Entry In Smooth Muscle Cells Research Articles

Xyloketal B exerts antihypertensive effect in renovascular hypertensive rats via the NO-sGC-cGMP pathway and calcium signaling.

Xyloketal B (Xyl-B) is a novel marine compound isolated from mangrove fungus Xylaria sp. (No 2508). We previously showed that Xyl-B promoted endothelial NO release and protected against atherosclerosis through the Akt/eNOS pathway. Vascular NO production regulates vasoconstriction in central and peripheral arteries and plays an important role in blood pressure control. In this study, we examined whether Xyl-B exerted an antihypertensive effect in a hypertensive rat model, and further explored the possible mechanisms underlying its antihypertensive action. Administration of Xyl-B (20 mg·kg-1·d-1, ip, for 12 weeks) significantly decreased the systolic and diastolic blood pressure in a two-kidney, two-clip (2K2C) renovascular hypertensive rats. In endothelium-intact and endothelium-denuded thoracic aortic rings, pretreatment with Xyl-B (20 μmol/L) significantly suppressed phenylephrine (Phe)-induced contractions, suggesting that its vasorelaxant effect was attributed to both endothelial-dependent and endothelial-independent mechanisms. We used SNP, methylene blue (MB, guanylate cyclase inhibitor) and indomethacin (IMC, cyclooxygenase inhibitor) to examine which endothelial pathway was involved, and found that MB, but not IMC, reversed the inhibitory effects of Xyl-B on Phe-induced vasocontraction. Moreover, Xyl-B increased the endothelial NO bioactivity and smooth muscle cGMP level, revealing that the NO-sGC-cGMP pathway, rather than PGI2, mediated the anti-hypertensive effect of Xyl-B. We further showed that Xyl-B significantly attenuated KCl-induced Ca2+ entry in smooth muscle cells in vitro, which was supposed to be mediated by voltage-dependent Ca2+ channels (VDCCs), and reduced ryanodine-induced aortic contractions, which may be associated with store-operated Ca2+ entry (SOCE). Taken together, these findings demonstrate that Xyl-B exerts significant antihypertensive effects not only through the endothelial NO-sGC-cGMP pathway but also through smooth muscle calcium signaling, including VDCCs and SOCE.

Smooth Muscle Cell Ca2+: Think Locally, Act Globally

Dynamic changes in intracellular Ca²⁺ levels in vascular smooth muscle cells are critically important for cardiovascular regulation. This Special Topic Issue highlights a series of expert opinion articles focused on this important subject. After a brief overview, novel discoveries surrounding smooth muscle cell Ca²⁺ influx via L-type and T-type channels are reviewed. Current work revealing the functional importance of dynamic Ca²⁺ signaling in the control of the parenchymal microvasculature and the emerging role of mitochondrial Ca²⁺ signaling and store-operated Ca²⁺ entry in smooth muscle cells is discussed. Finally, recent data describing a new target of localized Ca²⁺ signaling in arterial myocytes that is responsible for membrane depolarization is reviewed. Authors were encouraged to write in an opinionated and provocative manner with the hope of stimulating discussion in this area of research.

Regulation of noncapacitative calcium entry by arachidonic acid and nitric oxide in endothelial cells

Several peptides, including vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), activate the release of arachidonic acid (AA) and nitric oxide (NO) in endothelial cells (ECs). Both messengers are involved in EC proliferation and vascular permeability and control calcium homeostasis in different ways. Interestingly, it has been recently suggested that NO acts as a downstream mediator of AA-induced calcium entry in smooth muscle cells and isolated mouse parotid cells. In this paper, we have investigated the complex relationships that link intracellular calcium, AA, and NO in cultured endothelial cells. Using different experimental approaches, mainly simultaneous Ca2+ and NO fluorimetric confocal imaging, we provide evidence for a complex pathway leading to noncapacitative calcium entry (NCCE) in bovine aortic endothelial cells (BAECs). In particular, AA is able to induce NCCE through two different pathways: one dependent on eNOS recruitment and NO release, the other NO-independent. Finally, we show that NO increase is involved in the control of BAEC proliferation.

Blockade of calcium entry in smooth muscle cells by the antidepressant imipramine

The present study was designed to evaluate the effects of antidepressants on smooth muscle contractile activity. In rat aortic rings, the antidepressants imipramine, mianserin and sertraline provoked concentration-dependent inhibitions of the mechanical responses evoked by K + (30 mM) depolarization. These myorelaxant effects were not modified by the presence of glibenclamide or 80 mM K + in the bathing medium. Moreover, the vasodilator properties of imipramine were not affected by atropine, phentolamine and pyrilamine. Radioisotopic experiments indicated that imipramine failed to enhance 86 Rb outflow from prelabelled and perifused aortic rings whilst counteracting the increase in 45 Ca outflow provoked by a rise in the extracellular K + concentration. Simultaneous measurements of contractile activity and fura-2 fluorescence revealed that, in aortic rings, imipramine reduced the mechanical and fluorimetric response to K + challenge. In A7r5 smooth muscle cells, whole cell recordings further demonstrated that imipramine inhibited the inward Ca 2+ current. Under different experimental conditions, the ionic and relaxation responses to the antidepressants were reminiscent of those mediated by the Ca 2+ entry blocker verapamil. Lastly, it should be pointed out that imipramine exhibited a myorelaxant effect of similar amplitude on rat aorta and on rat distal colon. All together, these findings suggest that the myorelaxant properties of imipramine, and probably also setraline and mianserin, could result from their capacity to inhibit the voltage-sensitive Ca 2+ channels.

Nitrate tolerance: a unifying hypothesis.

) production, endo-thelial dysfunction, and the neurohormonal activation that followlong-term administration of organic nitrates. In this issue ofCirculation, we will try to integrate these observations withother separate, and, to a certain extent, antagonistic hypothesesthat have been proposed for the development of nitrate toler-ance.

Fibronectin Promotes Cell Cycle Entry in Smooth Muscle Cells in Primary Culture

Smooth muscle cell proliferation after arterial injury is regulated by growth factors and components of the extracellular matrix. We have previously demonstrated that fibronectin promotes a phenotypic modulation of freshly isolated rat smooth muscle cells from a contractile to a synthetic phenotype in primary culture and supports the ability of the cells to respond to growth factors. Here, we analyzed if fibronectin promotes cell cycle entry in freshly isolated rat aortic smooth muscle cells during primary culture. Cell cycle analysis showed that cells seeded on fibronectin remained in the G0/G1 phase of the cell cycle during the first 6 days of culture. During this period, there was an increased expression of cyclin D1 and p27KIP1 in the absence of exogenous growth factors. Addition of serum was followed by enhanced cyclin D1 expression, decreased p27KIP1 levels, hyperphosphorylation of Rb protein, induction of cyclin A and cyclin D3 expression, and cell cycle progression into S phase. The results indicate that fibronectin initiates cell cycle entry in freshly isolated smooth muscle cells by promoting the induction of cyclin D1 and thereby facilitates further cell cycle progression together with growth factors.

Expression of molecular mediators of apoptosis and their role in the pathogenesis of lower-extremity varicose veins

Purpose: In an earlier study, we observed a significant decrease in apoptosis in varicose veins, as compared with healthy veins, indicating that deregulated apoptosis plays a role in the pathogenesis of varicosities. In addition, significant differences were noted in the expression and subcellular localization of the cell cycle regulatory protein, cyclin D1 in varix tissues, as compared with controls. Because cell cycle checkpoint controls are linked to the signaling and execution of apoptotic cascades, we examined the expression of bcl-2 family members bax and bcl-x, known molecular mediators of apoptosis, and that of poly (ADP-ribose) polymerase (PARP), a downstream substrate of DNA cleavage. Methods: Twenty varicose vein specimens were retrieved from 20 patients (10 men, 10 women; mean age, 53.6 ± 4.7 years) undergoing lower-extremity varicose vein excision. Healthy greater saphenous vein segments (n = 27) were obtained from 27 patients (14 men, 13 women; mean age, 59.5 ± 2.4 years) undergoing infrainguinal arterial bypass grafting surgery. All tissues were distal portions. As per CEAP classification for chronic lower-extremity venous disease, most of the patients were in class 2 for clinical signs (n = 11); some patients were in class 3 (n = 4) or class 4 (n = 4), and only one patient was in class 5. Five 5-μm thick sections from formalin-fixed, paraffin-embedded specimens were used as a means of immunohistochemically localizing the expression of bax, bcl-x, and PARP, and 10 random high-power fields per section were evaluated by two independent reviewers blinded to the clinical findings. Statistical analyses were conducted by means of χ2, analysis of variance, Student and Fisher exact t tests with StatView software. Results: Immunoreactivity to pro-apoptotic bax was significantly higher in the normal veins (P <.001). Cytoplasmic expression of bcl-x was prominent in the cells of the vasa vasorum in both varicose and healthy veins. PARP expression was diminished in the varicose vein group, with 2.8 ± 0.7 (P =.01) and 1.4 ± 0.5 (P =.05) cells per high-power field in the intima and media, respectively. Neither bax nor PARP was noted in the adventitia of varicose veins, although their expression was detected in this layer of the control group (P <.001). Conclusion: The entry of smooth muscle cells into the apoptotic pathway may be regulated by the induction of bax in this model, because there is significant presence of this pro-apoptotic protein in healthy veins. Both bax and PARP are downregulated in varicose veins, as compared with healthy veins, and this may play a significant role in the pathogenesis of varicose veins. (J Vasc Surg 2001;33:1080-6.)

Fibronectin activates the p42/44 map kinase cascade and facilitates cell cycle entry of smooth muscle cells in primary culture

Fibronectin activates the p42/44 map kinase cascade and facilitates cell cycle entry of smooth muscle cells in primary culture

Capacitative calcium entry in smooth muscle cells from preglomerular vessels.

Calcium entry via voltage-gated L-type channels is responsible for at least half of the increase in cytosolic calcium ([Ca(2+)](i)) in afferent arterioles following agonist stimulation. We sought the presence of capacitative calcium entry in fresh vascular smooth muscle cells (VSMC) derived from rat preglomerular vessels. [Ca(2+)](i) was measured using fura-2 ratiometric fluorescence. Vasopressin V1 receptor agonist (V1R) (10(-7) M) increased [Ca(2+)](i) by approximately 100 nM. A calcium channel blocker (CCB), nifedipine or verapamil (10(-7) M), inhibited the response by approximately 50%. V1R in the presence of CCB increased [Ca(2+)](i) from 106 to 176 nM, confirming that calcium mobilization and/or entry may occur independent of voltage-gated channels. In nominally Ca(2+)-free buffer, V1R increased [Ca(2+)](i) from 94 to 129 nM, denoting mobilization; addition of CaCl(2) (1 mM) further elevated [Ca(2+)](i) to 176 nM, indicating a secondary phase of Ca(2+) entry. Similar responses were obtained when CCB was present in calcium-free buffer or when EGTA was present. In nominally Ca(2+)-free medium, the sarcoplasmic reticulum Ca(2+)-ATPase inhibitors (SRCAI), thapsigargin and cyclopiazonic acid (CPA), increased [Ca(2+)](i) from 97 to 128 and 143 nM, respectively, and to 214 and 220 nM, respectively, when 1 mM extracellular Ca(2+) was added. In the presence of verapamil, the results with CPA acid were nearly identical. In Ca(2+)-free buffer, the stimulatory effect of V1R or SRCAI on the Ca(2+)/fura signal was quenched by the addition of Mn(2+) (1 mM), demonstrating divalent cation entry. These studies provide evidence for capacitative (store- operated) calcium entry in VSMC freshly isolated from rat preglomerular arterioles.

Tyrosine kinase-dependent modulation of calcium entry in rabbit colonic muscularis mucosae.

We studied the role of tyrosine kinase in the regulation of Ca2+ entry in single smooth muscle cells of the rabbit colonic muscularis mucosae using the whole cell patch-clamp technique. Step depolarization to +10 mV from a holding potential of -60 mV produced inward currents that were abolished by 1 microM nifedipine, consistent with the activation of L-type Ca2+ channels. The tyrosine kinase inhibitors, genistein and tyrphostin B42, dose dependently inhibited these Ca2+ currents. The inactive analogue of tyrphostins, tyrphostin A1, did not affect the currents at concentrations of up to 100 microM. Conversely, the tyrosine phosphatase inhibitor, orthovanadate, enhanced peak Ca2+ currents by 30%. Spontaneous transient outward currents (STOCs) (50-600 pA) were elicited with high K+ in the pipette and at 0-mV holding potential. STOCs were activated due to release of Ca2+ from intracellular stores, required the presence of extracellular Ca2+ concentration, and were insensitive to nifedipine. Genistein abolished STOCs; however, in its presence, outward currents activated by caffeine or carbachol were not affected. The refilling of the Ca2+ stores was studied by first depleting intracellular Ca2+ with carbachol in Ca(2+)-free media followed by reperfusing with a Ca(2+)-containing solution for 3-5 min. Under these conditions, a second application of carbachol evoked an outward current due to Ca2+ release. However, this effect was abolished when the refilling of the stores was carried out in the presence of genistein. Carbachol-evoked currents were not attenuated when the refilling was examined in the presence of orthovanadate. Epidermal growth factor (200 ng/ml) enhanced Ca2+ currents by 60% and markedly increased STOCs by over 200%. Western blot analysis, using an anti-phosphotyrosine antibody, showed a tyrosine phosphorylated protein of 60 kDa in control conditions. This was markedly increased after treatment with epidermal growth factor and carbachol. These results suggest that 1) tyrosine kinase modulates the entry of Ca2+ through L-type channels and through nifedipine-resistant pathways involved in refilling of intracellular stores and 2) stimulation of the kinase by agonists enhances Ca2+ entry in the smooth muscle cells of the rabbit colonic muscularis mucosae.

Mechanism of Acetylcholine-Induced Constriction Enhanced by Endothelial Removal in Isolated, Perfused Canine Basilar Arteries

We examined the responses to intraluminally applied acetylcholine (ACh) in isolated and perfused canine basilar arteries by the stainless-steel cannula-inserting method. In control vessels with intact endothelium, ACh produced a slight vasodilatation followed by a long-lasting vasoconstriction in the absence of induced tone. Propranolol, a beta-adrenoceptor blocker, had no effect on the response to ACh. After endothelial removal with intraluminal saponin, the constrictor component of the response to ACh was significantly enhanced, and the constrictions to prostaglandin F2 alpha (PGF2 alpha) and potassium chloride (KCl) were significantly potentiated. After exposure to extraluminal oxyhemoglobin, the ACh-induced constriction was significantly augmented, whereas the dilator component of the response to calcium ionophore A23187 was significantly attenuated. The enhanced constriction to ACh after endothelial removal was significantly blocked by OKY-046 (a thromboxane synthetase inhibitor) and nimodipine (a dihydropyridine calcium antagonist), but was slightly though not significantly suppressed by AA-861 (a lipoxygenase inhibitor). The response to PGF2 alpha was not altered by OKY-046 or AA-861. These results suggest that the potentiation of ACh-induced constriction after endothelial removal may be mediated in part by thromboxane A2 (TXA2), linked with extracellular calcium entry in smooth muscle cells (SMC). This mechanism might be involved in the pathogenesis of cerebral vasospasm after subarachnoid hemorrhage (SAH).

Arterial smooth muscle cells in vivo: relationship between actin isoform expression and mitogenesis and their modulation by heparin.

Quiescent smooth muscle cells (SMC) in normal artery express a pattern of actin isoforms with alpha-smooth muscle (alpha SM) predominance that switches to beta predominance when the cells are proliferating. We have examined the relationship between the change in actin isoforms and entry of SMC into the growth cycle in an in vivo model of SMC proliferation (balloon injured rat carotid artery). alpha SM actin mRNA declined and cytoplasmic (beta + gamma) actin mRNAs increased in early G0/G1 (between 1 and 8 h after injury). In vivo synthesis and in vitro translation experiments demonstrated that functional alpha SM mRNA is decreased 24 h after injury and is proportional to the amount of mRNA present. At 36 h after injury, SMC prepared by enzymatic digestion were sorted into G0/G1 and S/G2 populations; only the SMC committed to proliferate (S/G2 fraction) showed a relative slight decrease in alpha SM actin and, more importantly, a large decrease in alpha SM actin mRNA. A switch from alpha SM predominance to beta predominance was present in the whole SMC population 5 d after injury. To determine if the change in actin isoforms was associated with proliferation, we inhibited SMC proliferation by approximately 80% with heparin, which has previously been shown to block SMC in late G0/G1 and to reduce the growth fraction. The switch in actin mRNAs and synthesis at 24 h was not prevented; however, alpha SM mRNA and protein were reinduced at 5 d in the heparin-treated animals compared to saline-treated controls. These results suggest that in vivo the synthesis of actin isoforms in arterial SMC depends on the mRNA levels and changes after injury in early G0/G1 whether or not the cells subsequently proliferate. The early changes in actin isoforms are not prevented by heparin, but they are eventually reversed if the SMC are kept in the resting state by the heparin treatment.

Pharmacological basis for the therapeutic applications of slow-channel blocking drugs.

Excitation-contraction coupling in cardiac muscle as well as in smooth muscle is mediated by the transmembrane fluxes of calcium. In the case of cardiac muscle, this transfer occurs through the slow-inward channel. Agents that selectively inhibit the myocardial slow-channel also block calcium entry in smooth muscle cells, particularly in arteries. Thus, such selective inhibitors of the slow channel, exemplified by verapamil, nifedipine, and diltiazem, produce a marked negative inotropic effect in cardiac muscle; in whole animals or in man, such a propensity is largely nullified or even reversed by the profound vasodilator effects of these compounds. The drugs known as calcium antagonists are chemically heterogeneous and they may exhibit associated pharmacological properties such as noncompetitive sympathetic inhibition while having varying potencies for inhibiting the calcium influx in smooth muscle and in the heart and nodal tissues. These similarities and differences influence the net electrophysiologic and hemodynamic effects of calcium antagonists in man. Electrophysiologically, the main effect is a depressant one on the AV node in which most agents of the class lengthen AV conduction and enhance refractoriness, a property that is relevant in the termination of PSVT and to the slowing of the ventricular response in atrial flutter and fibrillation. The effective refractory periods of atrial, ventricular, and His-Purkinje tissues or the bypass tracts are not altered by calcium antagonists, but conduction may be improved in ischemic tissues. On the surface ECG, the only effect is the short-term lengthening of the PR interval with no change in the QRS or Q-Tc intervals. The sinus frequency is variably affected relative to the competing influences of the direct effect, reflex response to hypotension and of sympathetic antagonism. The sinus node recovery time is affected little normally, but may be prolonged dramatically in the sick sinus syndrome. Hemodynamically, as a class of drugs, calcium antagonists produce a complex interplay of simultaneous changes in preload, afterload, contractility, coronary flow, and heart rate. The net hemodynamic effect that becomes apparent will be dependent on the agent used, on the cardiac condition and the level of ventricular function present, on the intactness of the autonomic nervous system, and on the route of drug administration. An appreciation of the electrophysiological and hemodynamic actions of calcium antagonists relative to their individual pharmacologic properties permits the rational choice of the appropriate agent in the control of a wide spectrum of cardiocirculatory disorders.

Mechanisms of action of transmitters and other substances on smooth muscle.

Mechanisms of action of transmitters and other substances on smooth muscle.