Decreased velocity of shortening in arterial smooth muscle from older (28- to 31-week-old) spontaneously hypertensive rats

An increased maximum velocity of shortening (Vmax) and increased shortening ability (delta Lmax) have been reported for caudal arterial smooth muscle from 16- to 18-week-old spontaneously hypertensive rats (SHR) compared with age-matched Wistar-Kyoto (WKY) control rats. It is known that hypertension results in hypertrophy of vascular smooth muscle. It is plausible that the faster Vmax of 16- to 18-week-old SHR arterial smooth muscle may slow down with age due to hypertrophy. The force-velocity (F-V) study done previously on caudal arterial strips from 16- to 18-week-old SHR and WKY rats was repeated on preparations from 28- to 31-week-old rats. An electromagnetic muscle lever was employed in recording force-velocity data. Analysis of these data revealed that the 28- to 31-week-old SHR (n = 7) mean F-V curve was not different from the 28- to 31-week-old WKY (n = 5) mean F-V curve (p greater than 0.05), and the shortening ability of 28- to 31-week-old SHR arterial muscle was significantly depressed compared with 28- to 31-week-old WKY arterial muscle (p less than 0.01). In conclusion, (i) although Vmax is faster in younger (16- to 18-week-old) SHR compared with age-matched WKY caudal arterial smooth muscle, SHR Vmax is not different from WKY Vmax in the older (28- to 31-week-old) rats. (ii) Shortening ability is greater in 16- to 18-week-old SHR caudal arterial strips compared with 16- to 18-week-old WKY strips, but is significantly depressed in 28- to 31-week-old SHR compared with 28- to 31-week-old WKY preparations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Morphological changes in cerebral vascular smooth muscle cells in stroke-prone spontaneously hypertensive rats (SHRSP)

The ultrastructure of the vascular smooth muscle cells of the middle cerebral artery in 6-month-old male stroke-prone spontaneously hypertensive rats (SHRSP) was studied by scanning (SEM) and transmission electron microscopy (TEM) and compared with that of age-matched normotensive Wistar Kyoto rats (WKY). Although the smooth muscle cells of WKY rats by SEM had a typical spindle shape and smooth surface texture, those of SHRSP were structurally modified by numerous surface invaginations and projections, bearing some structural resemblance to the myotendinous junction of skeletal muscle. Structural modifications affected more than half the surface of medial smooth muscle cells in SHRSP, but less than 0.6% of the surface of these cells in WKY rats. About 10% of medial smooth muscle cells were necrotic in SHRSP, but no necrotic cells were identified in WKY rats. By TEM, smooth muscle cells in SHRSP were shown to be irregular in profile with deep indentations of the plasma membrane and were surrounded by many layers of basal laminalike material. The present study suggests that most smooth muscle cells in the middle cerebral artery of SHRSP may be modified to adapt to chronic hypertension by increasing the junctional area between muscle cells and connective tissue and that some cells may undergo necrosis.     

Vasopressin-induced calcium increases in smooth muscle cells from spontaneously hypertensive rats

Cytosolic free Ca2+ concentrations [( Ca2+]i) were measured in smooth muscle cells (SMC) from spontaneously hypertensive rats (SHR) and age and sex matched Wistar-Kyoto rats (WKY). Resting levels of [Ca2+]i were 114 +/- 6 nM and 116 +/- 5 nM in SMC from WKY and SHR, respectively. Angiotensin II (AII) induced a dose-dependent large increases in [Ca2+]i in SMC. There were no significant differences in resting or AII-stimulated levels of [Ca2+]i when SMC from WKY and SHR were compared. Arg-vasopressin (AVP) caused a similar but smaller [Ca2+]i increase than AII in SMC. AVP caused larger [Ca2+]i increases in SMC from SHR than in SMC from WKY. Although concentrations of AVP higher than those ordinarily detected in plasma were necessary to obtain different responses between SHR and WKY, these differences may be related to the pathogenesis of hypertension.     

Calcium-requirement for activation of skinned vascular smooth muscle from spontaneously hypertensive (SHRSP) and normotensive control rats

The aim of this study was to clarify whether the increased vascular tone in spontaneous hypertension of rats is due to a change of the calcium-sensitivity of the contractile proteins themselves. In skinned rat tail artery rings from SHRSP and WKY rats the calcium-requirement for half maximal activation (3.6 × 10⁻⁶M Ca⁺⁺ for both rat strains) as well as relaxation half times (1.45 ± 0.43 min, SHRSP and 1.63 ± 0.48 min, WKY) were found to be identical. The extent of myosin phosphorylation in the contracted and in the relaxed state did not differ between SHRSP and WKY. It is concluded that changes at the level of the contractile proteins are not involved in the increased vascular tone of SHRSP essential hypertension.     

Nifedipine induces apoptosis in cultured vascular smooth muscle cells from spontaneously hypertensive rats

Apoptosis (programmed cell death) of smooth muscle cells (SMC) in blood vessels is an essential process involved in the control of vessel wall structure. Several antihypertensive drugs currently used in therapy may exert their pharmacological effects by promoting SMC apoptosis. The biochemical events which regulate SMC apoptosis in the vessel wall are complex, and not well understood. We therefore investigated whether treatment of cultured SMC from normotensive Wistar-Kyoto rats (WKY) and from spontaneously hypertensive rats (SHR) with selected antihypertensive drugs would induce SMC apoptosis. We treated aortic SMC from WKY and SHR in vitro with the L-type Ca2+ channel antagonist, nifedipine; with the nitric oxide donor, sodium nitroprusside (SNAP); with forskolin (an activator of adenylyl cyclase); or with thapsigargin (a selective inhibitor of the sarcoplasmic reticulum (SR), Ca2+-ATPase); and compared their apoptosis-promoting effects in SMC derived from the two strains of rats. SMC were derived from the thoracic aorta of 3-4-week-old WKY and SHR, and were used in passages 7-10. Apoptotic cells were detected by in-situ end labeling using the terminal deoxynucleotide transferase-mediated dUTP-nick end-labeling (TUNEL) method, and by morphological examination. We found that: 1) Treatment of cultured aortic SMC with the L-type Ca2+ channel antagonist, nifedipine (5 X 10(-5) M) for 24 hours induced a significantly higher level of apoptosis in SHR cells than in SMC from WKY. Cells from WKY, following exposure to nifedipine for 72 hours, exhibited a similar response to the cells from SHR treated for 24 hours. This was detectable by both morphological criteria as well as DNA labeling by the TUNEL technique. 2) Similar treatment of these cells with thapsigargin (1 x 10(-7) M) led to morphological alterations characteristic of apoptotic cells in SMC from both WKY and SHR, and cells from SHR but not WKY were labeled by the TUNEL technique at 24 hours. The TUNEL method did however identify cells from both WKY and SHR as apoptotic after 48 and 72 hours of treatment. 3) The addition of SNAP, or forskolin to the cultured SMC induced significant, but low levels of apoptosis in WKY SMC only. This selective apoptosis-promoting effect of nifedipine in SHR SMC may result from differences in the control of intracellular Ca2+ between the two strains of cells, or it may indicate that the signaling pathways which regulate apoptosis are different in SMC from the normotensive and the hypertensive rats. Our findings imply that SMC apoptosis may be a selective target for pharmacological intervention in hypertension.     

Urocortin II inhibits the apoptosis of mesenteric arterial smooth muscle cells via L-type calcium channels in spontaneously hypertensive rats.

Urocortin (UCN) II, a newly isolated corticotropinreleasing- factor (CRF) related peptide, has been found to have potent cardiovascular protective effects. To investigate the mechanisms of its vascular protective effects, we exposed mesenteric arterial smooth muscle cells (MASMC) from spontaneously hypertensive rats (SHR) to UCN II to observe the change in cell apoptosis using TUNEL assay and measured intracellular calcium concentration ([Ca2+]i) using confocal laser scanning microscope. In addition, effects of UCN II on L-type calcium currents (ICa,L) were also measured using whole-cell patch clamp. Our results showed that UCN II concentration-dependently, but time-independently inhibited cell apoptosis. Astressin 2B, a special CRF 2 receptor antagonist, had no influence on this inhibition. Hypoxia or Bay K8644, the L-type calcium channel activator, induced the apoptosis of MASMC from SHR. Pretreatment of the cells with UCN II diminished the effects of hypoxia or Bay K8644. UCN II was also observed to reduce [Ca2+]i increase induced by KCl or Bay K8644. UCN II concentration-dependently inhibited ICa,L, which was not affected by astressin 2B. It did not affect the activation of ICa,L, but markedly shifted the inactivation curve to the left. In conclusion, UCN II inhibits the apoptosis of MASMC from SHR via inhibiting L-type calcium channels.     

Inhibition by cinnarizine of the responses of smooth muscle from spontaneously hypertensive and normotensive rats

A comparison was made of the inhibition by cinnarizine, a calcium antagonist, of the contractile responses of aortic, carotid, and iliac arterial strips and vasa deferentia from 15- to 17-week-old spontaneously hypertensive rats (SHR) and their normotensive counterparts, Wistar Kyoto (WKY) rats. KCl-induced responses of the aorta from both strains of rats and carotid arteries from WKY only were more sensitive to inhibition than were responses to norepinephrine. No significant differences were observed in the inhibition of tissue responses from the two strains of rats with the exception of the K+-induced responses of carotid arterial strips from SHR which were significantly less sensitive to inhibition when compared with carotid strips from WKY.     

Alpha-lactorphin and beta-lactorphin improve arterial function in spontaneously hypertensive rats

alpha-lactorphin (Tyr-Gly-Leu-Phe) lowers blood pressure in conscious adult SHR. This tetrapeptide is originally released from milk protein alpha-lactalbumin by enzymatic hydrolysis. In order to evaluate the antihypertensive mechanisms of alpha-lactorphin, the effects of the tetrapeptide on vascular function were investigated in (30-35 weeks old) spontaneously hypertensive rats (SHR) with established hypertension and age-matched normotensive Wistar-Kyoto (WKY) rats in vitro. In addition, we studied the vascular effects of another structurally related tetrapeptide, beta-lactorphin (Tyr-Leu-Leu-Phe), which originates from milk protein beta-lactoglobulin. Endothelium-dependent relaxation to acetylcholine (ACh) was reduced in mesenteric arterial preparations of SHR as compared to those of WKY. In SHR, the ACh-induced relaxation was augmented by alpha-lactorphin or beta-lactorphin. The role of nitric oxide (NO) is suggested, since this improvement was abolished by the NO synthase (NOS) inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME). Simultaneous potassium channel inhibitor tetraethylammonium (TEA) elicited no additional effect on the ACh-induced relaxation. The cyclooxygenase inhibitor diclofenac did not attenuate the augmented ACh relaxation induced by alpha-lactorphin or beta-lactorphin, suggesting that endothelial vasodilatory prostanoids were not involved in the effect of the tetrapeptides. Endothelium-independent relaxation to the NO donor sodium nitroprusside (SNP) was augmented in mesenteric arterial preparations of SHR by simultaneous beta-lactorphin. The tetrapeptides did not alter vascular responses in mesenteric arteries from WKY. In conclusion, both alpha-lactorphin and beta-lactorphin improved vascular relaxation in adult SHR in vitro. The beneficial effect of alpha-lactorphin was directed towards endothelial function, whereas beta-lactorphin also enhanced endothelium-independent relaxation.     

自发性高血压大鼠和Wistar大鼠脑动脉平滑肌细胞膜电流的比较

目的:探讨自发性高血压大鼠(SHR)和Wistar大鼠脑动脉(BA)平滑肌细胞膜电流的异同。方法:应用全细胞膜片钳技术研究SHR和Wistar大鼠BA平滑肌细胞在电流密度、电流组成以及自发性瞬时外向K+电流(STOCs)特性的异同。结果:①当指令电压为0、+20、+40和+60mV时,SHR与Wistar大鼠BA平滑肌细胞间电流密度存在统计学差异(P<0.01)。②SHR与Wistar大鼠BA平滑肌细胞膜电流都对1 mmol/L电压依赖的K+通道(Kv)阻断剂4AP和1 mmol/L大电导Ca2+激活K+通道(BKCa)阻断剂TEA敏感。③SHR的STOCs发放频率和电流幅度都远大于Wistar大鼠。1 mmol/LTEA基本完全阻断STOCs通道电流,而4-AP对STOCs没有影响。结论:SHR和Wistar大鼠脑动脉平滑肌细胞的电流密度存在差异,两种平滑肌细胞外向电流都由BKCa和Kv通道组成。SHR大鼠平滑肌细胞更易诱发由BKCa通道介导的STOCs。     

Protein kinase C activity and reactivity to phorbol ester in vascular smooth muscle from spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto rats (WKY)

Protein kinase C (PKC) activity in aortic and renal arterial smooth muscle from SHR (20-23 wk male; mean arterial pressure = 178 mm Hg) and WKY (age/sex matched; mean arterial pressure = 126 mm Hg) was quantitated. Activity was greatest in the particulate fractions relative to the soluble fractions in all sources. The only difference between SHR and WKY was in the soluble fraction from SHR renal arteries, which had 2 fold more activity (255 pmol/mg/min) when compared with WKY (136 pmol/mg/min). This difference was not apparently related to force modulation, since the magnitude of isometric force development in renal arteries in response to phorbol 12,13-dibutyrate was not different between SHR and WKY. The magnitude of force developed in response to phorbol 12,13-dibutyrate and PKC activity in the particulate fraction was greatest in aorta vs. renal arteries in both WKY and SHR. These results suggest that regional vascular differences in the amount of PKC activity may exist which are not apparently related to a disease state (i.e., hypertension). These differences may be related to differential sensitivity to phorbol ester-mediated contractions in isolated smooth muscle.     

Proliferation kinetics of aortic smooth muscle cell populations: comparison of normotensive and spontaneously hypertensive rats

Abstract. An in vitro autoradiographic study of the proliferation of smooth muscle cells (SMC) from the aorta of normotensive and spontaneously hypertensive rats has been made. It was found, in primary culture, that SMC of spontaneously hypertensive rats entered proliferation at 2-2.5 days later than those from normotensive animals. As revealed by their very intensive labelling, a subpopulation of SMC with a high turnover rate was found in primary culture. In freshly isolated SMC from normotensive rat aorta, a subpopulation in S phase was detected, but we failed to detect it in aortae from spontaneously hypertensive rats. A difference in proliferative behaviour was also observed in subcultures of SMC from rats of both strains.     

Proliferation kinetics of aortic smooth muscle cell populations: comparison of normotensive and spontaneously hypertensive rats

An in vitro autoradiographic study of the proliferation of smooth muscle cells (SMC) from the aorta of normotensive and spontaneously hypertensive rats has been made. It was found, in primary culture, that SMC of spontaneously hypertensive rats entered proliferation at 2-2.5 days later than those from normotensive animals. As revealed by their very intensive labelling, a subpopulation of SMC with a high turnover rate was found in primary culture. In freshly isolated SMC from normotensive rat aorta, a subpopulation in S phase was detected, but we failed to detect it in aortae from spontaneously hypertensive rats. A difference in proliferative behaviour was also observed in subcultures of SMC from rats of both strains.     

Electron-microscopic studies on reaggregate cultures of vascular smooth muscle cells from normotensive and spontaneously hypertensive rats

Summary Vascular smooth muscle cells were taken from the aortae of the WKY (normotensive) and SHR (spontaneously hypertensive) strains of rat by enzymatic dispersion and put into reaggregate culture. Initially the cells became individual spheroids having average diameters of 10 m and surfaces that were either rough or smooth. The cells were far more complex than they appeared on their surfaces; after one day in culture, there was considerable internal variation in these cells. All the cells, whether WKY or SHR, lost the bulk of their cytoplasmic contents (including myofilaments, many mitochondria, and vesicular structures) in the early stages of culture and eventually became flattened. After 14 days in culture, these modified cells collected to form reaggregates that were commonly roughly spherical and several hundred m in diameter. These reaggregates consisted of peripheral regions made up of several layers of flattened cells overlying cores formed by glia-like networks of cells similar in cytological appearance to the cells at the periphery. The meshes formed in this way contained cellular debris derived from dead cells or extrusion of cellular contents. It appears that SHR cells are quicker to form reaggregates than are WKY cells. Yet the SHR cells retained a rounded conformation after five days, whereas the WKY cells were more flattened and formed a more discrete aggregate at this stage of culture. However, by the fourteenth day of culture, differences between the two cell strains were not so pronounced, as far as could be judged by observations made with scanning and transmission electron microscopy. Both WKY and SHR cells at 14 days appeared highly secretory, possessing large Golgi systems as well as numerous ER cisternae and mitochondria. SHR cells produced greater amounts of connective tissue at all stages of culture than did WKY cells, indicating that a similar difference may contribute to the hypertension which develops naturally in situ in SHR animals.     

Impaired UTP-induced relaxation in the carotid arteries of spontaneously hypertensive rats

Uridine 5′-triphosphate (UTP) has an important role as an extracellular signaling molecule that regulates inflammation, angiogenesis, and vascular tone. While chronic hypertension has been shown to promote alterations in arterial vascular tone regulation, carotid artery responses to UTP under hypertensive conditions have remained unclear. The present study investigated carotid artery responses to UTP in spontaneously hypertensive rats (SHR) and control Wistar Kyoto rats (WKY). Accordingly, our results found that although UTP promotes concentration-dependent relaxation in isolated carotid artery segments from both SHR and WKY after pretreatment with phenylephrine, SHR exhibited significantly lower arterial relaxation responses compared with WKY. Moreover, UTP-induced relaxation was substantially reduced by endothelial denudation and by the nitric oxide (NO) synthase inhibitor N^G-nitro-L-arginine in both SHR and WKY. The difference in UTP-induced relaxation between both groups was abolished by the selective P2Y₂ receptor antagonist AR-C118925XX and the cyclooxygenase (COX) inhibitor indomethacin but not by the thromboxane-prostanoid receptor antagonist SQ29548. Furthermore, we detected the release of PGE₂, PGF_2α, and PGI₂ in the carotid arteries of SHR and WKY, both at baseline and in response to UTP. UTP administration also increased TXA₂ levels in WKY but not SHR. Overall, our results suggest that UTP-induced relaxation in carotid arteries is impaired in SHR perhaps due to impaired P2Y₂ receptor signaling, reductions in endothelial NO, and increases in the levels of COX-derived vasoconstrictor prostanoids.