Tyrosine kinase-dependent calcium signaling in airway smooth muscle cells

Contractile agonists may stimulate mitogenic responses in airway smooth muscle by mechanisms that involve tyrosine kinases. The role of contractile agonist-evoked activation of tyrosine kinases in contractile signaling is not clear. We addressed this issue using cultured rat airway smooth muscle cells. In these cells, serotonin (5-HT, 1 microM) caused contraction (quantitated by a decrease in cell area), which was blocked by the tyrosine kinase inhibitor genistein (40 microM). Genistein and tyrphostin 23 (40 and 10 microM, respectively) significantly decreased 5-HT-evoked peak Ca(2+) responses, and the effect of genistein could be observed in the absence of extracellular Ca(2+). The specific inhibitor of mitogen-activated protein kinase kinase PD-98059 (30 microM) had no significant effect on peak Ca(2+) levels. Western analysis of cell extracts revealed that 5-HT caused a significant increase in tyrosine phosphorylation of proteins with molecular masses of approximately 70 kDa within 10 s of stimulation but no measurable tyrosine phosphorylation of the gamma isoform of phospholipase C (PLC-gamma). Tyrosine phosphorylation was inhibited by genistein. Furthermore, genistein (40 microM) significantly attenuated 5-HT-induced inositol phosphate production. We conclude that in airway smooth muscle contractile agonists acting on G protein-coupled receptors may activate tyrosine kinase(s), which in turn modulate calcium signaling by affecting, directly or indirectly, PLC-beta activity. It is unlikely that PLC-gamma or the mitogen-activated protein kinase pathway is involved in Ca(2+) signaling to 5-HT.     

Effect of serotonin on intracellular free calcium of rat cerebrovascular smooth muscle cells in culture

Smooth muscle cells were dissociated from conducting cerebral arteries of adult rats and maintained in culture for 2-4 days. The calcium-sensitive fluorescent probe, fura-2, was used to study the effect of the vasoconstrictor serotonin (5-HT) on the level of free intracellular Ca2+ in these cells. The baseline level of free intracellular calcium was 39 +/- 3.6 nM. In 74 out of 110 cells, 5-HT application transiently increased the free Ca2+ content. This effect was dose-dependent and was suppressed by nanomolar concentrations of the 5-HT2 receptor antagonist, ketanserin. The 5-HT induced rise in free intracellular calcium was not prevented by the presence of Co2+, La3+, or nifedipine, blockers of voltage-sensitive calcium channels. These results indicate that 5-HT mobilizes intracellular Ca2+ in cultured smooth muscle cells derived from the rat cerebrovasculature. The mobilization of intracellular Ca2+ appears to be triggered by a 5-HT2 type receptor, although further pharmacological experiments are required to verify this hypothesis.     

Bitter taste receptors on airway smooth muscle bronchodilate by localized calcium signaling and reverse obstruction

Bitter taste receptors (TAS2Rs) on the tongue probably evolved to evoke signals for avoiding ingestion of plant toxins. We found expression of TAS2Rs on human airway smooth muscle (ASM) and considered these to be avoidance receptors for inhalants that, when activated, lead to ASM contraction and bronchospasm. TAS2R agonists such as saccharin, chloroquine and denatonium evoked increased intracellular calcium ([Ca2(+)](i)) in ASM in a Gβγ-, phospholipase Cβ (PLCβ)- and inositol trisphosphate (IP?) receptor-dependent manner, which would be expected to evoke contraction. Paradoxically, bitter tastants caused relaxation of isolated ASM and dilation of airways that was threefold greater than that elicited by β-adrenergic receptor agonists. The relaxation induced by TAS2Rs is associated with a localized [Ca2(+)](i) response at the cell membrane, which opens large-conductance Ca2(+)-activated K(+) (BK(Ca)) channels, leading to ASM membrane hyperpolarization. Inhaled bitter tastants decreased airway obstruction in a mouse model of asthma. Given the need for efficacious bronchodilators for treating obstructive lung diseases, this pathway can be exploited for therapy with the thousands of known synthetic and naturally occurring bitter tastants.     

Acetylcholine-induced calcium signaling and contraction of airway smooth muscle cells in lung slices

The Ca(2+) signaling and contractility of airway smooth muscle cells (SMCs) were investigated with confocal microscopy in murine lung slices (approximately 75-microm thick) that maintained the in situ organization of the airways and the contractility of the SMCs for at least 5 d. 10--500 nM acetylcholine (ACH) induced a contraction of the airway lumen and a transient increase in [Ca(2+)](i) in individual SMCs that subsequently declined to initiate multiple intracellular Ca(2+) oscillations. These Ca(2+) oscillations spread as Ca(2+) waves through the SMCs at approximately 48 microm/s. The magnitude of the airway contraction, the initial Ca(2+) transient, and the frequency of the subsequent Ca(2+) oscillations were all concentration-dependent. In a Ca(2+)-free solution, ACH induced a similar Ca(2+) response, except that the Ca(2+) oscillations ceased after 1--1.5 min. Incubation with thapsigargin, xestospongin, or ryanodine inhibited the ACH-induced Ca(2+) signaling. A comparison of airway contraction with the ACH-induced Ca(2+) response of the SMCs revealed that the onset of airway contraction correlated with the initial Ca(2+) transient, and that sustained airway contraction correlated with the occurrence of the Ca(2+) oscillations. Buffering intracellular Ca(2+) with BAPTA prohibited Ca(2+) signaling and airway contraction, indicating a Ca(2+)-dependent pathway. Cessation of the Ca(2+) oscillations, induced by ACH-esterase, halothane, or the absence of extracellular Ca(2+) resulted in a relaxation of the airway. The concentration dependence of the airway contraction matched the concentration dependence of the increased frequency of the Ca(2+) oscillations. These results indicate that Ca(2+) oscillations, induced by ACH in murine bronchial SMCs, are generated by Ca(2+) release from the SR involving IP(3)- and ryanodine receptors, and are required to maintain airway contraction.     

Mechanisms of calcium signaling in smooth muscle cells explored with fluorescence confocal imaging

A rise in the intracellular concentration of ionized calcium ([Ca²⁺]_i) is a primary signal for contraction in all types of muscles. Recent progress in the development of imaging techniques, with special accent on fluorescence confocal microscopy, and new achievements in the synthesis of organelle- and ion-specific fluorochromes provide an experimental basis for studying the relationship between the structural organization of living smooth muscle cells (SMCs) and features of calcium signaling at the subcellular level. Applying fluorescent confocal imaging, patch-clamp recording, immunostaining, and flash photolysis techniques to freshly isolated SMCs, we have demonstrated that: (i) Ca²⁺ sparks are mediated by spontaneous clustered opening of ryanodine receptors (RyRs) and occur at the highest rate at preferred sites (frequent discharge sites, FDSs), the number of which depends on SMC type; (ii) FDSs are associated with sub-plasmalemmal sarcoplasmic reticulum (SR) elements, but not with polarized mitochondria; (iii) Ca²⁺ spark frequency increases with membrane depolarization in voltage-clamped SMCs or following neurotransmitter application to SMCs, in which the membrane potential was not controlled, leading to spark summation and resulting in a cell-wide increase in [Ca²⁺]_i and myocyte contraction; (iv) cross-talk between RyRs and inositol trisphosphate receptors (IP₃Rs) is an important determinant of the [Ca²⁺]_i dynamics and recruits neighboring Ca²⁺-release sites to generate [Ca²⁺]_i waves; (v) [Ca²⁺]_i waves induced by depolarization of the plasma membrane or by noradrenaline or caffeine, but not by carbachol (CCh), originate at FDSs; (vi) Ca²⁺-dependent K⁺ and Cl^- channels sense the local changes in [Ca²⁺]_i during a Ca²⁺ spark and thereby may couple changes in [Ca²⁺]_i within a microdomain to changes in the membrane potential, thus affecting the cell excitability; (vii) the muscarinic cation current (mI _cat) does not mirror changes in [Ca²⁺]_i, thus reflecting the complexity of [Ca²⁺]_i — muscarinic cationic channel coupling; (viii) RyR-mediated Ca²⁺ release, either spontaneous or caffeine-induced, does not augment mI _cat; (ix) intracellular flash release of Ca²⁺ is less effective in augmentation of mI _cat than flash release of IP₃, suggesting that IP₃ may sensitize muscarinic cationic channels to Ca²⁺; (x) intracellular flash release of IP₃ fails to augment mI _cat in SMCs, in which [Ca²⁺]_i was strongly buffered, suggesting that IP₃ exerts no direct effect on muscarinic cationic channel gating, and that these channels sense an increase in [Ca²⁺]_i rather than depletion of the IP₃-dependent Ca²⁺ store; and (xi) predominant expression of IP₃R type 1 in the peripheral SR provides a structural basis for a tight functional coupling between IP₃R-mediated Ca²⁺ release and muscarinic cationic channel opening.Neirofiziologiya/Neurophysiology, Vol. 36, Nos. 5/6, pp. 455–465, September–December, 2004.This revised version was published online in April 2005 with a corrected cover date and copyright year.     

Tyrosine kinase inhibitors block calcium channel currents in vascular smooth muscle cells.

Selective inhibitors of tyrosine kinases, tyrphostin 23 and genistein, produced concentration-dependent inhibition of voltage-operated calcium channel currents in vascular smooth muscle cells isolated from rabbit ear artery. The potency of these two structurally dissimilar inhibitors was similar to that reported for their action as inhibitors of tyrosine kinases. Daidzein, an inactive analogue of genistein, had little inhibitory effect on calcium channel currents at concentrations below 300 microM consistent with an action of these agents at a tyrosine kinase. However, tyrphostin 1, a reportedly less active tyrphostin derivative, also inhibited calcium channel currents with a potency similar to tyrphostin 23. These findings suggest that voltage-operated calcium channels in vascular smooth muscle may be modulated by endogenous tyrosine kinase(s) which display different sensitivities to inhibitors compared with the epidermal growth factor (EGF) receptor. Alternatively the possibility of direct blocking actions of these inhibitors at voltage-operated calcium channels cannot be excluded.     

Differential effects of intravenous anesthetics on capacitative calcium entry in human pulmonary artery smooth muscle cells

We assessed the roles of the protein kinase C (PKC) and the tyrosine kinase (TK) signaling pathways in regulating capacitative calcium entry (CCE) in human pulmonary artery smooth muscle cells (PASMCs) and investigated the effects of intravenous anesthetics (midazolam, propofol, thiopental, ketamine, etomidate, morphine, and fentanyl) on CCE in human PASMCs. Fura-2-loaded human PASMCs were placed in a dish (37 degrees C) on an inverted fluorescence microscope. Intracellular Ca2+ concentration ([Ca2+]i) was measured as the 340/380 fluorescence ratio in individual PASMCs. Thapsigargin, a sarcoplasmic reticulum Ca2+-adenosine triphosphatase inhibitor, was used to deplete intracellular Ca2+ stores after removing extracellular Ca2+. CCE was then activated by restoring extracellular Ca2+ (2.2 mM). The effects of PKC activation and inhibition, TK inhibition, and the intravenous anesthetics on CCE were assessed. Thapsigargin caused a transient increase in [Ca2+]i. Restoring extracellular Ca2+ caused a rapid peak increase in [Ca2+]i, followed by a sustained increase in [Ca2+]i; i.e., CCE was stimulated in human PASMCs. PKC activation attenuated (P < 0.05), whereas PKC inhibition potentiated (P < 0.05), both peak and sustained CCE. TK inhibition attenuated (P < 0.05) both peak and sustained CCE. Midazolam, propofol, and thiopental each attenuated (P < 0.05) both peak and sustained CCE, whereas ketamine, etomidate, morphine, and fentanyl had no effect on CCE. Our results suggest that CCE in human PASMCs is influenced by both the TK and PKC signaling pathways. Midazolam, propofol, and thiopental each attenuated CCE, whereas ketamine, etomidate, morphine, and fentanyl had no effect on CCE.     

Enhanced calcium signaling to bradykinin in airway smooth muscle from hyperresponsive inbred rats

Inbred Fischer 344 rats display airway hyperresponsiveness (AHR) in vivo compared with the normoresponsive Lewis strain. Fischer AHR has been linked with increased airway smooth muscle (ASM) contraction ex vivo and enhanced ASM cell intracellular Ca(2+) mobilization in response to serotonin compared with Lewis. To determine the generality of this association, we tested whether bradykinin (BK) also stimulates greater contraction of Fischer airways and greater Ca(2+) mobilization in Fischer ASM cells. Explants of Fischer intraparenchymal airways constricted faster and to a greater degree in response to BK than Lewis airways. BK also evoked higher Ca(2+) transients in Fischer than in Lewis ASM cells. ASM cell B(2) receptor expression was similar between the two strains. BK activated both phosphatidylinositide-specific phospholipase C (PI-PLC) and phosphatidylcholine-specific PLC to mobilize Ca(2+) in Fischer and Lewis ASM cells. PI-PLC activity, as measured by inositol polyphosphate accumulation, was similar in the two strains. PKC inhibition with GF109203X, Go6973, or Go6983 attenuated BK-mediated Ca(2+) transients in Fischer cells, whereas GF109203X potentiated while Go6976 and Go6983 did not affect Ca(2+) transients in Lewis cells. Enhanced Ca(2+) mobilization in ASM cells can arise from variations in PKC and may be an important component of nonspecific, innate AHR.     

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.     

Histone deacetylase inhibitors promote eNOS expression in vascular smooth muscle cells and suppress hypoxia‐induced cell growth

Hypoxia stimulates excessive growth of vascular smooth muscle cells (VSMCs) contributing to vascular remodelling. Recent studies have shown that histone deacetylase inhibitors (HDIs) suppress VSMC proliferation and activate eNOS expression. However, the effects of HDI on hypoxia‐induced VSMC growth and the role of activated eNOS in VSMCs are unclear. Using an EdU incorporation assay and flow cytometry analysis, we found that the HDIs, butyrate (Bur) and suberoylanilide hydroxamic acid (SAHA) significantly suppressed the proliferation of hypoxic VSMC lines and induced apoptosis. Remarkable induction of cleaved caspase 3, p21 expression and reduction of PCNA expression were also observed. Increased eNOS expression and enhanced NO secretion by hypoxic VSMC lines were detected using Bur or SAHA treatment. Knockdown of eNOS by siRNA transfection or exposure of hypoxic VSMCs to NO scavengers weakened the effects of Bur and SAHA on the growth of hypoxic VSMCs. In animal experiments, administration of Bur to Wistar rats exposed to hypobaric hypoxia for 28 days ameliorated the thickness and collagen deposition in pulmonary artery walls. Although the mean pulmonary arterial pressure (mPAP) was not obviously decreased with Bur in hypoxic rats, right ventricle hypertrophy index (RVHI) was decreased and the oxygen partial pressure of arterial blood was elevated. Furthermore, cell viability was decreased and eNOS and cleaved caspase 3 were induced in HDI‐treated rat pulmonary arterial SMCs. These findings imply that HDIs prevent hypoxia‐induced VSMC growth, in correlation with activated eNOS expression and activity in hypoxic VSMCs.     

Plasma membrane calcium pumps in smooth muscle: from fictional molecules to novel inhibitors

Plasma membrane Ca2+ pumps (PMCA pumps) are Ca2+-Mg2+ ATPases that expel Ca2+ from the cytosol to extracellular space and are pivotal to cell survival and function. PMCA pumps are encoded by the genes PMCA1, -2, -3, and -4. Alternative splicing results in a large number of isoforms that differ in their kinetics and activation by calmodulin and protein kinases A and C. Expression by 4 genes and a multifactorial regulation provide redundancy to allow for animal survival despite genetic defects. Heterozygous mice with ablation of any of the PMCA genes survive and only the homozygous mice with PMCA1 ablation are embryolethal. Some PMCA isoforms may also be involved in other cell functions. Biochemical and biophysical studies of PMCA pumps have been limited by their low levels of expression. Delineation of the exact physiological roles of PMCA pumps has been difficult since most cells also express sarco/endoplasmic reticulum Ca2+ pumps and a Na+-Ca2+-exchanger, both of which can lower cytosolic Ca2+. A major limitation in the field has been the lack of specific inhibitors of PMCA pumps. More recently, a class of inhibitors named caloxins have emerged, and these may aid in delineating the roles of PMCA pumps.     

心肌细胞钙信号研究进展

近年自激光共聚焦显微镜使用以来,结合膜片钳技术及分子生物学方法,在心肌细胞内的钙信号种类以及在心脏兴奋－收缩偶联研究方面取得了突破性进展。本文介绍了心肌细胞的钙信号研究进展,包括在心肌细胞内可以观察到的钙闪烁,钙微粒,钙波以及由心肌细胞膜上电除极而诱发的瞬时性钙增高等几种心脏细胞内钙变化的形式,意义以及局部调控兴奋－收给偶联的机制。     

Chemical signaling from colonic smooth muscle cells to DRG neurons in culture

Transductionmechanisms between target cells within the intestinal wall andperipheral terminals of extrinsic primary afferent neurons are poorlyunderstood. The purpose of this study was to characterize theinteractions between smooth muscle cells from the rat distal colon andlumbar dorsal root ganglion (DRG) neurons in coculture. DRG neuronsvisually appeared to make contact with several myocytes. We show thatbrief mechanical stimulation of these myocytes resulted inintracellular Ca²⁺ concentration([Ca²⁺]_i)transients that propagated into 57% of the contacting neurites. Directmechanical stimulation of DRG neurites cultured without smooth musclehad no effect. We also show that colonic smooth muscle cells expressmultiple connexin mRNAs and that these connexins formed functional gapjunctions, as evidenced by the intercellular transfer of Luciferyellow. Furthermore, thapsigargin pretreatment and neuronal heparininjection abolished the increase in neurite [Ca²⁺]_i,indicating that the neuronal Ca²⁺signal was triggered by inositol 1,4,5-trisphosphate-mediated Ca²⁺ release from intracellularstores. Our results provide evidence for intercellular chemicalcommunication between DRG neurites and intestinal smooth muscle cellsthat mediates the exchange of second messenger molecules betweendifferent cell types.     

Regulation of beta-adrenergic receptors and calcium channel agonist binding sites in cultured human embryonal smooth muscle cells

Recent electrophysiological studies with cell membrane patches of cardiac myocytes and an electrically excitable cell line derived from rat pituitary tumor suggested that voltage activated calcium channels must be phosphorylated to respond to membrane depolarization (Armstrong and Eckert 1986; Trautwein and Kameyama 1986). In view of the "phosphorylation hypothesis" we investigated the adenylate-cyclase activity, the characteristics of beta-adrenergic and calcium channel agonist binding sites in control and desensitized (exposure to isoproterenol) human embryonal cells (HEC), and in fragmented membrane preparations of canine coronary smooth muscle. Our results suggest that down-regulation of the membrane-bound beta-adrenergic receptors, induced by isoproterenol in human embryonal cells and also in adult canine vascular tissue, results in physical translocation of beta-adrenergic binding sites into the light membrane fraction. This phenomenon is accompanied with an increased intracellular concentration of cAMP in and an increased binding of the calcium channel agonist (3H) BAYK 8644 to both HEC and canine smooth muscle membrane preparations. It could be concluded that phosphorylation of beta-adrenergic receptors regulates not only the beta subcellular distribution of the beta receptors but also the availability of calcium channel agonist binding sites in the cellular membrane.     

Altered calcium signaling in colonic smooth muscle of type 1 diabetic mice

Seventy-six percent of diabetic patients develop gastrointestinal symptoms, such as constipation. However, the direct effects of diabetes on intestinal smooth muscle are poorly described. This study aimed to identify the role played by smooth muscle in mediating diabetes-induced colonic dysmotility. To induce type 1 diabetes, mice were injected intraperitoneally with low-dose streptozotocin once a day for 5 days. Animals developed hyperglycemia (>200 mg/dl) 1 wk after the last injection and were euthanized 7-8 wk after the last treatment. Computed tomography demonstrated decreased overall gastrointestinal motility in the diabetic mice. In vitro contractility of colonic smooth muscle rings from diabetic mice was also decreased. Fura-2 ratiometric Ca(2+) imaging showed attenuated Ca(2+) increases in response to KCl stimulation that were associated with decreased light chain phosphorylation in diabetic mice. The diabetic mice also exhibited elevated basal Ca(2+) levels, increased myosin phosphatase targeting subunit 1 expression, and significant changes in expression of Ca(2+) handling proteins, as determined by quantitative RT-PCR and Western blotting. Mice that were hyperglycemic for <1 wk also showed decreased colonic contractile responses that were associated with decreased Ca(2+) increases in response to KCl stimulation, although without an elevation in basal Ca(2+) levels or a significant change in the expression of Ca(2+) signaling molecules. These data demonstrate that type 1 diabetes is associated with decreased depolarization-induced Ca(2+) influx in colonic smooth muscle that leads to attenuated myosin light chain phosphorylation and impaired colonic contractility.