status. Because the encephalic renin-angiotensin system seems to be crucial in the development and/or maintenance of hypertension in SHR, we investigated the effect of antisense inhibition of TRH on that system and found that TRH antisense treatment significantly diminished the elevated diencephalic angiotensin II (Ang II) content in the SHR without any effect in control animals, suggesting that the Ang II system is involved in the TRH cardiovascular effects. To summarize, the central TRH system seems to be involved in the etiopathogenesis of hypertension in this model of essential hypertension.

The galanin receptor antagonist M40 blocks the central cardiovascular actions of the galanin N-terminal fragment (1–15)

It has been shown that galanin plays a role in central cardiovascular regulation. Galanin administered centrally induces an increase of heart rate and a weak vasodepressor response, whereas the N-terminal galanin fragment (1–15) elicits vasopressor effects and tachycardia. Furthermore, it has been shown that galanin-(1–15), but not galanin-(1–29), decreases the baroreceptor reflex sensitivity. Since these data demonstrate that both galanin and its N-terminal fragment (1–15) exert a different modulation on central cardiovascular control, the aim of this work has been to study if the specific galanin receptor antagonist Galanin-(1–12)-Pro-(Ala-Leu) 2-Ala]-amide (M40) could modulate their cardiovascular actions. Urethane anaesthetized rats were injected intracisternally and the changes in mean arterial pressure and heart rate were monitored. Two doses of M40 alone have been tested for their cardiovascular effects. With the dose of 1.0 nmol, a significant tachycardia was observed ( P<0.001), but 0.1 nmol was ineffective. This suggests a possible agonistic effect for the higher doses of M40. The galanin receptor antagonist M40 at the dose of 0.1 nmol failed to modify the weak vasodepressor effects and tachycardia induced by 3.0 nmol of galanin-(1–29). However, the same dose completely blocked the vasopressor and tachycardic responses elicited by 3.0 nmol of galanin-(1–15). These data show that M40 differentially counteracts the central cardiovascular responses of the galanin fragment and give a functional support for the existence of galanin receptor subtypes within the brainstem. Therefore, the present findings can be explained on the basis that the cardiovascular actions of galanin-(1–29) could be mediated by one type of galanin receptor, whereas a galanin receptor subtype that recognizes N-terminal fragments of galanin may mediate the actions of galanin-(1–15).

Modulatory effects of carbon monoxide on baroreflex activation in nucleus tractus solitarii of rats.

Recent studies suggest that carbon monoxide (CO), which is produced in significant quantities in many brain regions, may function as a neurotransmitter. Heme oxygenase catalyzes the metabolism of heme to CO and biliverdin; however, the physiological role of CO in central cardiovascular regulation was not well understood. In the present study, we evaluated the baroreflex response of CO in the nucleus tractus solitarii (NTS) of rats. Male Sprague-Dawley rats were anesthetized with urethane, and blood pressure and heart rate were monitored intra-arterially. Unilateral microinjection (60 nL) of hematin, a heme molecule cleaved by heme oxygenase to yield CO, into the NTS produced prominent dose-related depressor and bradycardic effects. Baroreflex responses were elicited by increasing doses of phenylephrine (10 to 30 microg/kg IV) before and after intra-NTS administration of zinc deuteroporphyrin 2,4-bis-glycol (ZnDPBG) (1 nmol), an inhibitor of heme oxygenase activity, or vehicle alone. The reflex bradycardia elicited by phenylephrine was significantly inhibited by pretreatment with ZnDPBG. Furthermore, the inhibitory effect of ZnDPBG on baroreflex activation was dose dependent. These results suggest CO formed by brain heme oxygenase plays a significant role in central cardiovascular regulation and that inhibition of heme oxygenase attenuated baroreflex activation.