Abstracts

Abstract

SymposiumRole of Nitric Oxide and Reactive Oxygen Species in the Brain Stem of Stroke-Prone Spontaneously Hypertensive Rats (SHRSP) in Central Nervous System Mechanisms of Hypertension. Y. Hirooka. Department of Cardiovascular Medicine, Kyushu University Graduate School of Medical Sciences, Fukuoka, Japan.Hypertension is a disorder of blood pressure regulation. Normally, blood pressure should be controlled by autonomic nervous system such as the baroreceptor reflex system, even if other local factors alter blood pressure. Therefore, abnormality of central nervous system mechanism(s) should be involved in hypertension. A decrease in nitric oxide (NO) and an increase in reactive oxygen species (ROS) contribute to the pathogenesis of hypertension and development of hypertensive vascular diseases. However, little is known regarding the role of NO or ROS in the brain stem, where the cardiovascular center (rostral ventrolateral medulla; RVLM) is located, in hypertension.We examined whether NO or ROS contribute to hypertensive mechanisms via the sympathetic nervous system. For this purpose, we transfected adenovirus vectors encoding endothelial NO synthase (AdeNOS) or Mn-superoxide dismutase (AdMnSOD) locally into the RVLM of SHRSP. Successful gene transfer was confirmed by immunohistochemistry or Western blot analysis. Enzymatic activity of expressed protein was confirmed by measurements of nitrite/nitrate (NOx) or SOD activity. Furthermore, we observed changes in blood pressure and heart rate using the radiotelemetry system in a conscious state. Urinary norepinephrine excretion was measured as a marker of sympathetic nerve activity.Expression levels of transfected gene peaked at day 7 to 10 after gene transfer. NOx levels were increased after AdeNOS transfection into the RVLM. SOD activity also was increased after AdMnSOD transfection. After transfection of AdeNOS into the RVLM, blood pressure and heart rate were decreased consistent with the time course of changes in expression levels of eNOS protein. In addition, the magnitude of the decrease in blood pressure and heart rate was significantly greater in SHRSP than in normotensive Wistar-Kyoto (WKY) rats. Urinary norepinephrine excretion was significantly greater in SHRSP than in WKY, indicating that enhanced sympathetic nerve activity is involved in hypertension of SHRSP. This was decreased in AdeNOS transfection in SHRSP. After transfection of AdMnSOD, blood pressure and heart rate were decreased only in SHRSP but not in WKY. Urinary norepinephrine excretion also was decreased after AdMnSOD transfection.Furthermore, thiobarbituric-acid reactive substances, a marker of ROS generation, were significantly greater in the brain stem of SHRSP than that of WKY. Electron spin resonance spectroscopy analysis also confirmed the increased ROS generation in the RVLM of SHRSP.Taken together, these results suggest that a decrease in NO activity and an increase in ROS generation in the RVLM contribute to hypertensive mechanisms of SHRSP. Further studies in this field are needed to obtain a better understanding of the mechanisms of hypertension and to develop an ideal treatment for hypertension.Sympathetic Activation in Vasculature of the Congenic Rat Strains for the Hypertension-Related Region on Chromosome 1. Y. Kobayashi,1 Hiromu Kawasaki,2 and T. Nabika.3 1Centre for Integrated Research in Science; 3Department of Functional Pathology, School of Medicine, Shimane University, Izumo, Japan; 2Department of Clinical Pharmaceutical Science, Graduate School of Natural Science and Technology, Okayama University, Okayama, Japan.Linkage studies have suggested that one of the rat quantitative trait loci (QTLs) for high blood pressure (BP) was located on chromosome 1. The phenotypes for this QTL, the serum noradrenaline (NA) concentration, the NA content in the thoracic aorta (TA), and the density of the sympathetic neurons in the supramesenteric arterioles (SMA) were evaluated in a congenic strain, WKY: SHRSP-(D1Wox29-D1Arb21)/Izm (abbreviated as WKYpch1.0), in which the QTL region for hypertension on chromosome 1 was transferred from SHRSP/Izm to WKY/Izm.BP was measured by the tail-cuff methods. Concentration of NA was measured by HPLC-ECD methods. Innervation of sympathetic nerves in the SMA was investigated with immunohistochemistry using an antineuropeptide Y (NPY) antibody. Many fibers containing NPY-like immunoreactivity were observed in the adventitia of arterioles. Amount of immunoreactive materials to a NPY antibody per area was analyzed with computer-based measurement. The number of the cross of NPY antibody-positive fibers with appropriate parallel lines was counted to evaluate the density of innervation. This study was conducted in line with the Guiding Principles for the Care and Use of Laboratory Animals approved by the Committee of Shimane University, School of Medicine.BP of WKYpch1.0 was significantly higher (~ 15 mmHg) than that of WKY/Izm at 20 weeks of age, but not at 7 or 11 weeks of age. Significantly higher BP was observed in SHRSP/Izm at 7 weeks of age and the hypertension became more severe at 11 and 20 weeks of age. The serum NA concentration of WKYpch1.0 did not differ significantly from that of SHRSP/Izm at week 7, which was significantly greater than that of WKY/Izm. NA concentrations in serum at 11 and 20 week of age were not different among the strains. The NA content in TA of SHRSP/Izm was significantly greater than that of WKY/Izm at 7 weeks of age. In WKYpch1.0, the NA content in TA at week 7 tended to be greater than that of WKY/Izm, though the difference was not significant. In SMA, the density of the NA neurons was significantly greater in WKYpch1.0 when compared with SHRSP/Izm and WKY/Izm at week 5. Amount of NPY-like materials in SMA was higher in SHRSP/Izm compared with that in WKYpch1.0 or WKY/Izm.Thus, increase of innervated fibers does not depend on the amount of the materials. Activation of sympathetic system of the vasculature of WKYpch1.0 may contribute to the serum catecholamine changes in a prehypertensive period. Along this line, hyperreaction of WKYpch1.0 on sympathetic activity to environmental stress and abolishment of the enhancement of BP of the congenic rats after guanethidine treatment at postnatal period were reported (Cui et al., Clin Exp Pharmacol Physiol 2003;30:464.). In conclusion, involvement of physiological and morphological alterations in the peripheral sympathetic nervous system in a prehypertensive stage in the pathogenesis of hypertension in this congenic strain was suggested.Supported in part from Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science and Smoking Research Foundation, Japan.Role of Rho/Rho-Kinase in the Brain Stem in Blood Pressure Regulation. K. Ito, Y. Hirooka, H. Shimokawa, A. Takeshita, and K. Sunagawa. Department of Cardiovascular Medicine, Kyushu University Graduate School of Medical Sciences, Fukuoka, Japan.The Rho/Rho-kinase pathway in the neurons is involved in the maintenance of dendritic spines, which form the postsynaptic contact sites of excitatory synapse. Recent studies suggest that morphological changes of dendritic spines occur rapidly and are associated with glutamate sensitivity. We speculated that Rho/Rho-kinase pathway in the nucleus tractus solitarii (NTS) may affect the synaptic transmission and contribute to cardiovascular regulation. Therefore, the aim of the present study was to elucidate the role of the Rho/Rho-kinase pathway in the NTS in blood pressure regulation.We performed microinjection of Y-27632, a specific Rho-kinase inhibitor into the bilateral NTS of Wistar-Kyoto rats (WKY) or spontaneously hypertensive rats (SHR). In addition, we transfected adenovirus vectors encoding dominant negative Rho-kinase (AdDNRhoK) into the bilateral NTS of both strains. Blood pressure and heart rate (HR) were measured by femoral artery catheter in the unconscious state or using radiotelemetry system in the conscious state. The sympathetic nerve activity (SNA) was evaluated by recordings of renal SNA or urinary norepinephrine excretion of 24 h. We evaluated the effects of Rho-kinase into the NTS on baroreflex function. Reflex changes of HR were elicited by intravenous infusion of either phenylephrine or sodium nitroprusside after the AdDNRhoK transfection in the conscious state. We compared the magnitude of decreases in blood pressure evoked by microinjection of glutamate into the NTS with or without Y-27632.Microinjection of Y-27632 or the gene transfer of AdDNRhoK into the NTS decreased blood pressure, HR, and SNA (renal SNA or urinary norepinephrine excretion) in both strains. However, the magnitude of decrease in these alterations was significantly larger in SHR than in WKY. The maximum gain of baroreflex control of HR was attenuated in SHR compared with WKY before the gene transfer. Transfection of AdDNRhoK significantly augmented the maximum gain both in WKY and SHR. The extent of this augmentation, however, was greater in SHR than in WKY. When lower doses of glutamate were microinjected, the magnitude of decrease in blood pressure was significantly reduced in SHR compared with WKY. The magnitude of decreases in blood pressure evoked by coinjection of glutamate and Y-27632 was significantly greater compared with only glutamate injection in both strains.Our results suggest that Rho-kinase in the NTS contributes to the regulation of blood pressure and baroreflex function via a neural mechanism. The activation of this pathway may contribute to the hypertensive mechanisms and impairment of baroreflex function of SHR. Furthermore, these effects may result from changes of glutamate sensitivity.