Expression of smooth muscle cell-specific proteins in neural progenitor cells induced by agonists of G protein-coupled receptors and transforming growth factor-beta.

Neural progenitor cells isolated from the embryonic cerebral cortex are well known to differentiate into neurons and glial cells, but recent reports have demonstrated differentiation into smooth muscle cells (SMCs) under the influence of fetal bovine serum. In this study, we report that agonists for G protein-coupled receptors (GPCRs), including endothelin, lysophosphatidic acid and carbachol, effectively promote the expression of SMC-specific proteins in the presence of transforming growth factor-beta (TGF-beta). Incubation of neural progenitor cells with agonists for GPCRs or TGF-beta alone induced the expression of an SMC-specific protein, alpha-smooth muscle actin (SMA), and their combination resulted in incremental increase. Stimulation with combinations of each GPCR agonist and TGF-beta increased the numbers of large, flat cells with thick actin fibers and also caused expression of other SMC marker proteins. Endothelin and TGF-beta enhanced SMA promoter-luciferase reporter activity at different times after stimulation. The mutation of TGF-beta control element of SMA promoter constructs decreased TGF-beta-enhanced luciferase activity but not endothelin-stimulated activity. Transfection of active forms of RhoA and its effector, mDia, strongly enhanced SMA promoter activity, and a dominant negative form of RhoA inhibited endothelin-stimulated promoter activity but not TGF-beta-stimulated activity. Whereas endothelin consistently activated RhoA, TGF-beta did not, and a specific inhibitor of TGF-beta type I receptor blocked TGF-beta-enhanced SMA promoter activity, suggesting involvement of Smad phosphorylation. These results suggest that separate signaling pathways of G protein and TGF-beta cooperatively promote the expression of SMC-specific proteins in neural progenitor cells.

Comparison of inhibitory effects of Y-27632, a Rho kinase inhibitor, in strips of small and large mesenteric arteries from spontaneously hypertensive and normotensive Wistar-Kyoto rats.

Since Y-27632, a specific inhibitor of Rho kinase, decreases the blood pressure in spontaneously hypertensive rats (SHR), it is suggested that Rho kinase is involved in the pathophysiology of hypertension. However, the effects of Y-27632 on isolated resistance arteries have never been determined. This study aimed to examine the possible role of the Rho/Rho kinase pathway during arterial contraction in isolated resistance arteries from SHR. The profile of arterial relaxant effects of Y-27632 was compared in endothelium-denuded strips of small and large mesenteric arteries from 13-week-old SHR and normotensive Wistar-Kyoto rats (WKY). The addition of 10(-6) mol/l norepinephrine (NE) to the strips of small arteries caused an initial peak followed by a tonic contraction in both strains. There was no difference between the two strains in either the initial peak or the tonic contraction. The addition of Y-27632 (0.3-3 micromol/l) to the tonic contraction of these strips caused a concentration-dependent relaxation in both strains. The relaxation was greater in SHR than in WKY. Similar results were observed in strips of large arteries. The relaxant effects of Y-27632 were greater in the large artery than in the small artery. Y-27632 also induced a concentration-dependent relaxation in strips precontracted with 65.9 mmol/l K+ depolarization. In both arteries, this relaxation was greater in SHR. The relaxant effects of Y-27632 were greater in the K+-contracted strips than in the NE-contracted strips. We conclude that Y-27632 shows the greater relaxant effects on the SHR arteries, and the effects are more evident in the large artery and in the K+-contracted strips.

Ca2+ buffering function of the sarcoplasmic reticulum is increased in the carotid artery from spontaneously hypertensive rats.

To clarify whether the Ca2+ uptake function of the sarcoplasmic reticulum (SR) during arterial contraction is altered in hypertension, the effects of cyclopiazonic acid (CPA) and thapsigargin, which inhibit SR Ca2+-ATPase, on the contractile responses to Bay k 8644, an agonist of L-type Ca2+ channels, were compared in endothelium-denuded strips of carotid arteries from 13-week-old spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). The addition of Bay k 8644 (1-300 nM) to the strips caused a concentration-dependent contraction that was larger in SHR than in WKY. The contractile responses to Bay k 8644 were augmented by CPA (10 microM) or thapsigargin (100 nM) in both strains. This augmentation was greater in SHR. Each of CPA and thapsigargin induced a relatively transient contraction, and both of these contractions were larger in SHR than in WKY. The basal 45Ca influx in this artery was larger in SHR than in WKY. The addition of caffeine (1-20 mM) caused a transient contraction that was larger in SHR than in WKY. Our results indicate that 1) the large Ca2+ influx during rest in the SHR carotid artery is strongly buffered by Ca2+ uptake into the superficial SR; and 2) the Ca2+ uptake function of the SR during the contraction with Bay k 8644 was increased in SHR compared with WKY. We conclude that the SHR carotid artery has an increased total capacity of SR for Ca2+ storage as an attempt to compensate for the large Ca2+ influx.

Increased Ca2+ buffering function of sarcoplasmic reticulum in small mesenteric arteries from spontaneously hypertensive rats.

We compared the Ca2+ buffering function of the superficial sarcoplasmic reticulum (SR) during rest and during contraction in endothelium-denuded strips of small mesenteric arteries from 13-week-old spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). The addition of caffeine (1-20 mM) caused a transient contraction in both strains, and the contraction was significantly larger in SHR. When the SR Ca2+ buffering function was eliminated by cyclopiazonic acid (CPA; 10 microM) or thapsigargin (100 nM), both of which inhibit SR Ca2+-ATPase, or by ryanodine (10 microM), which depletes the SR Ca2+, there was a larger contraction in SHR than in WKY, suggesting that the Ca2+ buffering function of the SR during rest is more important in SHR than in WKY. Judging from the augmenting effects of these three agents on the contractile responses to Bay k 8644 (1-300 nM), an agonist of L-type Ca2+ channels, or norepinephrine (10(-9)-10(-4) M), an alpha-adrenoceptor agonist, the effects were significantly greater in SHR than in WKY. We conclude that 1) the Ca2+ influx during rest and during stimulation with Bay k 8644 or norepinephrine is strongly buffered by Ca2+ uptake into the superficial SR in the small mesenteric arteries from SHR and WKY; and 2) these Ca2+ buffering functions are increased in SHR because of the larger capacity of SR for Ca2+ storage.

Contribution of sarcoplasmic reticulum Ca2+ to the activation of Ca2+ -activated K+ channels in the resting state of arteries from spontaneously hypertensive rats.

OBJECTIVE: Localized release of Ca2+ from the sarcoplasmic reticulum (SR) toward the plasmalemma, sometimes visualized as Ca2+ sparks, can activate Ca2+-activated K+ (KCa) channels. We have already reported that the addition of charybdotoxin (ChTX), a blocker of KCa channels, to the resting state of arteries from spontaneously hypertensive rats (SHR) caused a powerful contraction, suggesting that KCa channels were active in the resting state. This study aimed to determine whether the Ca2+ responsible for activity of KCa channels was derived from SR. METHODS: Possible mechanisms underlying the ChTX-induced contractions were examined in endothelium-denuded strips of femoral, mesenteric, small mesenteric and carotid arteries from 13-week-old SHR and normotensive Wistar-Kyoto (WKY) rats by using selective inhibitors of the Ca2+ spark process. RESULTS: ChTX (100 nmol/l) induced a contraction in the SHR arteries. The ChTX-induced contractions were increased by a moderate membrane depolarization by 15.9 mmol/l K+ and were abolished by nifedipine (100 nmol/l). When SR Ca2+ was depleted by treatment of the strips with ryanodine (10 mumol/l) plus caffeine (20 mmol/l) or with thapsigargin (100 nmol/l), the ChTX-induced contraction was decreased in femoral, mesenteric and small mesenteric arteries and was almost abolished in the carotid artery. A similar phenomenon can be observed in arteries from WKY rats after a moderate membrane depolarization. In both SHR and WKY rats, SR Ca2+-dependent ChTX-induced contraction always represents 20-30% of the maximal K+-induced contraction. CONCLUSIONS: We conclude that activation of KCa channels depended upon influx of Ca2+ through L-type Ca2+ channels and release of Ca2+ from the SR, suggesting that recycling of entering Ca2+ from the superficial SR toward the plasmalemma sufficiently elevated Ca2+ near these channels to activate them.