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1.
Bladder and vascular smooth muscle cells cultured from four rat strains (WKY, SHR, WKHA, WKHT) differing in rates of nerve growth factor (NGF) production were used to determine whether a relationship exists between intracellular calcium and NGF secretion. Basal cytosolic calcium was related to basal NGF secretion rates in bladder and vascular smooth muscle cells from all four strains with the exception of WKHT bladder muscle cells. Thrombin is a calcium-mobilizing agent and increases NGF production from vascular but not bladder smooth muscle cells. Strain differences were found in the magnitude of the calcium peak induced by thrombin in vascular smooth muscle cells, but these differences did not correlate with NGF secretion. Thrombin caused a calcium response in bladder smooth muscle cells without influencing NGF production. Quenching the calcium transient with a calcium chelator had no effect on thrombin-inducted NGF secretion rates in vascular smooth muscle cells. Thus, basal intracellular calcium may establish a set point for NGF secretion from smooth muscle. In addition, transient elevations in cytosolic calcium were unrelated to the induction of NGF output. 相似文献
2.
A N Gurkovskaia N I Gokina M F Shuba L I Kazak I S Chekman 《Fiziologicheski? zhurnal》1989,35(6):50-53
The effects of dibasol on spontaneous electrical and contractile activities as well as on the reactions evoked by hyperkalemic solution and noradrenaline were studied in smooth muscle of rabbit portal vein. It was shown that dibasol blocked the potential-operated influx Ca2+ into smooth muscle cells. The noninactivating calcium channels were found to be more sensitive to dibasol than inactivating ones. Significant part of the tonic contraction induced by noradrenaline was resistant to dibasol suggesting its weak effect on Ca2+ influx through calcium channels operated by alpha 1-adrenoceptors. It is supposed that vasodilative effect of dibasol is associated with blocking the influx Ca2+ through potential-operated noninactivating calcium channels into smooth muscle cells. 相似文献
3.
Detrusor smooth muscle cells of the guinea-pig are functionally coupled via gap junctions in situ and in cell culture 总被引:8,自引:0,他引:8
Neuhaus J Wolburg H Hermsdorf T Stolzenburg JU Dorschner W 《Cell and tissue research》2002,309(2):301-311
Intercellular communication between smooth muscle cells is crucial for contractile behaviour in normal and pathologically altered urinary bladder. Since the study of coupling is difficult in situ, we established cell cultures of bladder smooth muscle cells to analyse coupling mechanisms. Microinjection of Lucifer yellow demonstrated syncytia composed of only a few to several dozen cells. Electron-microscopic examination of freeze-fracture specimens and ultrathin sections revealed that the dye-coupling was based on typical gap junction formation between the cultured smooth muscle cells. Furthermore, we were able to demonstrate gap junctions within the tissue fragments from which the primary cultures were grown. By Western blotting, we found connexin-43-positive protein bands both in native tissue probes from the guinea-pig urinary bladder and in smooth muscle cell cultures. Extracellular electrical stimulation of single cells evoked calcium transients, as visualized by fura-2 ratiofluorimetry. Calcium waves propagated throughout the syncytia with a declining amplitude, showing that the calcium signal was not regenerative. Therefore, the calcium signal was probably transmitted by a diffusible factor. These findings correlated well with the dye-coupling that we found between detrusor smooth muscle cells in situ. The use of smooth muscle cell cultures therefore seems to be a feasible approach for studying coupling behaviour in vitro. 相似文献
4.
Calcium ionophores, such as the A23187, cause endothelium-dependent relaxation of arterial strips with intact endothelium, whereas the effect of the ionophore should result from the combination of a relaxation caused by the endothelium-dependent factors and of a contraction of the smooth muscles. In addition, the application of a calcium ionophore to a strip of pig coronary arteries without endothelium does not change cytosolic free calcium concentration and force developed by the smooth muscle cells. To explain these paradoxes, the hypothesis that active calcium extrusion would match the entry of extracellular calcium caused by the ionophore was tested. We see that the sodium-calcium exchanger extrudes calcium that enters the smooth muscle cells in the absence of the ionophore. This exchanger is efficient enough to expel the increased influx of calcium created by the additional calcium carriers formed by the ionophore. This explains the inefficiency of calcium ionophores to increase cytosolic free calcium of smooth muscle cells and consequently, the fact that the ionophore does not cause a contraction of a strip without endothelium. This makes evident that a calcium ionophore fully relaxes, in an endothelium-dependent manner. an intact strip of porcine coronary artery. 相似文献
5.
Stable and functional expression of the calcium channel alpha 1 subunit from smooth muscle in somatic cell lines.
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E Bosse R Bottlender T Kleppisch J Hescheler A Welling F Hofmann V Flockerzi 《The EMBO journal》1992,11(6):2033-2038
Voltage-activated calcium channels are membrane spanning proteins that allow the controlled entry of Ca2+ into the cytoplasm of cells. The principal channel forming subunit of an L-type calcium channel is the alpha 1 subunit. Transfection of Chinese hamster ovary (CHO) cells with complementary DNA encoding the calcium channel alpha 1 subunit from smooth muscle led to the expression of functional calcium channels which bind calcium channel blockers and show the voltage-dependent activation and slow inactivation and unitary current conductance characteristic of calcium channels in smooth muscle. The currents mediated by these channels are sensitive towards dihydropyridine-type blockers and agonists indicating that the calcium channel blocker receptor sites were present in functional form. The smooth muscle alpha 1 subunit cDNA alone is sufficient for stable expression of functional calcium channels with the expected kinetic and pharmacological properties in mammalian somatic cells. 相似文献
6.
The conditions and efficacy of transfection of vascular cells in primary culture using DEAE-dextran, calcium phosphate and lipofectin have been investigated using chloramphenicol acetyltransferase and luciferase as reporter genes. Subsequently factor VIII was expressed in endothelial and smooth muscle cells. Both reporter genes could be expressed after transfection of umbilical vein endothelial cells, umbilical artery smooth muscle cells and fibroblasts. The expression of both reporter genes in endothelial and smooth muscle cells was highest using lipofectin. After transfection of smooth muscle cells with both full-length and mutant factor VIII genes, factor VIII activity and antigen were secreted into the culture medium, the secretion remaining stable to serial cell passage. The secretion of factor VIII from transfected smooth muscle cells was confirmed by the immunoprecipitation of [35S]methionine labelled protein. Endothelial cells also were successfully transfected with the mutant factor VIII gene. 相似文献
7.
Stretch-elicited intracellular calcium ([Ca(2+)](i)) changes in individual smooth muscle cells in a ring of aorta were measured simultaneously with the force developed by the ring. A phasic increase in [Ca(2+)](i) was observed in 30% of the cells and a sustained one in 10%. Depletion of intracellular calcium store by thapsigargin and caffeine decreased phasic and increased sustained calcium responses. The inhibition of calcium entry either by stretching the aorta in a calcium-free medium or by the inhibition of stretch-activated, non-selective cationic channels by 5 microM GsMtx-4 toxin, decreased the proportion of sustained [Ca(2+)](i) responses but increased transient responses. In this condition, a third of the cells responded to stretch by a bursts of [Ca(2+)](i) spikes. The decrease of calcium influx triggered the generation of burst of calcium spikes after the application of stretch steps to the vascular wall. We conclude that progressive recruitment of smooth muscle cells is the mechanism underlying the force-generating part of the myogenic response. Two types of stretch-elicited calcium responses were observed during the recruitment of the smooth muscle cells. One was a phasic calcium discharge generated by the sarcoplasmic reticulum. The second was a tonic response produced by the activation of the stretch-sensitive cationic channels allowing extracellular Ca(2+) entry. 相似文献
8.
Ca2+ dynamics in a population of smooth muscle cells: modeling the recruitment and synchronization
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Many experimental studies have shown that arterial smooth muscle cells respond with cytosolic calcium rises to vasoconstrictor stimulation. A low vasoconstrictor concentration gives rise to asynchronous spikes in the calcium concentration in a few cells (asynchronous flashing). With a greater vasoconstrictor concentration, the number of smooth muscle cells responding in this way increases (recruitment) and calcium oscillations may appear. These oscillations may eventually synchronize and generate arterial contraction and vasomotion. We show that these phenomena of recruitment and synchronization naturally emerge from a model of a population of smooth muscle cells coupled through their gap junctions. The effects of electrical, calcium, and inositol 1,4,5-trisphosphate coupling are studied. A weak calcium coupling is crucial to obtain a synchronization of calcium oscillations and the minimal required calcium permeability is deduced. Moreover, we note that an electrical coupling can generate oscillations, but also has a desynchronizing effect. Inositol 1,4,5-trisphosphate diffusion does not play an important role to achieve synchronization. Our model is validated by published in vitro experiments obtained on rat mesenteric arterial segments. 相似文献
9.
Jasbir D. Upadhyaya Nisha Singh Anurag S. Sikarwar Raja Chakraborty Sai P. Pydi Rajinder P. Bhullar Shyamala Dakshinamurti Prashen Chelikani 《PloS one》2014,9(10)
Activation of bitter taste receptors (T2Rs) in human airway smooth muscle cells leads to muscle relaxation and bronchodilation. This finding led to our hypothesis that T2Rs are expressed in human pulmonary artery smooth muscle cells and might be involved in regulating the vascular tone. RT-PCR was performed to reveal the expression of T2Rs in human pulmonary artery smooth muscle cells. Of the 25 T2Rs, 21 were expressed in these cells. Functional characterization was done by calcium imaging after stimulating the cells with different bitter agonists. Increased calcium responses were observed with most of the agonists, the largest increase seen for dextromethorphan. Previously in site-directed mutational studies, we have characterized the response of T2R1 to dextromethorphan, therefore, T2R1 was selected for further analysis in this study. Knockdown with T2R1 specific shRNA decreased mRNA levels, protein levels and dextromethorphan-induced calcium responses in pulmonary artery smooth muscle cells by up to 50%. To analyze if T2Rs are involved in regulating the pulmonary vascular tone, ex vivo studies using pulmonary arterial and airway rings were pursued. Myographic studies using porcine pulmonary arterial and airway rings showed that stimulation with dextromethorphan led to contraction of the pulmonary arterial and relaxation of the airway rings. This study shows that dextromethorphan, acting through T2R1, causes vasoconstrictor responses in the pulmonary circuit and relaxation in the airways. 相似文献
10.
Calcium channels of amphibian stomach and mammalian aorta smooth muscle cells. 总被引:8,自引:0,他引:8
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Whole-cell and single-channel calcium currents were studied using single smooth muscle cells enzymatically-isolated from stomach of Amphiuma tridactylum and from guinea-pig aorta. These cells have a high specific resistance and can sustain calcium action potentials after suppression of potassium currents. Dialyzed Amphiuma smooth muscle cells had calcium currents which were stable for several hours whereas the calcium currents of aortic cells ran down quickly. Single channel calcium currents in cell-attached patches behaved similarly for the two cell types. Calcium channel conductance in 110 mM barium was 12 pS and the mean open time was 1.4 ms at a nominal membrane potential of +10 mV. Exposure of both cell types to BAY K8644 resulted in a dramatic prolongation of the calcium channel open times and a shift in the probability of opening to more negative potentials. Low-threshold calcium channels were not identified in the extensively studied amphibian cells. High-threshold calcium channels therefore appear to be the primary pathway for the calcium influx that produces contraction in these smooth muscle cells. 相似文献
11.
Summary The intracellular localization of calcium by means of cytochemical techniques was studied in smooth muscle cells of mouse intestine. When the lead acetate method according to Carasso and Favard (1966) was used calcium was found in mitochondria and sarcoplasmic reticulum and occasionally between the myofilaments. The active ATP-dependent accumulation of calcium into cell structures was investigated by the oxalate method (Heumann and Zebe, 1967). After appropriate treatment the only structures of smooth muscle cells which contained calcium oxalate (identified by microprobe analysis) were elements of the sarcoplasmic reticulum.The results are discussed in relation to the role of calcium in the control of muscle activity during the contraction-relaxation cycle.The electron probe microanalysis was carried out at SIEMENS (Berlin) in collaboration with Dr. von Muschwitz. I thank Miss M. Schlatter for her skillful assistance. The investigation was supported by the Deutsche Forschungsgemeinschaft. 相似文献
12.
13.
We investigated whether cyclic stretch affects TRPC4 or TRPC6 expression and calcium mobilization in cultured vascular smooth muscle cells. In aortic and mesenteric smooth muscle cells isolated from male Sprague-Dawley rats, TRPC4 expression was decreased after 5 h stretch and remained suppressed through 24 h stretch. After removal of the stretch stimulus, TRPC4 expression recovered within 2 h. Stretch did not affect TRPC6 expression. Stretch also decreased capacitative calcium entry, while agonist-induced calcium influx was increased. Similar results were obtained in primary aortic smooth muscle cells. TRPC4 mRNA levels were not decreased in response to mechanical strain. TRPC4 downregulation was also achieved by increasing extracellular calcium and was attenuated by gadolinium and MG132, suggesting that TRPC4 protein is regulated by intracellular calcium concentration and/or the ubiquitin-proteasome pathway. These data suggest that stretch-induced downregulation of TRPC4 protein expression and capacitative calcium entry may be a protective mechanism to offset stretch-induced increases in intracellular calcium. 相似文献
14.
OBJECTIVE: The purpose of this study was to evaluate the contribution of capacitative calcium influx to intracellular calcium levels during agonist-induced stimulation of vascular smooth muscle cells. METHODS: Aortic vascular smooth muscle cells (A7r5) were loaded with Indo-1 and intracellular calcium transients were measured. Cells were challenged with either arginine vasopressin (0. 5 microM) or thapsigargin (1 microM). Lanthanum (1 mM) was used to block capacitative calcium influx through store-operated channels. Calcium traces were analyzed for basal, peak and plateau responses. Recordings were derivatized and integrated to gain additional information. Nonlinear regression provided a time constant that describes restoration of ionic equilibrium involving both sequestration and extrusion pathways. RESULTS: Stimulation of cells with thapsigargin produced a non-L-type calcium influx that was attenuated by lanthanum. Cells excited with vasopressin exhibited a rapid calcium increase followed by a gradual decrease to a plateau level. Lanthanum pretreatment prior to stimulation caused no significant change in baseline, peak or plateau calcium levels as compared to control. Lanthanum caused no significant change in maximal calcium release rate, calcium integrals or time constant as compared to control. CONCLUSIONS: Capacitative calcium entry can occur in vascular smooth muscle cells, but does not appear to contribute significantly to the vasopressin response. 相似文献
15.
Fritsch RM Saur D Kurjak M Oesterle D Schlossmann J Geiselhöringer A Hofmann F Allescher HD 《The Journal of biological chemistry》2004,279(13):12551-12559
16.
Budel S Schuster A Stergiopoulos N Meister JJ Bény JL 《American journal of physiology. Heart and circulatory physiology》2001,281(3):H1156-H1162
We tested the hypothesis that the cytosolic free calcium concentration in endothelial cells is under the influence of the smooth muscle cells in the coronary circulation. In the left descending branch of porcine coronary arteries, cytosolic free calcium concentration ([Ca(2+)](i)) was estimated by determining the fluorescence ratio of two calcium probes, fluo 4 and fura red, in smooth muscle and endothelial cells using confocal microscopy. Acetylcholine and potassium, which act directly on smooth muscle cells to increase [Ca(2+)](i), were found to indirectly elevate [Ca(2+)](i) in endothelial cells; in primary cultures of endothelial cells, neither stimulus affected [Ca(2+)](i), yet substance P increased the fluorescence ratio twofold. In response to acetylcholine and potassium, isometric tension developed by arterial strips with intact endothelium was attenuated by up to 22% (P < 0.05) compared with strips without endothelium. These findings suggest that stimuli that increase smooth muscle [Ca(2+)](i) can indirectly influence endothelial cell function in porcine coronary arteries. Such a pathway for negative feedback can moderate vasoconstriction and diminish the potential for vasospasm in the coronary circulation. 相似文献
17.
Sarcoplasmic reticulum (SR) is abundant in uterine smooth muscle cells. The functional role of this organelle in the regulation of uterine myocytes is not fully understood. The data available in the literature suggest that SR plays a dual role: as a source of calcium and as a calcium sink shaping calcium transients produced by membrane depolarisation and uterotonic agonists. Advances in digital imaging techniques including confocal microscopy of isolated living cells, and the development of methods for direct measurement of intraluminal calcium, has triggered a substantial increase in the number of publications elucidating the role of intracellular stores in calcium signalling. In this paper we review the literature and our own work on the SR calcium store in uterine smooth muscle cells. 相似文献
18.
J Thyberg L Palmberg 《Biology of the cell / under the auspices of the European Cell Biology Organization》1987,60(2):125-132
Cultured arterial smooth muscle cells go through a transition from a contractile to a synthetic phenotype. Morphologically, the transition includes a reduction in size of the myofilament bundles and the formation of an extensive rough endoplasmic reticulum and a large Golgi complex. Functionally, it leads to loss of contractility, onset of cellular proliferation, and secretion of extracellular matrix components. This change in differentiated characteristics under in vitro conditions has attracted attention because of its resemblance to the modification of the smooth muscle cells that occurs in vivo during atherogenesis. Here, transmission electron microscopy and [3H]-thymidine autoradiography were used to study the role of calcium ions in the control of phenotypic properties and growth of cultivated rat aortic smooth muscle cells. The calcium antagonist nisoldipine was found to lack distinct effect on the structural reorganization of the cells, but showed a moderate prohibitory effect on the start of DNA synthesis early in primary culture. In growth-arrested secondary cultures, nisoldipine inhibited induction of DNA synthesis by serum or platelet-derived growth factor (PDGF). The agent's effect was inversely related to the concentration of calcium ions in the extracellular medium and was partially counteracted by the calcium agonist BAY K 8644. In contrast, W-7, an antagonist of the calcium-binding protein calmodulin, potentiated the effect of nisoldipine and, at higher concentrations, inhibited induction of DNA synthesis in itself. The results suggest that the mitogenic stimulation of arterial smooth muscle cells involves a flux of calcium ions through the plasma membrane and requires participation of calmodulin. 相似文献
19.
Comparative study of the effect of calmodulin inhibitors trifluoperazine, W-12, and W-13 and the TRPV1 channel blocker capsazepine on receptor-dependent calcium metabolism in smooth muscle cells of the rat aorta and on the contraction of the isolated rat aorta was performed. Trifluoperazine almost completely abolishes an increase in free cytoplasmic calcium concentration in smooth muscle cells isolated from the rat aorta and smooth muscle cells of the A7r5 line in response to serotonin and does not affect cellular reaction to vasopressin and angiotensin II. W-12 and W-13 also do not attenuate responses to vasopressin and angiotensin II and reduces by two times free cytoplasmic calcium concentration elevation in response to serotonin. The efficiency of calcium metabolism suppression by calmodulin inhibitors correlates with the degree of inhibition of the aorta contractile response to serotonin. It was demonstrated that the inhibitory action of calmodulin antagonists on calcium metabolism in smooth muscle cells and the contractility of the isolated rat aorta during the activation of serotonin vasoconstrictive receptors are realized by a TRPV1-independent mechanism. It was demonstrated in experiments in vivo that trifluoperazine does not influence hypotensive reaction in rats (normally observed in response to intravenous serotonin injection), but removes the hypertensive effect of this neurotransmitter in rats after chronic introduction of dexamethasone. The results obtained confirm the hypothesis (that we previously stated) about the direct involvement of calmodulin in signal transmission from vasoconstrictive serotonin receptors. 相似文献
20.
Summary Smooth muscle cells from rat vas deferens were studied by electron microscopy. Vesicular and tubular membranous structures containing an electron-opaque material were found in the smooth muscle cells. Similar structures were also found in a subfraction (F3) of microsomes of vas deferens smooth muscle which was shown to be rich in both plasma membrane and putative endoplasmic reticulum markers. Treatment of the tissues with calcium-free Krebs solution containing EGTA prior to fixation eliminated almost completely the presence of these dense-cored membranous structures (DMS), whereas incubation of the subcellular membrane fraction with EGTA solution had no effect on the appearance of the DMS. Plasma membrane infoldings were found in the smooth muscle cells extending well into their interior. Horseradish peroxidase penetrates vesicles in a location similar to that of DMS in smooth muscle cells, suggesting that some of the DMS may be connected to the extracellular space. We conclude that the dense-core material within the DMS is calcium dependent. We also suggest that some of the DMS represent infoldings of the plasma membrane extending into the cell's interior. 相似文献