Invited review: mechanisms of calcium handling in smooth muscles.

The concentration of cytoplasmic Ca(2+) regulates the contractile state of smooth muscle cells and tissues. Elevations in global cytoplasmic Ca(2+) resulting in contraction are accomplished by Ca(2+) entry and release from intracellular stores. Pathways for Ca(2+) entry include dihydropyridine-sensitive and -insensitive Ca(2+) channels and receptor and store-operated nonselective channels permeable to Ca(2+). Intracellular release from the sarcoplasmic reticulum (SR) is accomplished by ryanodine and inositol trisphosphate receptors. The impact of Ca(2+) entry and release on cytoplasmic concentration is modulated by Ca(2+) reuptake into the SR, uptake into mitochondria, and extrusion into the extracellular solution. Highly localized Ca(2+) transients (i.e., sparks and puffs) regulate ionic conductances in the plasma membrane, which can provide feedback to cell excitability and affect Ca(2+) entry. This short review describes the major transport mechanisms and compartments that are utilized for Ca(2+) handling in smooth muscles.     

Invited review: regulation of myosin phosphorylation in smooth muscle.

Phosphorylation of the regulatory light chains of myosin II (rMLC) by the Ca(2+)/calmodulin-dependent myosin light-chain kinase (MLCK) and dephosphorylation by a type 1 phosphatase (MLCP), which is targeted to myosin by a regulatory subunit (MYPT1), are the predominant mechanisms of regulation of smooth muscle tone. The activities of both enzymes are modulated by several protein kinases. MLCK is inhibited by the Ca(2+)/calmodulin-dependent protein kinase II, whereas the activity of MLCP is increased by cGMP and perhaps also cAMP-dependent protein kinases. In either case, this results in a decrease in the Ca(2+) sensitivity of rMLC phosphorylation and force production. The activity of MLCP is inhibited by Rho-associated kinase, one of the effectors of the monomeric GTPase Rho, and protein kinase C, leading to an increase in Ca(2+) sensitivity. Hence, smooth muscle tone appears to be regulated by a network of activating and inactivating intracellular signaling cascades.     

Serotonin-induced cytosolic free calcium transients in cultured vascular smooth muscle cells

Serotonin induced a transient elevation in the levels of cytosolic calcium in cultured rat vascular smooth muscle cells. Ketanserin, a selective antagonist of serotonin 2 receptors, dose-dependently inhibited the elevation of cytosolic calcium induced by serotonin, and ultimately unmasked a serotonin-induced decrease in the levels of cytosolic calcium. These observations show that serotonin has direct and dual effects, that is, it increases and decreases cytosolic free calcium concentrations in vascular smooth muscle cells, in culture. Knowledge of such events is important because serotonergic inhibitors may prove to be useful drugs for treating clinical hypertension and vasospastic disorders.     

Invited review: the circle of life: cell cycle regulation in airway smooth muscle.

Severe asthma is characterized by increased airway smooth muscle (ASM) mass, due predominantly to ASM hyperplasia. Diverse stimuli, which include growth factors, plasma- or inflammatory cell-derived mediators, contractile agonists, cytokines, and extracellular matrix proteins, induce ASM proliferation. Mitogens act via receptor tyrosine kinase, G protein-coupled receptors, or cytokine receptors, to activate p21ras and stimulate two parallel signaling pathways in ASM cells, namely, the extracellular signal-regulated kinase (ERK) or the phosphatidylinositol 3-kinase (PI3K) pathways. ERK and PI3K regulate cell cycle protein expression and thus modulate cell cycle traversal. ERK activation and downstream effectors of PI3K, such as Rac1 and Cdc42, stimulate expression of cyclin D1, a key regulator of G(1) progression in the mammalian cell cycle. In addition, PI3K activates 70-kDa ribosomal S6 kinase, an enzyme that also regulates the translation of many cell cycle proteins, including the elongation factor E2F. The present review examines the mitogens and critical signal transduction pathways that stimulate ASM cell proliferation. Further study in this area may reveal new therapeutic targets to abrogate ASM hyperplasia in diseases such as asthma and chronic obstructive pulmonary disease.     

Chloride-dependent calcium transients induced by angiotensin II in vascular smooth muscle cells

Cl^– is essential for the vasoconstrictive response to angiotensin II (ANG II). In vascular smooth muscle cells (VSMC), we determined whether ANG II-induced transient increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) is Cl^– dependent. After incubating the cells at different extracellular Cl^– concentration ([Cl^–]_e) for 40 min, the ANG II-induced Ca²⁺ transients at 120 meq/l Cl^– were more than twice those at either 80 or 20 meq/l Cl^–. Replacing Cl^– with bicarbonate or gluconate yielded similar results. In addition, after removal of extracellular Ca²⁺, ANG II-induced as well as platelet-derived growth factor-induced Ca²⁺ release exhibited Cl^– dependency. The difference of Ca²⁺ release with high vs. low [Cl^–]_e was not affected by acutely altering [Cl^–]_e 1 min before administration of ANG II when [Cl^–]_i was yet to be equilibrated with [Cl^–]_e. Pretreatment of a Cl^– channel inhibitor, 5-nitro-2-(3-phenylpropylamino)benzoic acid, increased ANG II-induced Ca²⁺ release and entry at 20 meq/l Cl^– but did not alter those at 120 meq/l Cl^–. However, after equilibration, a reduced [Cl^–]_e did not affect thapsigargin-induced Ca²⁺ release, suggesting that Cl^– may not affect the size of intracellular Ca²⁺ stores. Nevertheless, at high [Cl^–], the peak increase of inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃] induced by ANG II was approximately sixfold that at low [Cl^–]. Thus the Cl^–-dependent effects of ANG II on Ca²⁺ transients may be mediated, at least in part, by a Cl^–-dependent Ins(1,4,5)P₃ accumulation in VSMC. anion; inositol 1,4,5-trisphosphate; Ca²⁺ release     

Spatial and temporal patterns of intracellular calcium in colonic smooth muscle

Summary Intracellular calcium [Ca²⁺] _i measurements in cell suspension of gastrointestinal myocytes have suggested a single [Ca²⁺] _i transient followed by a steady-state increase as the characteristic [Ca²⁺] _i response of these cells. In the present study, we used digital video imaging techniques in freshly dispersed myocytes from the rabbit colon, to characterize the spatiotemporal pattern of the [Ca²⁺] _i signal in single cells. The distribution of [Ca²⁺] _i in resting and stimulated cells was nonhomogeneous, with gradients of high [Ca²⁺] _i present in the subplasmalemmal space and in one cell pole. [Ca²⁺] _i gradients within these regions were not constant but showed temporal changes in the form of [Ca²⁺] _i oscillations and spatial changes in the form of [Ca²⁺] _i waves. [Ca²⁺] _i oscillations in unstimulated cells (n = 60) were independent of extracellular [Ca²⁺] and had a mean frequency of 12.6 +1.1 oscillations per min. The baseline [Ca²⁺], was 171 ± 13 nm and the mean oscillation amplitude was 194 ± 12 nm. Generation of [Ca²⁺] _i waves was also independent of influx of extracellular Ca²⁺. [Ca²⁺] _i waves originated in one cell pole and were visualized as propagation mostly along the subplasmalemmal space or occasionally throughout the cytoplasm. The mean velocity was 23 +3 m per sec (n = 6). Increases of [Ca²⁺] _i induced by different agonists were encoded into changes of baseline [Ca²⁺] _i and the amplitude of oscillations, but not into their frequency. The observed spatiotemporal pattern of [Ca²⁺] _i regulation may be the underlying mechanism for slow wave generation and propagation in this tissue. These findings are consistent with a [Ca²⁺] _i regulation whereby cell regulators modulate the spatiotemporal pattern of intracellularly generated [Ca²⁺] _i oscillations.The authors thank Debbie Anderson for excellent technical assistance with the electron microscopy and Dr. M. Regoli for providing the NK-1 agonist [Sar⁹,Met(O₂)¹¹]-SP. This work was supported by National Institutes of Health Grants DK 40919 and DK 40675 and Veterans Administration Grant SMI.     

Inhibition of calcium transients in cultured vascular smooth muscle cells by pertussis toxin

Effects of pertussis toxin on Ca2+ transients in rat arterial smooth muscle cells in primary culture were monitored, using quin 2-microfluorometry. In the presence or the absence of extracellular Ca2+, norepinephrine, histamine, caffeine and high extracellular K+ induced elevations in cytosolic Ca2+ concentration. Cytosolic Ca2+ elevations induced by norepinephrine and histamine were inhibited by pretreatment of the cells with pertussis toxin, time- and dose-dependently. However, elevations induced by caffeine and K+-depolarization were unaffected by the pretreatment with this toxin. Thus, it is suggested that GTP binding protein, a pertussis toxin substrate and involved in the receptor-mediated cytosolic Ca2+ transients, is not involved in transient elevations in cytosolic Ca2+ induced by caffeine and K+-depolarization in cultured vascular smooth muscle cells.     

Charge-coupled device imaging of rapid calcium transients in cultured arterial smooth muscle cells

Transient changes in the concentration of intracellular free calcium are associated with the transduction of primary signals and the subsequent employment of Ca2+ as a second messenger in a multitude of cell types. These transients, typically monitored with the calcium-sensitive fluorescent dye Fura-2, are known to occur with a time course in the order of seconds. In order to accurately monitor such rapid changes in intracellular free calcium concentration in both single cells and simultaneously in several cells in a single field, we have developed a digital fluorescence imaging system based on a charge-coupled device (CCD) camera. We report here on the detailed kinetics of calcium increases in cultured arterial swine smooth muscle cells in response to the agonist ATP.     

Agonist-induced calcium transients in cultured smooth muscle cells: measurements with fura-2 loaded monolayers

Elevation of cytosolic Ca2+ in response to depolarization and various receptor agonists was measured in several types of cultured smooth muscle cells (DDT1, A10, rabbit aorta) loaded with the either quin-2 or fura-2, and assayed either in suspension or in monolayer cultures attached to plastic cover slips. Agonists (norepinephrine, vasopressin) induced both the release of intracellular Ca2+ and the influx of extracellular Ca2+. Agonist-induced Ca2+ influx was not blocked by dihydropyridines, and depolarization did not induce Ca2+ influx. However, in fura-2 loaded monolayers of PC12 cells, depolarization did induce dihydropyridine-sensitive Ca2+ influx. Thus cultured smooth muscle cells appear to express receptor-operated Ca2+ channels, but not functional voltage-operated Ca2+ channels.     

Imaging of calcium transients in skeletal muscle fibers.

Epifluorescence images of Ca2+ transients elicited by electrical stimulation of single skeletal muscle fibers were studied with fast imaging techniques that take advantage of the large fluorescence signals emitted at relatively long wavelengths by the dyes fluo-3 and rhod-2 in response to binding of Ca2+ ions, and of the suitable features of a commercially available CCD video camera. The localized release of Ca2+ in response to microinjection of InsP3 was also monitored to demonstrate the adequate space and time resolutions of the imaging system. The time resolution of the imager system, although limited to the standard video frequency response, still proved to be adequate to investigate the fast Ca2+ release process in skeletal muscle fibers at low temperatures.     

Mechanical transients of single toad stomach smooth muscle cells. Effects of lowering temperature and extracellular calcium

Smooth muscle's slow, economical contractions may relate to the kinetics of the crossbridge cycle. We characterized the crossbridge cycle in smooth muscle by studying tension recovery in response to a small, rapid length change (i.e., tension transients) in single smooth muscle cells from the toad stomach (Bufo marinus). To confirm that these tension transients reflect crossbridge kinetics, we examined the effect of lowering cell temperature on the tension transient time course. Once this was confirmed, cells were exposed to low extracellular calcium [( Ca2+]o) to determine whether modulation of the cell's shortening velocity by changes in [Ca2+]o reflected the calcium sensitivity of one or more steps in the crossbridge cycle. Single smooth muscle cells were tied between an ultrasensitive force transducer and length displacement device after equilibration in temperature-controlled physiological saline having either a low (0.18 mM) or normal (1.8 mM) calcium concentration. At the peak of isometric force, after electrical stimulation, small, rapid (less than or equal to 1.8% cell length in 3.6 ms) step stretches and releases were imposed. At room temperature (20 degrees C) in normal [Ca2+]o, tension recovery after the length step was described by the sum of two exponentials with rates of 40-90 s-1 for the fast phase and 2-4 s-1 for the slow phase. In normal [Ca2+]o but at low temperature (10 degrees C), the fast tension recovery phase slowed (apparent Q10 = 1.9) for both stretches and releases whereas the slow tension recovery phase for a release was only moderately affected (apparent Q10 = 1.4) while unaffected for a stretch. Dynamic stiffness was determined throughout the time course of the tension transient to help correlate the tension transient phases with specific step(s) in the crossbridge cycle. The dissociation of tension and stiffness, during the fast tension recovery phase after a release, was interpreted as evidence that this recovery phase resulted from both the transition of crossbridges from a low- to high-force producing state as well as a transient detachment of crossbridges. From the temperature studies and dynamic stiffness measurements, the slow tension recovery phase most likely reflects the overall rate of crossbridge cycling. From the tension transient studies, it appears that crossbridges cycle slower and have a longer duty cycle in smooth muscle. In low [Ca2+]o at 20 degrees C, little effect was observed on the form or time course of the tension transients.(ABSTRACT TRUNCATED AT 400 WORDS)     

Interleukin-1beta induces calcium transients and enhances basal and store operated calcium entry in human myometrial smooth muscle

We have previously reported increased protein expression of sarcoplasmic reticulum calcium ATPase (SERCA) 2b in myometrium from women in labor at term, but the stimulus for this change is unknown. Proinflammatory cytokines have been implicated in the cascade of events leading to preterm and term labor, and we hypothesize that interleukin (IL)-1beta may induce changes in key calcium homeostatic mechanisms and, in turn, augment myometrial contractility before labor. The aim of the present study was to investigate the long-term effects of IL-1beta on SERCA 2b protein expression, calcium mobilization from intracellular stores, and store-operated calcium entry. Myometrial biopsies were obtained, with consent, from women undergoing elective cesarean section at term. Primary-cultured human myometrial smooth muscle (HMSM) cells were exposed to IL-1beta (10 ng/ml) for 24 h or to culture medium alone (control). Cells were subsequently used in Western blot studies or loaded with fura-2 to assess calcium dynamics using fluorescent digital imaging. The present study clearly demonstrated that IL-1beta significantly increased SERCA 2b protein expression in HMSM cells. Cyclopiazonic acid-induced calcium transients were also augmented, predominantly by activation of lanthanum-sensitive, store-operated calcium entry. HMSM cell excitability was enhanced, as evidenced by increased basal calcium entry and the initiation of spontaneous calcium transients in 37% of IL-1beta-treated cells. IL-1beta modulation of calcium mobilization may be an important mechanism in the cascade of events preparing the pregnant uterus for labor.     

Effects of capacitative calcium entry on agonist-induced calcium transients in A7r5 vascular smooth muscle cells

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.     

Invited review: arteriolar smooth muscle mechanotransduction: Ca(2+) signaling pathways underlying myogenic reactivity.

The smooth muscle of arterioles responds to an increase in intraluminal pressure with vasoconstriction and with vasodilation when pressure is decreased. Such myogenic vasoconstriction provides a level of basal tone that enables arterioles to appropriately adjust diameter in response to neurohumoral stimuli. Key in this process of mechanotransduction is the role of changes in intracellular Ca(2+). However, it is becoming clear that considerable complexity exists in the spatiotemporal characteristics of the Ca(2+) signal and that changes in intracellular Ca(2+) may play roles other than direct effects on the contractile process via activation of myosin light-chain phosphorylation. The involvement of Ca(2+) may extend to modulation of ion channels and release of Ca(2+) from the sarcoplasmic reticulum, alterations in Ca(2+) sensitivity, and coupling between cells within the vessel wall. The purpose of this brief review is to summarize the current literature relating to Ca(2+) and the arteriolar myogenic response. Consideration is given to coupling of Ca(2+) changes to the mechanical stimuli, sources of Ca(2+), involvement of ion channels, and spatiotemporal aspects of intracellular Ca(2+) signaling.     

Temporal and spatial dynamics underlying capacitative calcium entry in human colonic smooth muscle

Angiotensin converting enzyme (ACE) has been shown to be involved in regulation of apoptosis in nonintestinal tissues. This study examined the role of ACE in the modulation of intestinal adaptation utilizing ACE knockout mice (ACE-/-). A 60% small bowel resection (SBR) was used, since this model results in a significant increase in intestinal epithelial cell (EC) apoptosis as well as proliferation. Baseline villus height, crypt depth, and intestinal EC proliferation were higher, and EC apoptosis rates were lower in ACE-/- compared with ACE+/+ mice. After SBR, EC apoptosis rates remained significantly lower in ACE-/- compared with ACE+/+ mice. Furthermore, villus height and crypt depth after SBR continued to be higher in ACE-/- mice. The finding of a lower bax-to-bcl-2 protein ratio in ACE-/- mice may account for reduced EC apoptotic rates after SBR in ACE-/- compared with ACE+/+ mice. The baseline higher rate of EC proliferation in ACE-/- compared with ACE+/+ mice may be due to an increase in the expression of several EC growth factor receptors. In conclusion, ACE appears to have an important role in the modulation of intestinal EC apoptosis and proliferation and suggests that the presence of ACE in the intestinal epithelium has a critical role in guiding epithelial cell adaptive response.     

Invited review: focal adhesion and small heat shock proteins in the regulation of actin remodeling and contractility in smooth muscle.

Smooth muscle cells are able to adapt rapidly to chemical and mechanical signals impinging on the cell surface. It has been suggested that dynamic changes in the actin cytoskeleton contribute to the processes of contractile activation and mechanical adaptation in smooth muscle. In this review, evidence for functionally important changes in actin polymerization during smooth muscle contraction is summarized. The functions and regulation of proteins associated with "focal adhesion complexes" (membrane-associated dense plaques) in differentiated smooth muscle, including integrins, focal adhesion kinase (FAK), c-Src, paxillin, and the 27-kDa small heat shock protein (HSP27) are described. Integrins in smooth muscles are key elements of mechanotransduction pathways that communicate with and are regulated by focal adhesion proteins that include FAK, c-Src, and paxillin as well as proteins known to mediate cytoskeletal remodeling. Evidence that functions of FAK and c-Src protein kinases are closely intertwined is discussed as well as evidence that focal adhesion proteins mediate key signal transduction events that regulate actin remodeling and contraction. HSP27 is reviewed as a potentially significant effector protein that may regulate actin dynamics and cross-bridge function in response to activation of p21-activated kinase and the p38 mitogen-activated protein kinase signaling pathway by signaling pathways linked to integrin proteins. These signaling pathways are only part of a large number of yet to be defined pathways that mediate acute adaptive responses of the cytoskeleton in smooth muscle to environmental stimuli.     

Intracellular free calcium transients induced by norepinephrine in rat aortic smooth muscle cells in primary culture

In cultured rat arterial smooth muscle cells treated with quin 2, cytosolic Ca2+ transients induced by norepinephrine were recorded microfluorometrically. In the presence or absence of extracellular Ca2+, norepinephrine induced transient and dose-dependent elevations in cytosolic Ca2+, with a similar time course, the peak levels being observed at 2 min. These transient elevations in cytosolic Ca2+ were dose-dependently inhibited by alpha-adrenergic antagonists, the order of potency being prazosin greater than phentolamine greater than yohimbine, irrespective of the presence of extracellular Ca2+. We propose that with or without extracellular Ca2+, norepinephrine activates mainly alpha-1 adrenoceptors leading to a release of Ca2+ from intracellular stores. This would explain the transient elevation in cytosolic Ca2+ in rat aortic vascular smooth muscle cells in primary culture.     

Invited review: cGMP-dependent protein kinase signaling mechanisms in smooth muscle: from the regulation of tone to gene expression.

cGMP is a second messenger that produces its effects by interacting with intracellular receptor proteins. In smooth muscle cells, one of the major receptors for cGMP is the serine/threonine protein kinase, cGMP-dependent protein kinase (PKG). PKG has been shown to catalyze the phosphorylation of a number of physiologically relevant proteins whose function it is to regulate the contractile activity of the smooth muscle cell. These include proteins that regulate free intracellular calcium levels, the cytoskeleton, and the phosphorylation state of the regulatory light chain of smooth muscle myosin. Other studies have shown that vascular smooth muscle cells (VSMCs) that are cultured in vitro may cease to express PKG and will, coincidentally, acquire a noncontractile, synthetic phenotype. The restoration of PKG expression to the synthetic phenotype VSMC results in the cells acquiring a more contractile phenotype. These more recent studies suggest that PKG controls VSMC gene expression that, in turn, regulates phenotypic modulation of the cells. Therefore, the regulation of PKG gene expression appears to be linked to phenotypic modulation of VSMC. Because several vascular disorders are related to the accumulation of synthetic, fibroproliferative VSMC in the vessel wall, it is likely that changes in the activity of the nitric oxide/cGMP/PKG pathway is involved the development of these diseases.