Alpha2-adrenoceptor and adenosine A1 receptor within the nucleus tractus solitarii in hypertension development.

Alpha2-adrenoceptor and A1 adenosine receptor systems within the nucleus tractus solitarii (NTS) play an important role in cardiovascular control. Deregulation of these systems may result in an elevated sympathetic tone, one of the root causes of neurogenic hypertension. The dorsomedial/dorsolateral and subpostremal NTS subnuclei of spontaneously hypertensive rats (SHR) show density changes in both receptors, even at 15 days of age, prior to the onset of hypertension. In addition, adenosine A1 receptors have been specifically reported to modulate alpha2-adrenoceptors in several brain regions, including the NTS, via a PLC-dependent pathway involving cross regulation between sympathetic neurons and astrocytes. The physiological cross talk between these receptor systems is also deregulated in SHR suggesting that alpha2-adrenoceptor and A1 adenosine receptor might be germane to the development of hypertension. In this review, we will focus on these systems within the NTS during development, pointing out some interesting modulations in processes, and chemical changes within specific subnuclei of NTS circuitry, that might have implications for neurogenic hypertension.

α2-Adrenergic receptor distribution and density within the nucleus tractus solitarii of normotensive and hypertensive rats during development.

The nucleus tractus solitarii (NTS), located in the brainstem, is one of the main nuclei responsible for integrating different signals in order to originate a specific and orchestrated autonomic response. Antihypertensive drugs are well known to stimulate alpha(2)-adrenoceptor (alpha(2R)) in brainstem cardiovascular regions to induce reduction in blood pressure. Because alpha(2R) impairment is present in several models of hypertension, the aim of the present study was to investigate the distribution and density of alpha(2R) binding within the NTS of Wistar Kyoto (WKY) and spontaneously hypertensive (SHR) rats during development (1,15,30 and 90 day-old) by an in vitro autoradiographical study. The NTS shows heterogeneous distribution of alpha(2R) in dorsomedial/dorsolateral, subpostremal and medial/intermediate subnuclei. Alpha(2R) increased from rostral to caudal dorsomedial/dorsolateral subnuclei in 30 and 90 day-old SHR but not in WKY. Alpha(2R) decreased from rostral to caudal subpostremal subnucleus in 15, 30 and 90 day-old SHR but not in WKY. Medial/intermediate subnuclei did not show any changes in alpha(2R) according to NTS levels. Furthermore, alpha(2R) are decreased in SHR as compared with WKY in all NTS subnuclei and in different ages. Surprisingly, alpha(2R) impairment was also found in pre-hypertensive stages, specifically in subpostremal subnucleus of 15 day-old rats. Finally, alpha(2R) decrease from 1 to 90 day-old rats in all subnuclei analyzed. This decrease is different between strains in rostral dorsomedial/dorsolateral and caudal subpostremal subnuclei within the NTS. In summary, our results highlight the importance of alpha(2R) distribution within the NTS regarding the neural control of blood pressure and the development of hypertension.

Modulation of tyrosine hydroxylase, neuropeptide y, glutamate, and substance p in Ganglia and brain areas involved in cardiovascular control after chronic exposure to nicotine.

Considering that nicotine instantly interacts with central and peripheral nervous systems promoting cardiovascular effects after tobacco smoking, we evaluated the modulation of glutamate, tyrosine hydroxylase (TH), neuropeptide Y (NPY), and substance P (SP) in nodose/petrosal and superior cervical ganglia, as well as TH and NPY in nucleus tractus solitarii (NTS) and hypothalamic paraventricular nucleus (PVN) of normotensive Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR) after 8 weeks of nicotine exposure. Immunohistochemical and in situ hybridization data demonstrated increased expression of TH in brain and ganglia related to blood pressure control, preferentially in SHR, after nicotine exposure. The alkaloid also increased NPY immunoreactivity in ganglia, NTS, and PVN of SHR, in spite of decreasing its receptor (NPY1R) binding in NTS of both strains. Nicotine increased SP and glutamate in ganglia. In summary, nicotine positively modulated the studied variables in ganglia while its central effects were mainly constrained to SHR.

Plasticity of opioid receptors in the female periaqueductal gray: multiparity-induced increase in the activity of genes encoding for mu and kappa receptors and a post-translational decrease in delta receptor expression.

The periaqueductal gray (PAG) has been reported as a potential site for opioid regulation of behavioral selection. Opioid-mediated behavioral and physiological responses differ between nulliparous and multiparous females. This study addresses the effects of multiple reproductive experiences on µ-, κ- and δ-opioid receptor (Oprm1, Oprk1, and Oprd1 respectively) gene activity and µ, κ and δ protein expression (MOR, KOR and DOR respectively) in the PAG of the female rats. This was done by evaluating the opioid gene expression using real-time (RT-PCR) and quantification of each protein receptor by Western blot analysis. The RT-PCR results show that multiple reproductive experiences increase Oprm1 and Oprk1 gene expression. Western blot analysis revealed increased MOR and KOR while DOR protein was decreased in multiparous animals. Taken together, these data suggest that multiple reproductive experiences influence both gene activity and opioid receptor expression in the PAG. Post-translational mechanisms seem particularly relevant for DOR expression. Thus, opioid transmission in the PAG might be modulated by different mechanisms of multiparity-induced plasticity according to the opioid receptor type.

Gene expression profiling of cultured cells from brainstem of newborn spontaneously hypertensive and Wistar Kyoto rats.

The spontaneously hypertensive rat (SHR) is a good model to study several diseases such as the attention-deficit hyperactivity disorder, cardiopulmonary impairment, nephropathy, as well as hypertension, which is a multifactor disease that possibly involves alterations in gene expression in hypertensive relative to normotensive subjects. In this study, we used high-density oligoarrays to compare gene expression profiles in cultured neurons and glia from brainstem of newborn normotensive Wistar Kyoto (WKY) and SHR rats. We found 376 genes differentially expressed between SHR and WKY brainstem cells that preferentially map to 17 metabolic/signaling pathways. Some of the pathways and regulated genes identified herein are obviously related to cardiovascular regulation; in addition there are several genes differentially expressed in SHR not yet associated to hypertension, which may be attributed to other differences between SHR and WKY strains. This constitute a rich resource for the identification and characterization of novel genes associated to phenotypic differences observed in SHR relative to WKY, including hypertension. In conclusion, this study describes for the first time the gene profiling pattern of brainstem cells from SHR and WKY rats, which opens up new possibilities and strategies of investigation and possible therapeutics to hypertension, as well as for the understanding of the brain contribution to phenotypic differences between SHR and WKY rats.

Differential regulation of the renin-angiotensin system by nicotine in WKY and SHR glia.

Given that (1) the renin-angiotensin system (RAS) is compartmentalized within the central nervous system in neurons and glia (2) the major source of brain angiotensinogen is the glial cells, (3) the importance of RAS in the central control of blood pressure, and (4) nicotine increases the probability of development of hypertension associated to genetic predisposition; the objective of the present study was to evaluate the effects of nicotine on the RAS in cultured glial cells from the brainstem and hypothalamus of Wistar Kyoto (WKY) and spontaneously hypertensive (SHR) rats. Ligand binding, real-time PCR and western blotting assays were used to compare the expression of angiotensinogen, angiotensin converting enzyme, angiotensin converting enzyme 2 and angiotensin II type1 receptors. We demonstrate, for the first time, that there are significant differences in the basal levels of RAS components between WKY and SHR rats in glia from 1-day-old rats. We also observed that nicotine is able to modulate the renin-angiotensin system in glial cells from the brainstem and hypothalamus and that the SHR responses were more pronounced than WKY ones. The present data suggest that nicotine effects on the RAS might collaborate to the development of neurogenic hypertension in SHR through modulation of glial cells.

Effects of bilateral adrenalectomy on systemic kainate-induced activation of the nucleus of the solitary tract. Regulation of blood pressure and local neurotransmitters.

Glutamatergic transmission through metabotropic and ionotropic receptors, including kainate receptors, plays an important role in the nucleus of the solitary tract (NTS) functions. Glutamate system may interact with several other neurotransmitter systems which might also be influenced by steroid hormones. In the present study we analyzed the ability of systemic kainate to stimulate rat NTS neurons, which was evaluated by c-Fos as a marker of neuronal activation, and also to change the levels of NTS neurotransmitters such as GABA, NPY, CGRP, GAL, NT and NO by means of quantitative immunohistichemistry combined with image analysis. The analysis was also performed in adrenalectomized and kainate stimulated rats in order to evaluate a possible role of adrenal hormones on NTS neurotransmission. Male Wistar rats (3 month-old) were used in the present study. A group of 15 rats was submitted either to bilateral adrenalectomy or sham operation. Forty-eight hours after the surgeries, adrenalectomized rats received a single intraperitoneal injection of kainate (12 mg/kg) and the sham-operated rats were injected either with saline or kainate and sacrificed 8 hours later. The same experimental design was applied in a group of rats in order to register the arterial blood pressure. Systemic kainate decreased the basal values of mean arterial blood pressure (35%) and heart rate (22%) of sham-operated rats, reduction that were maintained in adrenalectomized rats. Kainate triggered a marked elevation of c-Fos positive neurons in the NTS which was 54% counteracted by adrenalectomy. The kainate activated NTS showed changes in the immunoreactive levels of GABA (143% of elevation) and NPY (36% of decrease), which were not modified by previous ablation of adrenal glands. Modulation in the levels of CGRP, GAL and NT immunoreactivities were only observed after kainate in the adrenalectomized rats. Treatments did not alter NOS labeling. It is possible that modulatory function among neurotransmitter systems in the NTS might be influenced by steroid hormones and the implications for central regulation of blood pressure or other visceral regulatory mechanisms control should be further investigated.

Adenosine modulates alpha2-adrenergic receptors within specific subnuclei of the nucleus tractus solitarius in normotensive and spontaneously hypertensive rats.

Adenosine is known to modulate neuronal activity within the nucleus tractus solitarius (NTS). The modulatory effect of adenosine A1 receptors (A1R) on alpha2-adrenoceptors (Adr2R) was evaluated using quantitative radioautography within NTS subnuclei and using neuronal culture of normotensive (WKY) and spontaneously hypertensive rats (SHR). Radioautography was used in a saturation experiment to measure Adr2R binding parameters (Bmax, Kd) in the presence of 3 different concentrations of N6-cyclopentyladenosine (CPA), an A1R agonist. Neuronal culture confirmed our radioautographic results. [3H]RX821002, an Adr2R antagonist, was used as a ligand for both approaches. The dorsomedial/dorsolateral subnucleus of WKY showed an increase in Bmax values (21%) induced by 10 nmol/L of CPA. However, the subpostremal subnucleus showed a decrease in Kd values (24%) induced by 10 nmol/L of CPA. SHR showed the same pattern of changes as WKY within the same subnuclei; however, the modulatory effect of CPA was induced by 1 nmol/L (increased Bmax, 17%; decreased Kd, 26%). Cell culture confirmed these results, because 10(-5) and 10(-7) mol/L of CPA promoted an increase in [3H]RX821002 binding of WKY (53%) and SHR cells (48%), respectively. DPCPX, an A1R antagonist, was used to block the modulatory effect promoted by CPA with respect to Adr2R binding. In conclusion, our study shows for the first time an interaction between A1R that increases the binding of Adr2R within specific subnuclei of the NTS. This may be important in understanding the complex autonomic response induced by adenosine within the NTS. In addition, changes in interactions between receptors might be relevant to understanding the development of hypertension.

Nicotine modulates the renin-angiotensin system of cultured neurons and glial cells from cardiovascular brain areas of Wistar Kyoto and spontaneously hypertensive rats.

Considering the importance of the renin-angiotensin system (RAS) for the central control of blood pressure and that nicotine increases the probability of development of hypertension associated to genetic predisposition, our aims are (1) to determine RAS in cultured neurons and glia from the brainstem and hypothalamus of spontaneously hypertensive (SHR) and Wistar Kyoto (WKY) rats; (2) to analyze the possibility of nicotine to interact with brain RAS; and (3) to hypothesize any contribution of nicotine and RAS to the development of neurogenic hypertension. This study demonstrated physiological differences in RAS between cultured neuronal and glial cells from the brainstem and hypothalamus of SHR and WKY neonate rats. Our study also featured evidences of direct modulation of the RAS by nicotine in neurons and glia of brainstem and hypothalamus, which seems to be differential between the two rat strains. Such modulation gives us a clue about the mechanisms possibly involved in the genesis of neurogenic hypertension in vivo, for example, increase in angiotensin II type 1 receptor binding and decrease in angiotensin-converting enzyme 2. In conclusion, we demonstrated that neuronal and glial RAS from the brainstem and hypothalamus of SHR differ from WKY rats and nicotine differentially modulates the brain RAS in SHR and WKY.

Chronic nicotine administration. Analysis of the development of hypertension and glutamatergic neurotransmission.

Among numerous neurotransmitters involved in central cardiovascular control, glutamate is one of the most studied transmitters that are related to nicotine considering its release and its postsynaptic regulation. However, there are no conclusive studies about nicotine effects on glutamatergic system and its relevance on hypertension development, which can help to understand the role of these two systems in that pathology. In this context, the objective of the present study is to evaluate the effects of systemic chronic nicotine exposure on hypertension development as well as the interaction between nicotine and the glutamatergic system in normotensive and neurogenic hypertensive rats. By means of high performance liquid chromatograph, immunohistochemistry, in situ hybridization and binding techniques, glutamatergic system was evaluated in SHR and Wistar Kyoto (WKY) rats treated with nicotine, delivered subcutaneously through nicotine pellets, for 8 weeks. The most important findings in this study were that (1) moderate doses of nicotine accelerated the onset and increased blood pressure in SHR but not in WKY rats, (2) the nicotine dosage and time of treatment employed did not affect body weight, (3) chronic nicotine treatment differentially affected glutamatergic system in normotensive and hypertensive rats, and (4) spontaneously hypertensive rats seem to be more sensitive to peripherally administered nicotine than Wistar Kyoto rats considering blood pressure and glutamatergic neurotransmission changes. In conclusion, we have demonstrated that a moderate dose of nicotine accelerates the onset and exacerbates hypertension in the SHR and that might be, at least in part, related to the modulation of glutamatergic neurotransmission.

Differential expression of nNOS mRNA and protein in the nucleus tractus solitarii of young and aged Wistar-Kyoto and spontaneously hypertensive rats.

OBJECTIVE: To examine neuronal nitric oxide synthase (nNOS) mRNA and protein in the nucleus tractus solitarii (NTS) and paraventricular hypothalamic nucleus (PVN) of Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR) during their life span. METHODS: By means of in situ hybridization and immunohistochemistry, we evaluated nNOS mRNA and protein in the NTS and PVN of 15-day- and 1-, 2-, 4-, 8- and 12-month-old SHR and WKY rats. RESULTS: Two patterns of nNOS expression were observed in two subnuclei of the NTS: medial (NTSm) and central (NTSce). NTSce of the SHR exhibited higher nNOS mRNA and protein expression in all ages analyzed when compared to the age-matched WKY. Increased amounts of nNOS mRNA and protein were seen in the NTSm only during the early life of SHR (15 days to 4 months) when compared to WKY, suggesting a special role of this circuitry before the establishment of hypertension. No changes were seen in nNOS mRNA and protein expression in the PVN of the SHR in comparison to the WKY in all periods. nNOS analysis during the life span showed either a decrease or no change in nNOS mRNA expression in NTS or PVN associated with increased nNOS protein at some analyzed periods, suggesting the differential regulation of nNOS mRNA and protein during aging. CONCLUSIONS: Data suggest that different NTS subnuclei exhibit nNOS changes in different phases of the life of SHR and this might be important during the development of hypertension in these animals.

Effects of ethanol consumption on vasopressin and neuropeptide Y immunoreactivity and mRNA expression in peripheral and central areas related to cardiovascular regulation.

Results from previous studies have demonstrated that ethanol influences central neural mechanisms involved in the control of blood pressure. We studied the effects of ethanol consumption on vasopressin and neuropeptide Y immunoreactivity and mRNA expression in the nucleus tractus solitarius and paraventricular hypothalamic nucleus, as well as in the petrosal and nodose ganglia of rats. The ethanol-fed rats received liquid diet ad libitum containing 37.5% ethanol-derived calories (6.7% volume/volume), and the pair-fed rats received the same volume of diet containing isocaloric amounts of maltose-dextrin substituted for ethanol for 3 or 28 days. Arterial blood pressure was evaluated in a separate group of rats, which was unchanged by 3 days, but elevated by 21% after 28 days of ethanol consumption. Vasopressin immunoreactivity and mRNA signal were not detected in the ganglia, nor were they changed in the nucleus tractus solitarius and paraventricular hypothalamic nucleus, by 3 days of ethanol consumption. However, after 28 days of ethanol liquid diet consumption, vasopressin-positive terminals were decreased in the nucleus tractus solitarius and vasopressin immunoreactivity cell bodies and mRNA signal were decreased in the paraventricular hypothalamic nucleus. Neuropeptide Y-immunoreactive terminals were increased in the nucleus tractus solitarius only after 28 days of ethanol liquid diet consumption, but they were decreased in the paraventricular hypothalamic nucleus in rats treated with ethanol for 3 or 28 days. We concluded that the levels of both vasopressin and neuropeptide Y neurotransmitters are changed by long-term ethanol consumption in the neuronal pathways related to control of blood pressure.

Systemic site of action for pressor effect of angiotensin II injected into the fourth cerebral ventricle of rats?

Angiotensin II (ANG II) causes a systemic pressor effect when injected into the cerebral ventricles. In the rat fourth ventricle, the effective doses for the ANG II pressor effect are over 100 times larger than in the systemic circulation. Considering the discrepancy of doses, the possibility that ANG II may reach the systemic circulation and promote pressor effects, following injection into the fourth ventricle, was investigated. The effects on blood pressure of different vasoactive peptides that produce pressor responses when injected into the central nervous system were compared. Dose-response curves were obtained for intravenous or fourth cerebroventricular injections of ANG II, lysyl-vasopressin (LVP), bradykinin (BK), or endothelin-1 (ET-1). The ED50 ratios for intracerebroventricular/intraveneous injections were 110 for ANG II, 109 for LVP, 0.01 for BK, and approximately 0.4 for ET-1. In cross-circulation preparations, pressor responses occurred in the donor rat following injection into the fourth cerebral ventricle of the recipient animal, showing that effective doses of ANG II, administered to the fourth cerebral, reach the systemic circulation. The same results were obtained for the microinjection of 4 nmol of LVP into the fourth cerebral ventricle of recipient animals. High-performance reverse-phase liquid chromatography analyses of arterial blood showed that approximately 1% of the [125I]ANG II injected into the fourth cerebral ventricle may be recovered from the systemic circulation a few seconds after the microinjection. The systemic administration of the ANG II receptor antagonist losartan blocked the response to ANG II injected into the fourth ventricle whereas antagonist administration in the same ventricle did not. Angiotensin injections into the lateral ventricle produced pressor responses that were reduced by antagonist administration to the same ventricle but not by systemic administration of the antagonist. The data suggest that the pressor effect resulting from ANG II or LVP injections into the fourth cerebral ventricle may be due to the action of this peptide in the systemic circulation. On the other hand, the pressor effect due to ANG II microinjection into the lateral ventricle apparently results from the direct stimulation of central periventricular structures.

Acute changes in 3H-PAC and 125I-PYY binding in the nucleus tractus solitarii and hypothalamus after a hypertensive stimulus.

Activation of alpha-2-adrenergic and neuropeptide Y (NPY) receptors in the nucleus tractus solitarii (NTS) induces hypotension and bradycardia. On the contrary, activation of angiotensin II (Ang II) receptors leads to hypertension. Acute changes in binding parameters of alpha-2-adrenergic, NPY and Ang II receptors were evaluated in the NTS and paraventricular hypothalamic nucleus (PVN) of rats after a hypertensive stimulus employing quantitative receptor autoradiography. Saturation experiments showed a decrease in the number (Bmax) of alpha-2-adrenergic binding sites in the NTS 6 hours after coarctation-induced hypertension. Furthermore, the affinity of NPY receptors was diminished as seen by the increase in the KD value of 125I-PYY. Tyrosine hydroxylase and NPY immunoreactivities were increased in the NTS and ventral medulla. Binding of 125I-Ang II was not changed in the NTS. Binding of all ligands analyzed was not altered in the PVN. The results suggest an acute down-regulation of alpha-2-adrenergic and NPY receptors involved with hypotension in response to hypertensive stimulus, which might be related to an increased availability of catecholamines and NPY in the NTS.