Debora R. Fior-Chadi

Change in the expression of NPY receptor subtypes Y1 and Y2 in central and peripheral neurons related to the control of blood pressure in rats following experimental hypertension

Neuropeptide Y (NPY) is known to participate in central mechanisms of blood pressure control. However, variations on the expression of its receptors in response to a hypertensive challenge are not well defined, specially when considering that Y1 and Y2 often mediate opposite responses. In this study we have employed in situ hybridization to analyze changes in mRNA expression of NPY receptor subtypes Y1 and Y2 in the nucleus tractus solitarii (NTS), paraventricular nucleus of the hypothalamus (PVN) and petrosal and nodose ganglions 2 h, 3 and 7 days after aortic coarctation induced hypertension. Quantification by image analysis showed significant differences between sham-operated and aortic-coarcted hypertensive rats. Y1 receptor mRNA expression was increased (39%) in petrosal ganglion, 3 days after surgery. Y2 receptor mRNA expression was increased (143%) in the NTS of hypertensive compared with sham rats 2 h after surgery. Y2 receptor mRNA was decreased (62%) in the nodose ganglion of hypertensive compared with sham rats 2 h after surgery. No change was seen in Y1 and Y2 mRNA expression in the PVN in any analyzed period. The data suggest that NPY Y1 and Y2 receptors might participate in the mechanisms involved in the establishment/maintenance of hypertension induced by aortic coarctation. Acute changes seem to be involved with the adaptation to the new hypertensive state.

Adenosine A1 receptor distribution in the nucleus tractus solitarii of normotensive and spontaneously hypertensive rats

Summary.Adenosine acts at many sites to modulate neuronal activity. The nucleus tractus solitarii (NTS) is a major brain site in cardiovascular control. The present study was undertaken for a detailed analysis of the distribution of A1 adenosine receptor (A1R) in the NTS of spontaneously hypertensive rats (SHR) and Wistar Kyoto (WKY), using in vitro autoradiography with [3H]DPCPX. The density of [3H]DPCPX in the whole NTS decreased according to the rostral-caudal levels. This high level of [3H]DPCPX binding at rostral sites is due to an specific label of the dorsomedial/dorsolateral subnuclei. On the other hand, analysis of subpostremal subnucleus, showed opposite results. The density of [3H]DPCPX binding in the subpostremal NTS increased according to the rostral-caudal levels. Furthermore, it was observed an increased [3H]DPCPX binding in the SHR compared with WKY. The results show a complex pattern of A1R distribution in the NTS, which highlight the powerful modulatory actions mediated by adenosine in the NTS barosensitive neurons.

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.

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.

Adenosine Modulates α2-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 α2-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.

Quantitative autoradiography of adrenergic, neuropeptide Y and angiotensin II receptors in the nucleus tractus solitarii and hypothalamus of rats with experimental hypertension

Catecholamines, neuropeptide Y (NPY) and angiotensin II (Ang II) are known to participate in the central control of blood pressure. However, the modulation of these neurotransmitter receptors in response to a hypertensive stimulus is not appropriately established. The purpose of the present study was to examine binding parameters of alpha(2)-adrenergic, NPY and Ang II receptors in the nucleus tractus solitarii (NTS) and paraventricular hypothalamic nucleus (PVN) following a hypertensive stimulus in the aortic-coarcted rat by means of quantitative receptor autoradiography. No changes were seen in binding parameters of alpha(2)-adrenergic and NPY receptors in the NTS of the hypertensive rat compared to control. However, an increased affinity (54%) of noradrenaline competing for 3H-PAC was seen in the PVN. Moreover, an increased binding (49%) of 125I-PYY was also observed in the PVN. The affinity of Ang II for 125I-Sar(1)Ile(8)-Ang II binding sites was also increased (57%) in the NTS of the hypertensive rat. No changes in the binding parameters of radioactive Ang II were observed in the PVN. The results suggest that systems involved with hypertension like Ang II in the NTS and catecholamines in the PVN might collaborate in the development/maintenance of high blood pressure in the aortic-coarcted rat.

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.

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.

Adenosine modulates alpha2-adrenergic receptors through a phospholipase C pathway in brainstem cell culture of rats.

Adenosine acts in the nucleus tractus solitarii (NTS), one of the main brain sites related to cardiovascular control. In the present study we show that A(1) adenosine receptor (A(1R)) activation promotes an increase on alpha(2)-adrenoceptor (Alpha(2R)) binding in brainstem cell culture from newborn rats. We investigated the intracellular cascade involved in such modulatory process using different intracellular signaling molecule inhibitors as well as calcium chelators. Phospholipase C, protein kinase Ca(2+)-dependent, IP(3) receptor and intracellular calcium were shown to participate in A(1R)/Alpha(2R) interaction. In conclusion, this result might be important to understand the role of adenosine within the NTS regarding autonomic cardiovascular control.

Decreases in the expression of CGRP and galanin mRNA in central and peripheral neurons related to the control of blood pressure following experimental hypertension in rats

Calcitonin gene-related peptide (alpha CGRP) and galanin (GAL) are peptides known to participate in central mechanisms of blood pressure control. Nonetheless, variations in the synthesis of the peptides in response to a hypertensive challenge are not well described, specially using a model, which allows acute and chronic analyses. In this study, we have employed in situ hybridization to analyse changes in mRNA expression of alpha CGRP and GAL in the nucleus tractus solitarii (NTS), hypothalamic paraventricular nucleus (PVN) as well as petrosal and nodose ganglia after aortic coarctation-induced hypertension in rats. Acute (2h) and chronic (3 and 7 days) analyses were performed in order to evaluate the involvement of both peptides in different periods of hypertension. The analysis of relative mRNA levels showed significant differences between sham-operated and aortic coarcted hypertensive rats. alpha CGRP mRNA expression was decreased 2h (40%) and 3 days (42%) in nodose and petrosal ganglia, respectively, after coarctation. No changes in CGRP mRNA signal were seen in the NTS and PVN in the analysed periods. GAL mRNA expression was decreased in the NTS (19%) and PVN (55%), 3 and 7 days, respectively, after coarctation-induced hypertension. No changes in GAL mRNA expression were observed in petrosal and nodose ganglia following aortic coarctation. Data suggest that alpha CGRP and GAL may participate in the mechanisms involved in the establishment/maintenance of hypertension induced by aortic coarctation. Acute changes might be involved with the adaptation to the hypertensive state, while changes at the chronic phase might be related to counteraction of hypertension.