Unique endothelin receptor binding in kidneys of ETB receptor deficient rats

The study investigated whether the amelioration of endothelial dysfunction by candesartan (2 mg.kg-1.day-1; 10 wk) in spontaneously hypertensive rats (SHR) was associated with modification of hepatic redox system. Systolic arterial pressure (SAP) was higher (P < 0.05) in SHR than in Wistar-Kyoto rats (WKY) and was reduced (P < 0.05) by candesartan in both strains. Acetylcholine (ACh) relaxations were smaller (P < 0.05) and contractions induced by ACh + NG-nitro-l-arginine methyl ester (l-NAME) were greater (P < 0.05) in SHR than in WKY. Treatment with candesartan enhanced (P < 0.05) ACh relaxations in SHR and reduced (P < 0.05) ACh + l-NAME contractions in both strains. Expression of aortic endothelial nitric oxide synthase (eNOS) mRNA was similar in WKY and SHR, and candesartan increased (P < 0.05) it in both strains. Aortic mRNA expression of the subunit p22phox of NAD(P)H oxidase was higher (P < 0.05) in SHR than in WKY. Treatment with candesartan reduced (P < 0.05) p22phox expression only in SHR. Malonyl dialdehyde (MDA) levels were higher (P < 0.05), and the ratio reduced/oxidized glutathione (GSH/GSSG) as well as glutathione peroxidase activity (GPx) were lower (P < 0.05) in liver homogenates from SHR than from WKY. Candesartan reduced (P < 0.05) MDA and increased (P < 0.05) GSH/GSSG ratio without affecting GPx. Vessel, lumen, and media areas were bigger (P < 0.05) in SHR than in WKY. Candesartan treatment reduced (P < 0.05) media area in SHR without affecting vessel or lumen area. The results suggest that hypertension is not only associated with elevation of vascular superoxide anions but with alterations of the hepatic redox system, where ANG II is clearly involved. The results further support the key role of ANG II via AT1 receptors for the functional and structural vascular alterations produced by hypertension.     

Reduced connexin-43 expression in the aorta of prehypertensive rats

Genetic component represents an important factor in the development of hypertension, which is known to be associated with changes in expression of vascular gap junction protein connexin 43 (Cx43). The aim of the study was to examine the distribution and expression of Cx43 in the aortic endothelium of adult normotensive Wistar rats (W), borderline hypertensive rats (BHR) and spontaneously hypertensive rats (SHR). Rings of the thoracic aorta were processed for immunofluorescence and Western blot analysis of endothelial Cx43 and for electron microscopy. Both, BHR and SHR exhibited significantly increased blood pressure vs. W (132+/-2 mm Hg and 185+/-3 mm Hg vs. 110+/-2 mm Hg). Reduced Cx43 immunofluorescence was observed in the endothelium of BHR and these alterations were more pronounced in SHR. Western blot analysis showed significant suppression of Cx43 expression in the aorta of both BHR (p<0.05) and SHR (p<0.001) vs. W. Electron microscopy revealed local subcellular alterations of interendothelial connections in BHR including extended tight junctions. These alterations were more frequent and marked in SHR. The results indicate that connexin 43 expression is reduced in the aortic endothelium already in prehypertensive period, which may affect cell-to-cell communication and thus participate in acceleration of hypertensive disease.     

Changes in angiotensin receptors expression play a pivotal role in the renal damage observed in spontaneously hypertensive rats

The renal renin-angiotensin system plays a central role in the development of hypertension. The aim of this work was to verify the expression of angiotensin II receptors AT(1)R and AT(2)R in the microsomal fraction of renal cortex and correlate this with the development of hypertension and renal damage in spontaneously hypertensive rats (SHR) using Wistar-Kyoto rats (WKY) as controls. AT(1)R expression increased (126%) and AT(2)R expression decreased (66%) in 4-wk-old SHR; AT(2) expression decreased in 14-wk-old SHR (61%) compared with respective age-matched WKY. These modifications were correlated to the increase in protein kinase C activity and decrease in protein kinase A activity. Four-week-old SHR showed large accumulations of macrophages in kidney glomerulus and the tubulointerstitial area, dense cortical collagen deposition, and arterial proliferative changes in the walls of arterioles and medium-sized vessels. Similar modifications were also observed in 14-wk-old SHR. Four-week-old SHR treated with losartan (30 mg·kg(-1)·day(-1)) or hydralazine (15 and 30 mg·kg(-1)·day(-1)) by gavage for 10 wk did not develop hypertension. The decrease in AT(2)R expression and renal damage observed in SHR remained even after treatment with hydralazine. On the other hand, losartan treatment prevented the modifications observed in 14-wk-old SHR, indicating that renal injuries are caused specifically by AT(1) rather than an increase in blood pressure. Our results indicate that the imbalance in AT(1)R and AT(2)R expression is associated with an inflammatory process that contributes to renal injury in adult SHR and to the development of hypertension.     

Time course of vascular arginase expression and activity in spontaneously hypertensive rats

There is growing evidence that vascular arginase plays a role in pathophysiology of vascular diseases. We recently reported high arginase activity/expression in young adult hypertensive spontaneously hypertensive rats (SHR). The aim of the present study was to characterize the time course of arginase pathway abnormalities in SHR and to explore the contributing role of hemodynamics and inflammation. Experiments were conducted on 5, 10, 19 and 26-week-old SHR and their age-matched control Wistar Kyoto (WKY) rats. Arginase activity as well as expression of arginase I, arginase II, endothelial and inducible NOS were determined in aortic tissue extracts. Levels of L-arginine, NO catabolites and IL-6 (a marker of inflammation) were measured in plasma. Arginase activity/expression was also measured in 10-week-old SHR previously treated with hydralazine (20 mg/kg/day, per os, for 5 weeks). As compared to WKY, SHR exhibited high vascular arginase I and II expression from prehypertensive to established stages of hypertension. However, a mismatch between expression and activity was observed at the prehypertensive stage. Arginase expression was not related either to plasma IL-6 levels or to expression of NOS. Prevention of hypertension by hydralazine significantly blunted arginase upregulation and restored arginase activity. Importantly, arginase activity and blood pressure (BP) correlated in SHR. In conclusion, our results demonstrate that arginase upregulation precedes blood pressure rising and identify elevated blood pressure as a contributing factor of arginase dysregulation in genetic hypertension. They also demonstrated a close relationship between arginase activity and BP, thus making arginase a promising target for antihypertensive therapy.     

Up-regulation of myocardial connexin-43 in spontaneously hypertensive rats fed red palm oil is most likely implicated in its anti-arrhythmic effects

The purpose of this study was to test our hypothesis that red palm oil (RPO) intake may affect abnormalities of myocardial connexin-43 (Cx43) and protein kinase Cε (PKCε) signaling, and consequently the propensity of the spontaneously hypertensive rat heart (SHR) heart to arrhythmias. SHR and Wistar-Kyoto (WKY) rats fed a standard rat chow plus red palm oil (200?μL/day) for 5?weeks were compared with untreated rats. Cytosolic but not particulate PKCε expression as well as Cx43-mRNA, total Cx43 proteins, and its phoshorylated forms were increased, and disordered localization of Cx43 was attenuated in the left ventricle of RPO-fed SHR compared with untreated rats. These alterations were associated with suppression of early post-ischemic-reperfusion-related ventricular tachycardia and electrically inducible ventricular fibrillation. However, the treatment dose of RPO caused down-regulation of myocardial Cx43, but did not alter its cell membrane distribution or overall PKCε expression in WKY rats. It was, however, associated with poor arrhythmia protection, suggesting overdosing. Results indicate that SHR benefit from RPO intake, particularly because of its apparent anti-arrhythmic effects. This protection can be, in part, attributed to the preservation of cell-to-cell communication via up-regulation of myocardial Cx43, but not with PKCε activation.     

Endothelium-derived relaxing factors in the kidney of spontaneously hypertensive rats

Acetylcholine (ACh)-induced vasodilation is mainly due to endothelium-derived nitric oxide (EDNO) and hyperpolarizing factor (EDHF). To explore the mechanisms underlying attenuated endothelium-dependent vasodilation in hypertensive arteries, we measured the EDNO released from isolated kidneys of spontaneously hypertensive rats (SHR) using a sensitive chemiluminescence assay system of NO. ACh-induced renal vasodilation was significantly smaller in SHR than in the normotensive control, Wistar-Kyoto rats (WKY). However, ACh-induced NO release did not differ between SHR and WKY (10⁻⁷ M: SHR +37 ± 2 [SE] vs. WKY +32 ± 4 fmol/min/g kidney). Perfusion with a 20 mEq/L high-K⁺ buffer, which is reported to inhibit action of EDHF, significantly reduced ACh-induced vasorelaxation in WKY but not in SHR, resulting in identical renal perfusion pressure in SHR and wKY under these conditions. These results indicate that attenuated ACh-induced vasorelaxation in the SHR kidney may be attributed to a decrease in EDHF rather than that in EDNO.     

Diabetes reduces aortic endothelial gap junctions in ApoE-deficient mice: simvastatin exacerbates the reduction.

We examined the endothelial gap junctions in diabetic hyperlipidemic mice. Male apolipoprotein E (apoE)-deficient mice were made diabetic by streptozotocin. Three weeks later, the animals were treated with simvastatin for 2 weeks. The expression of aortic gap junctions in the non-diabetic (n=10), untreated diabetic (n=10), and simvastatin-treated diabetic animals (n=6) was analyzed. There was a >4-fold increase in serum cholesterol level and >50% increase in plaque areas in the diabetic mice, regardless of simvastatin treatment. Western blotting of aortae showed reduced expression of connexin37 (Cx37) and Cx40 in the diabetic mice, which were further decreased in the simvastatin-treated diabetic mice. Immunoconfocal microscopy showed that endothelial gap junctions made of Cx37 and Cx40 were both reduced in the untreated diabetic mice compared with the non-diabetic mice (decrease: Cx37, 41%; Cx40, 42%; both p<0.01). The reduction was greater in the simvastatin-treated mice (decrease in treated diabetic vs non-diabetic: Cx37, 61%; Cx40, 79%; both p<0.01; decrease in treated diabetic vs untreated diabetic: Cx37, 34%; Cx40, 63%; both p<0.01). Cx37 and Cx40 were decreased in the endothelium of plaque surface. Cx43 appeared in the medial layer and inner layer of the intima. All three connexins were rarely expressed in monocytes/macrophages inside the plaques. In conclusion, in apoE-deficient mice, streptozotocin-induced diabetes is associated with downregulation of endothelial Cx37 and Cx40 gap junctions. Short-term treatment with simvastatin exacerbates the downregulation.     

Flow regulates intercellular communication in HAEC by assembling functional Cx40 and Cx37 gap junctional channels

Direct cell-to-cell transfer of ions and small signaling molecules via gap junctions plays a key role in vessel wall homeostasis. Vascular endothelial gap junctional channels are formed by the connexin (Cx) proteins Cx37, Cx40, and Cx43. The mechanisms regulating connexin expression and assembly into functional channels have not been fully identified. We investigated the dynamic regulation of endothelial gap junctional intercellular communication (GJIC) by fluid flow and the participation of each vascular connexin in functional human endothelial gap junctions in vitro. Human aortic endothelial cells (HAEC) were exposed for 5, 16, and 24 h to physiological flows in a parallel-plate flow chamber. Connexin protein expression and localization were evaluated by immunocytochemistry, and functional GJIC was evaluated by dye injection. Connexin-mimetic peptide inhibitors were used to assess the specific connexin composition of functional channels. HAEC monolayers in culture exhibited baseline functional communication at a striking low level despite abundant expression of Cx43 and Cx40 localized at cell-to-cell appositions. Upon exposure to flow, GJIC by dye spread demonstrated a significant time-dependent increase from baseline levels, reaching 7.5-fold in 24 h. Inhibition studies revealed that this response was mediated primarily by Cx40, with lesser contributions of the other two vascular connexins assembled into functional homotypic and/or heterotypic channels. This is the first study to demonstrate that flow simultaneously and differentially regulates expression of the Cx37, Cx40, and Cx43 proteins and their involvement in the augmentation of intercellular communication by dye transfer in human endothelial cells in vitro.     

Gap junctional communication underpins EDHF-type relaxations evoked by ACh in the rat hepatic artery

Synthetic peptides homologous to the Gap 26 and Gap 27 domains of the first and second extracellular loops of the major vascular connexins (Cx37, Cx40, and Cx43) have been used to investigate the role of gap junctions in endothelium-derived hyperpolarizing factor (EDHF)-type relaxations of the rat hepatic artery. These peptides were designated 37,40Gap 26, 43Gap 26, 37,43Gap 27, and 40Gap 27, according to connexin specificity. When administered at 600 microM, none of the peptides individually affected maximal EDHF-type relaxations to ACh. By contrast, at 300 microM each, paired peptide combinations targeting more than one connexin subtype attenuated relaxation by up to 50%, and responses were abolished by the triple peptide combination 43Gap 26 + 40Gap 27 + 37,43Gap 27. In parallel experiments with A7r5 cells expressing Cx40 and Cx43, neither 43Gap 26 nor 40Gap 27 affected intercellular diffusion of Lucifer yellow individually but, in combination, significantly attenuated dye transfer. The findings confirm that functional cell-cell coupling may depend on more than one connexin subtype and demonstrate that direct intercellular communication via gap junctions constructed from Cx37, Cx40, and Cx43 underpins EDHF-type responses in the rat hepatic artery.     

Connexin 43 mediates endothelium-derived hyperpolarizing factor-induced vasodilatation in subcutaneous resistance arteries from healthy pregnant women

The role of gap junctions in endothelium-derived hyperpolarizing factor (EDHF)-mediated relaxation of human arteries was assessed using connexin mimetic peptides (CMPs) designated (37,43)Gap27, (40)Gap27, and (43)Gap26 according to homology with the major vascular connexins (Cx37, Cx40, and Cx43). Resistance arteries were obtained from subcutaneous fat biopsies of healthy pregnant women undergoing elective cesarean section. Endothelium-dependent vasodilatation to bradykinin (BK) was assessed using wire myography. N(omega)-nitro-l-arginine methyl ester (l-NAME) and indomethacin (nitric oxide synthase and cyclooxygenase inhibitors, respectively) attenuated maximal relaxation to BK (R(max)) by approximately 50%. Coincubation with l-NAME, indomethacin, and the combined CMPs ((37,43)Gap27, (40)Gap27, and (43)Gap26) almost abolished relaxation to BK (R(max) = 12.2 +/- 3.7%). In arteries incubated with l-NAME and indomethacin, the addition of either (37,43)Gap27 or (40)Gap27 had no significant effect on R(max), whereas (43)Gap26 caused marked inhibition (R(max) = 21 +/- 6.4%, P = 0.005 vs. l-NAME plus indomethacin alone) that was similar to that of the triple combination. Endothelium-independent vasorelaxation was unaffected by CMPs, l-NAME, or indomethacin. Immunohistochemistry demonstrated Cx37, Cx40, and Cx43 expression in the endothelium and vascular smooth muscle. In pregnant women, EDHF-mediated vasorelaxation of subcutaneous resistance arteries is dependent on Cx43 and gap junctions.     

Role of connexin37 and connexin40 in vascular development

Mice lacking both connexin37 (Cx37) and connexin40 (Cx40), gap junction proteins expressed in vascular endothelium, die perinatally with pronounced vascular abnormalities. Early vasculogenesis proceeds normally, but by E18.5 Cx37(-/-)Cx40(-/-) animals display vessel dilatation and congestion as well as localized hemorrhages in skin, testis, intestines, and lungs. Abnormal vascular channels are present in the testis, often forming cavernous hemangioma-like defects. Unusually large, distended vessels are also present in the submucosa and lamina propria of the intestine. Ablation of Cx40 has a greater effect on endothelial dye-transfer than ablation of Cx37, and the effect of Cx40 ablation is age-dependent. Only in embryonic aortas lacking both Cx37 and Cx40 is there a complete loss of endothelial coupling. Surprisingly, elimination of Cx40 results in a large drop in aortic endothelial Cx37 on western blots, and deletion of Cx37 also reduces endothelial Cx40 levels. In contrast, in the medial layer, both Cx37 and Cx43 increase when Cx40 is ablated. These studies indicate that Cx37 and Cx40 are collectively critical for endothelial communication and provide evidence of an important role for gap junctions in vascular development. In addition, Cx37 and Cx40 appear to be mutually dependent on each other for normal expression in vascular endothelium.     

Expression of connexin 37, 40 and 43 in rat mesenteric arterioles and resistance arteries

Connexins are the protein constituents of gap junctions which mediate intercellular communication in most tissues. In arterioles gap junctions appear to be important for conduction of vasomotor responses along the vessel. Studies of the expression pattern of connexin isoforms in the microcirculation are sparse. We investigated the expression of the three major vascular connexins in mesenteric arterioles (diameter <50 micro m) from male Sprague-Dawley rats, since conducted vasomotor responses have been described in these vessels. The findings were compared with those obtained from upstream small resistance arteries. Indirect immunofluorescence techniques were used on whole mounts of mesenteric arterioles and on frozen sections of resistance arteries (diameter approximately 300 micro m). Mesenteric arterioles expressed Cx40 and Cx43 in the endothelial layer, and Cx37 was found in most but not all vessels. Connexins were not demonstrated in the media. In resistance arteries endothelial cells expressed Cx37, Cx40 and Cx43. Ultrastructural studies of mesenteric arterioles confirmed that gap junction plaques between endothelial cells are present, whereas myoendothelial, or smooth muscle cell gap junctions could not be demonstrated. The findings suggest that smooth muscle cells in mesenteric arterioles may not be well coupled and favour that conducted vasomotor responses in these vessels are propagated through the endothelial cell layer.     

The effect of anti-hypertensive drug treatment on brown adipocyte diameter and locule distribution in rats

To determine the effects of sympathoplegic anti-hypertensive drug treatment on brown adipose tissue morphology, groups of adult male Wistar-Kyoto (WKY) and spontaneous hypertensive (SHR) rats were orally administered a solution containing 2.3 microM reserpine, 0.5 M hydralazine and 1.68 mM hydrochlorothiazide ad libitum or tap water and brown adipocyte diameter and extent of fat loculization were determined 3 weeks later. Pulse rates of rats were significantly greater in SHR than WKY and were unaffected by treatment, while drug treatment resulted in significant decreases in mean arterial pressure of both groups at the end of the study. Mean adipocyte diameters were smaller in untreated SHR than WKY and drug treatment of both groups was associated with increases in adipocyte diameter and cytological change from physiologically more active to less active cells. These drug-induced alterations in BAT morphology area consistent with decreased sympathetic activity and suggest that thermogenic capacity of brown adipose tissue may be pharmacologically modified, thereby altering an animal's capacity for energy expenditure.     

Role of Connexin37 and Connexin40 in Vascular Development

Mice lacking both connexin37 (Cx37) and connexin40 (Cx40), gap junction proteins expressed in vascular endothelium, die perinatally with pronounced vascular abnormalities. Early vasculogenesis proceeds normally, but by E18.5 Cx37^?/?Cx40^?/?animals display vessel dilatation and congestion as well as localized hemorrhages in skin, testis, intestines, and lungs. Abnormal vascular channels are present in the testis, often forming cavernous hemangioma-like defects. Unusually large, distended vessels are also present in the submucosa and lamina propria of the intestine. Ablation of Cx40 has a greater effect on endothelial dye-transfer than ablation of Cx37, and the effect of Cx40 ablation is age-dependent. Only in embryonic aortas lacking both Cx37 and Cx40 is there a complete loss of endothelial coupling. Surprisingly, elimination of Cx40 results in a large drop in aortic endothelial Cx37 on western blots, and deletion of Cx37 also reduces endothelial Cx40 levels. In contrast, in the medial layer, both Cx37 and Cx43 increase when Cx40 is ablated. These studies indicate that Cx37 and Cx40 are collectively critical for endothelial communication and provide evidence of an important role for gap junctions in vascular development. In addition, Cx37 and Cx40 appear to be mutually dependent on each other for normal expression in vascular endothelium.     

Alteration in sialidase and other glycosidase activities in the kidney of spontaneously hypertensive rats: persistence after preventive treatment with hydralazine

Because kidney microangiopathy with capillary basement membrane thickening has been reported in spontaneous hypertension, we have studied the activities of three lysosomal glycosidases able to degrade the carbohydrate moieties of basement membrane constituents in the kidney cortex of 12-week-old spontaneously hypertensive rats (SHR) and age-matched normotensive Wistar Kyoto rats (WKY). These activities were also determined in SHR and WKY treated from 6 to 12 weeks of age with hydralazine (mean dose, 18 mg/kg per day in drinking water). Sialidase specific activity on sialyl-alpha 2-3-[3H]lactitol was markedly decreased in the kidney of untreated SHR, 40% activity remaining relative to that found in untreated age-matched WKY (p less than 0.001). beta-Galactosidase specific activity on p-nitrophenyl-beta-D-galactoside was also decreased, 86% activity remaining relative to that found in untreated WKY (p less than 0.001). Glucosyl-galactosyl-hydroxylysyl glucohydrolase specific activity on glucosyl-galactosyl-hydroxylysine was equally diminished, 74% activity remaining relative to that found in untreated age-matched WKY (p less than 0.001). In contrast, the activities of two control glycosidases inactive on the carbohydrate moieties of basement membrane constituents, alpha-glucosidase assayed with p-nitrophenyl-alpha-D-glucoside as substrate and beta-glucosidase assayed with p-nitrophenyl-beta-D-glucoside as substrate, were significantly increased. All the alterations in enzyme activities observed in the kidney of SHR were also present in the long-term treated normotensive SHR. No effect of the hydralazine treatment on the three enzyme activities investigated could be demonstrated in the WKY. Thus the alterations observed in the kidneys of SHR appear to be independent of blood pressure level.     

Myocardial NOS activity and connexin-43 expression in untreated and omega-3 fatty acids-treated spontaneously hypertensive and hereditary hypertriglyceridemic rats

The purpose of this study is to investigate myocardial nitric oxide synthase (NOS) activity and connexin-43 (Cx43) expression in young and old spontaneously hypertensive rats (SHR), adult hereditary hypertriglyceridemic (HTG) rats, and age-matched healthy rats without and with omega-3 PUFA supplementation for 2 months. Results showed that comparing to healthy rats the myocardial NOS activity was significantly increased in young SHR (8.2 ± 1.16 vs. 1.37 ± 0.67 pmol/min/mg) as well as old SHR (3.21 ± 0.75 vs. 2.22 ± 0.56 pmol/min/mg) and to much lesser extent in HTG rats, i.e., 1.87 ± 0.42 vs. 1.34 ± 0.1 pmol/min/mg. In parallel, there was a significant decline of total and phosphorylated forms of Cx43 in both groups of SHR while not in HTG rat hearts in which phosphorylated form of Cx43 was increased. Elevated NOS activity was suppressed (P < 0.05) in young and old SHR supplemented with omega-3 PUFA and it was associated with up-regulation of Cx43. In contrast to SHR, elevation of NOS activity in HTG rat hearts was not affected by treatment with omega-3 PUFA. However, increase of phosphorylated form of Cx43 was suppressed. In conclusion, there is an inverse relationship between myocardial NOS activity and Cx43 expression in SHR while not HTG rat hearts and omega-3 PUFA modulate both NOS activity and Cx43 expression. Whether over-expression of inducible NOS might account for down-regulation of myocardial Cx43 and whether its up-regulation is associated with an increase of endothelial NOS should be explored in further study.     

Distribution of connexin37, connexin40 and connexin43 in the aorta and coronary artery of several mammals

Intercellular communication between cells of the vessel wall is established by a combination of diffusion and convection of humoral and endothelial factors in the extracellular fluid or by direct intercellular contacts present in the form of gap junctions composed of proteins called connexins. At least connexin (Cx)37, Cx40 and Cx43 are expressed in the vessel wall, but disparate findings with regard to the cell specific localisation of connexins in the vasculature indicate that the distribution of connexins may be species and vessel specific. Moreover, differences in expression exist between cells in culture and tissue sections. We performed an inventory immunohistochemical study on the localisation of Cx37, Cx40 and Cx43 on tissue sections of the bovine, micropig and rat aorta and coronary system, which represent morphologically and functionally different types of vessels in the arterial system. We could observe Cx40 labelling most commonly, although with various intensities, between endothelial and smooth muscle cells of the species studied, with the exception of rat aortic smooth muscle cells. The distribution of Cx43 is more differentiated and mostly confined to smooth muscle cells, although it can be detected scarcely between endothelial cells. Cx37, when detectable, is predominantly expressed between endothelial cells in a heterogeneous pattern. We conclude that Cx40 is the constitutive vascular gap junction protein in situ and guarantees cell coupling between cells in the vessel wall. The differentiated distribution of both Cx37 and Cx43 suggests they are involved in more dynamic processes. Accepted: 12 October 1999     

Hippocampal levels of dynorphin A (1–8) in neonatal and 16-week-old spontaneously hypertensive rats: Comparisons with DOCA-salt hypertension

In this study the possible role of hippocampal dynorphin in the development of hypertension in spontaneously hypertensive rats (SHR) was investigated by determining dynorphin A (1–8) (DN A (1–8)) levels in hippocampus in 16 week old SRH, Wistar Kyoto (WKY) controls and SHR treated with antihypertensive drugs as well as DOCA-salt hypertensive Sprague Dawley (SD) rats, using radioimmunoassay (RIA). We found that DN A (1–8) was decreased significantly in both dorsal (–68%) and ventral (–58%) hippocampus in SHR compared with WKY rats. Treatment with hydralazine and guanethidine (25 mg/kg/24 hr of each drug in drinking water) for 8 weeks to prevent the development of hypertension in young SHR had no effect on this low hippocampal dynorphin level. We failed to find significant changes in hippocampal DN A (1–8) level in DOCA-salt hypertensive rats. The low level of hippocampal dynorphin existed before the development of hypertension in 6 day neonatal SHR (–73%). Hippocampal Met-enkephalin was unchanged in all experimental groups except for a slight decrease in neonatal SHR. The results establish a genetic difference in the hippocampal dynorphin system of SHR compared with WKY, the significance of which, for the development of hypertension, remains to be investigated.     

缝隙连接在同型半胱氨酸介导的自发性高血压大鼠血管平滑肌细胞增殖中的作用

目的:探讨缝隙连接(GJ)是否参与同型半胱氨酸(Hcy)介导的自发性高血压(SHR)大鼠血管平滑肌细胞(VSMCs)增殖及可能的分子机制。方法:原代培养SHR VSMCs,细胞分四组:①对照组,②Hcy组,③缝隙连接阻断剂(18α-GA)组和④Hcy+18α-GA组。MTT法及流式细胞仪检测细胞的增殖活性,免疫荧光技术观察细胞中Cx43、Cx40蛋白表达及定位,Western blot法检测细胞中Cx43、Cx40蛋白表达,染料示踪分子传递法(划痕标记染料传输法)检测细胞的缝隙连接功能。结果:①与对照组相比,Hcy组MTT法测得A值及细胞周期测得S值增高(P0.05),18α-GA组降低(P0.05);与Hcy组比,TGFβ-1+18α-GA组A值及S值均降低(P0.05)。②免疫荧光技术检测细胞中Cx43、Cx40蛋白表达呈阳性,两者共定位于胞浆。③与对照组相比,Hcy组Cx43、Cx40蛋白表达增强(P0.05),18α-GA组Cx43、Cx40表达均减弱(P0.05);与Hcy组比,Hcy+18α-GA组Cx43、Cx40表达均减弱(P0.05)。④与对照组相比,Hcy组缝隙连接功能明显增强(P0.05),18α-GA组缝隙连接功能明显减弱(P0.05),与Hcy组比,Hcy+18α-GA组缝隙连接功能显著降低(P0.05)。结论:Hcy通过上调SHR VSMCs Cx43和Cx40的蛋白表达,引起缝隙连接通讯功能的增强,促进了SHR VSMCs增殖。