Ca2+-dependent rapid Ca2+ sensitization of contraction in arterial smooth muscle

Ca(2+) ion is a universal intracellular messenger that regulates numerous biological functions. In smooth muscle, Ca(2+) with calmodulin activates myosin light chain (MLC) kinase to initiate a rapid MLC phosphorylation and contraction. To test the hypothesis that regulation of MLC phosphatase is involved in the rapid development of MLC phosphorylation and contraction during Ca(2+) transient, we compared Ca(2+) signal, MLC phosphorylation, and 2 modes of inhibition of MLC phosphatase, phosphorylation of CPI-17 Thr38 and MYPT1 Thr853, during alpha(1) agonist-induced contraction with/without various inhibitors in intact rabbit femoral artery. Phenylephrine rapidly induced CPI-17 phosphorylation from a negligible amount to a peak value of 0.38+/-0.04 mol of Pi/mol within 7 seconds following stimulation, similar to the rapid time course of Ca(2+) rise and MLC phosphorylation. This rapid CPI-17 phosphorylation was dramatically inhibited by either blocking Ca(2+) release from the sarcoplasmic reticulum or by pretreatment with protein kinase C inhibitors, suggesting an involvement of Ca(2+)-dependent protein kinase C. This was followed by a slow Ca(2+)-independent and Rho-kinase/protein kinase C-dependent phosphorylation of CPI-17. In contrast, MYPT1 phosphorylation had only a slow component that increased from 0.29+/-0.09 at rest to the peak of 0.68+/-0.14 mol of Pi/mol at 1 minute, similar to the time course of contraction. Thus, there are 2 components of the Ca(2+) sensitization through inhibition of MLC phosphatase. Our results support the hypothesis that the initial rapid Ca(2+) rise induces a rapid inhibition of MLC phosphatase coincident with the Ca(2+)-induced MLC kinase activation to synergistically initiate a rapid MLC phosphorylation and contraction in arteries with abundant CPI-17 content.     

Aging impairs Ca2+ sensitization pathways in gallbladder smooth muscle

Calcium sensitization is an important physiological process in agonist-induced contraction of smooth muscle. In brief, calcium sensitization is a pathway that leads to smooth muscle contraction independently of changes in [Ca²⁺]_i by mean of inhibition of myosin light chain phosphatase. Aging has negative impacts on gallbladder contractile response due to partial impairment in calcium signaling and alterations in the contractile machinery. However, information regarding aging-induced alterations in calcium sensitization is scanty. We hypothesized that the calcium sensitization system is negatively affected by age. To investigate this, gallbladders were collected from adult (4 months old) and aged (22–24 months old) guinea pigs. To evaluate the contribution of calcium sensitization pathways we assayed the effect of the specific inhibitors Y-27632 and GF109203X on the “in vitro” isometric gallbladder contractions induced by agonist challenges. In addition, expression and phosphorylation (as activation index) of proteins participating in the calcium sensitization pathways were quantified by Western blotting. Aging reduced bethanechol- and cholecystokinin-evoked contractions, an effect associated with a reduction in MLC20 phosphorylation and in the effects of both Y-27632 and GF109203X. In addition, there was a drop in ROCK I, ROCK II, MYPT-1 and PKC expression and in the activation/phosphorylation of MYPT-1, PKC and CPI-17 in response to agonists. Interestingly, melatonin treatment for 4 weeks restored gallbladder contractile responses due to re-establishment of calcium sensitization pathways. These results demonstrate that age-related gallbladder hypocontractility is associated to alterations of calcium sensitization pathways and that melatonin treatment exerts beneficial effects in the recovery of gallbladder contractility.     

Calmodulin stimulation of 45Ca2+ transport and protein phosphorylation in cholinergic synaptic vesicles.

Cholinergic synaptic vesicles isolated from the electric organ of Torpedo californica exhibit ATP-dependent uptake of 45Ca2+ that is stimulated by exogenous calmodulin. ATP-independent uptake also occurs, but it is only weakly stimulated by calmodulin. Saturating calmodulin decreased the Michaelis constant for ATP-dependent 45Ca2+ uptake from 52 +/- 0.4 to 12 +/- 0.2 microM and increased the maximal velocity from 3.4 +/- 0.3 to 5.2 +/- 0.5 nmol/mg of protein per min. The dose-response curve for calmodulin-dependent stimulation showed a maximal increase of 3.5-fold in the uptake rate; 0.2 microM calmodulin gave half-maximal stimulation. The activity of the vesicle-associated ATPase was unaffected. Incubation of vesicles with [gamma-32P]ATP and Ca2+ resulted in phosphorylation of four polypeptides of molecular weights about 64,000, 58,000, 54,000, and 41,000 when calmodulin was added. Vesicles that were previously phosphorylated and purified exhibited 2-fold enhanced ATP-independent uptake of 45Ca2+. Cyclic AMP could not substitute for calmodulin. The calcium transport system of the cholinergic synaptic vesicle is regulated by a calcicalmodulin-dependent protein kinase that is vesicle-associated.     

Ionic mechanisms and Ca2+ handling in airway smooth muscle.

Asthma is a disease characterised by reversible contraction of airway smooth muscle. Many signalling pathways are now known to underlie that contraction, almost all of which revolve around Ca(2+) handling. Ca(2+) homeostasis in turn is governed by a wide variety of ionic mechanisms, which are still poorly understood. The present review will briefly summarise those mechanisms that have been recognised for decades, but will then devote considerable attention to several novel ionic signalling mechanisms such as capacitative Ca(2+) entry, the reverse mode of the Na(+)/Ca(2+) exchanger, the role of Cl(-) channels in the release of internal Ca(2+) and that of ryanodine receptors in the refilling of the sarcoplasmic reticulum, as well as the regulation of the monomeric G-protein Rho by ionic mechanisms. Lastly, evidence will be provided that Ca(2+)-dependent contraction may be driven by spatial and temporal heterogeneities in the intracellular Ca(2+) concentration (i.e. Ca(2+) waves/oscillations) rather than by an increase in the global steady state intracellular Ca(2+) concentration.     

Promoting effects of IL-13 on Ca2+ release and store-operated Ca2+ entry in airway smooth muscle cells

Th2 cytokine interleukin (IL)-13 plays a central role in the pathogenesis of allergic asthma. IL-13 exhibits a direct effect on airway smooth muscle cells (ASMCs) to cause airway hyperresponsiveness. IL-13 has been demonstrated to regulate Ca²⁺ signaling in ASMCs, but the underlying mechanisms are not fully understood. Store-operated Ca²⁺ entry (SOCE) plays an important role in regulating Ca²⁺ signaling and cellular responses of ASMCs, whether IL-13 affects SOCE in ASMCs has not been reported. In this study, by using confocal Ca²⁺ fluorescence imaging, we found that IL-13 (10 ng/ml) treatment increased basal intracellular Ca²⁺ ([Ca²⁺]_i) level, Ca²⁺ release and SOCE induced by SERCA inhibitor thapsigargin in rat bronchial smooth muscle cells. The glucocorticoid dexamethasone and the short-acting β2 adrenergic agonist (β2 agonist) salbutamol suppressed IL-13-augumented basal [Ca²⁺]_i, Ca²⁺ release and SOCE, whereas the long-acting β2 agonist salmeterol had no effect on altered Ca²⁺ signaling in IL-13-treated ASMCs. Membrane-permeable cAMP analog dibutyryl-cAMP (db-cAMP) similarly decreased Ca²⁺ release and SOCE induced by thapsigargin in IL-13-treated ASMCs, confirmed a role of cAMP/PKA signaling pathway in the regulation of SOCE. IL-13 promoted the proliferation of ASMCs stimulated by serum; this effect was inhibited by nonspecific Ca²⁺ channel blockers SKF-96365 and NiCl₂, by salmeterol, but not by salbutamol and dexamethasone. IL-13 treatment did not change the expression of SOC channel-associated molecules STIM1, Orai1 and TRPC1 at mRNA level. Our findings identified a promoting effect of IL-13 on Ca²⁺ release and SOCE in ASMCs, which partially contributes to its effect on the proliferation of ASMCs; the differences of glucocorticoids and β2 agonists in inhibiting Ca²⁺ signal and proliferation potentiated by IL-13 suggest that these therapies of asthma may have distinct effect on the relief of airway contraction and remodeling in bronchial asthma.     

Relationship between Ca2+-affinity and shielding of bulk water in the Ca2+-pump from molecular dynamics simulations

The sarcoplasmic reticulum Ca(2+)-ATPase transports two Ca(2+) per ATP hydrolyzed from the cytoplasm to the lumen against a large concentration gradient. During transport, the pump alters the affinity and accessibility for Ca(2+) by rearrangements of transmembrane helices. In this study, all-atom molecular dynamics simulations were performed for wild-type Ca(2+)-ATPase in the Ca(2+)-bound form and the Gln mutants of Glu771 and Glu908. Both of them contribute only one carboxyl oxygen to site I Ca(2+), but only Glu771Gln completely looses the Ca(2+)-binding ability. The simulations show that: (i) For Glu771Gln, but not Glu908Gln, coordination of Ca(2+) was critically disrupted. (ii) Coordination broke at site II first, although Glu771 and Glu908 only contribute to site I. (iii) A water molecule bound to site I Ca(2+) and hydrogen bonded to Glu771 in wild-type, drastically changed the coordination of Ca(2+) in the mutant. (iv) Water molecules flooded the binding sites from the lumenal side. (v) The side chain conformation of Ile775, located at the head of a hydrophobic cluster near the lumenal surface, appears critical for keeping out bulk water. Thus the simulations highlight the importance of the water molecule bound to site I Ca(2+) and point to a strong relationship between Ca(2+)-coordination and shielding of bulk water, providing insights into the mechanism of gating of ion pathways in cation pumps.     

Regulation of airway smooth muscle cell contractility by Ca2+ signaling and sensitivity

Airway smooth muscle cell contraction is regulated by changes in intracellular Ca2+ concentration ([Ca2+]i) and the responsiveness of the airway smooth muscle cell to this Ca2+. The mechanism controlling [Ca2+]i primarily involves agonist-induced release of Ca2+ from internal stores to generate Ca2+ oscillations. The extent of contraction correlates with the persistence and frequency of these Ca2+ oscillations. The maintenance of the Ca2+ oscillations requires Ca2+ influx, but membrane depolarization appears to have a minor role in initiating or sustaining contraction. Contraction also requires agonist-induced Ca2+ sensitization, which is mediated mainly by decreases in myosin light-chain phosphatase activity. Although it is not clear if airway hyperresponsiveness associated with asthma results from the specific modulation of these Ca2+-based regulatory mechanisms, bronchodilators relax airways by both attenuating the Ca2+ oscillations and by decreasing the Ca2+ sensitivity.     

Angiotensin II activates intermediate-conductance Ca2+ -activated K+ channels in arterial smooth muscle cells

Angiostensin II (Ang II) regulates the migration and proliferation of vascular smooth muscle cells. Recent studies indicate that intermediate-conductance Ca2+ -activated K+ (IKca) channels have an important role in cell migration and proliferation. It is not known, however, whether the action of Ang II is linked to IKca channel regulation. Here, we investigated the modulation of IKca channels by Ang II in artery smooth muscle cells. Functional IKca channel expression in cultured embryonic rat aorta smooth muscle (A10) cells was studied using the patch-clamp technique. These cells predominantly express IKca channels. In contrast, large-conductance Ca2+ -activated K+ (BKca) currents were rarely observed in excised patches. Ang II increased the IKca current in a contration-dependent manner. Losartan (1.0 microM), an AT1 selective antagonist, abolished the activation of IKca channels by Ang II. Pretreatment with 100 microM myristoylated protein kinase C inhibitor peptide 20-28 or 10 microM GF109203X completely abolished the AngII-induced activation of IKca currents, whereas the action of Ang II was not prevented in the presence of 100 microM Rp-cyclic 3', 5'-hydrogen phosphotiate adenosine triethylammonium, a protein kinase A inhibitor, or 1.0 microM KT-5823, a protein kinase G inhibitor. A membrane permeant analogue of diacylglycerol 1, 2-dioctanoyl-sn-glycerol (10 microM) induced the activation of IKca currents. These data suggest that Ang II activates IKca channels through the activation of protein kinase C, and the AT1 receptor is involved in the regulation of these channels.     

Actions of Ca2+ antagonists on two types of Ca2+ channels in rat aorta smooth muscle cells in primary culture

Mechanisms of blockade of two types of Ca2+ channels by the organic Ca2+ antagonists, nicardipine, diltiazem, verapamil, and flunarizine, were examined in rat aorta smooth muscle cells in primary culture by using the whole-cell voltage-clamp method. T-type Ca2+ current (T-type ICa) was isolated by an internal perfusion of 5 mM F-, which irreversibly suppressed the L-type ICa, without affecting T-type ICa. L-type ICa was isolated by setting a holding potential at -60 mV, at which most of the T-type Ca2+ channels were inactivated. L-type ICa is halved by 0.1 microM nicardipine, 3.0 microM diltiazem, 0.6 microM verapamil, and 0.1 microM flunarizine, whereas T-type ICa is halved by the same drugs at 0.6, 30, 30, and 0.1 microM, respectively. Diltiazem and verapamil accelerated the decay of L-type ICa and cumulatively blocked L-type ICa during repetitive step depolarizations elicited every 30 seconds ("use-dependent block"). Diltiazem and verapamil neither changed the decay of T-type ICa nor showed a use-dependent block of T-type ICa. Nicardipine and flunarizine blocked both L- and T-type ICa from the first depolarization step after drug treatment ("tonic block") and shifted their steady-state inactivation curves to the left. The estimated binding constants of nicardipine and flunarizine for the inactivated state of T-type Ca2+ channels (48 and 19 nM, respectively) were smaller than those for the resting state of L-type Ca2+ channels (160 and 90 nM, respectively). A low concentration (0.1 microM) of nicardipine initially potentiated T-type ICa and then reduced it. We conclude from these results that 1) nicardipine and flunarizine block not only the resting state but, more preferentially, the inactivated state of both the L- and T-type Ca2+ channels; 2) verapamil and diltiazem preferentially act on the open state of the L-type Ca2+ channel and on the resting and inactivated state of the T-type Ca2+ channel; and 3) the T-type Ca2+ channel of the rat aorta smooth muscle cells appears to be more sensitive to nicardipine and flunarizine than does the L-type Ca2+ channel at around the resting membrane potential.     

Peroxynitrite inhibits Ca2+-activated K+ channel activity in smooth muscle of human coronary arterioles

We examined the hypothesis that ONOO-, a product of the interaction between superoxide (O2*-) and nitric oxide (NO), inhibits calcium-activated K+ (KCa) channel activity in vascular smooth muscle cells (VSMCs) of human coronary arterioles (HCAs), thereby reducing hyperpolarization-mediated vasodilation. HCAs were dissected from right atrial appendages. The interaction of ONOO- with microvessels was determined by immunohistochemistry using a nitrotyrosine antibody. Strong staining was observed in arteries exposed to authentic ONOO- or to sodium nitroprusside (SNP)+xanthine (XA)+xanthine oxidase (XO). Dilation to 10(-8) mol/L bradykinin (BK) was abolished in vessels exposed to ONOO- (-2.5+/-8%; P<0.05) but not DC-ONOO- (65+/-8%). Reduced dilation to BK was also observed after application of XO and SNP. Dilation to NS1619 (KCa channel opener) was reduced in endothelial denuded arterioles treated with ONOO-. In isolated VSMCs, whole-cell peak K+ current density was reduced by ONOO- (control 65+/-15 pA/pF; ONOO- 42+/-9 pA/pF; P<0.05). Iberiotoxin had no further effect on whole-cell K+ current. In inside-out patches, ONOO- but not DC-ONOO- decreased open state probability (NP(o)) of KCa channel by 50+/-12%. O2*- generated by XA+XO had no effect on BK-induced dilation and NP(o) of KCa channels. These results suggest that ONOO-, but not O2*-, inhibits KCa channel activity in VSMCs possibly by a direct effect. This mechanism may contribute to impaired EDHF-mediated dilation in conditions such as ischemia/reperfusion where increased activity of NO synthase occurs in the presence of excess of O2*-.     

Cytosolic Ca2+ attenuates ANF-induced cyclic GMP response in vascular smooth muscle cells

Despite a high density of atrial natriuretic factor (ANF) receptors, cultured vascular smooth muscle cells of the spontaneously hypertensive rat (SHR) manifest a blunted cyclic GMP (cGMP) response to ANF. We explored the role of cytosolic free Ca2+ ([Ca2+]i) in the ANF-induced cGMP response of cultured aortic vascular smooth muscle cells from SHR and two normotensive rat strains: Wistar-Kyoto (WKY) and American Wistar. Exposure to 500 nmol/l A23187 in Ca2+-containing but not in Ca2+-deficient medium resulted in a decline in the ANF-induced cGMP response at maximal ANF concentration (500 nmol/l; SHR from 1004 +/- 98 to 423 +/- 67, P less than 0.001; WKY from 1791 +/- 209 to 625 +/- 90, P less than 0.001; American Wistar from 1496 +/- 125 to 559 +/- 96 fmol/10(6) cells/4 min, P less than 0.001). The same phenomenon was observed by depolarization with 50 mmol/l KCl in Ca2+-containing medium. There were no significant differences among the rat strains in basal levels of [Ca2+]i. If Ca2+ plays a role in the blunted cGMP response to ANF in vascular smooth muscle cells of the SHR, this effect may be exerted by a distinct pool of the ion in the submembrane domain which is associated with the particulate guanylate cyclase system.     

Evidence for a Ca(2+)-gated ryanodine-sensitive Ca2+ release channel in visceral smooth muscle.

Although a role for the ryanodine receptor (RyR) in Ca2+ signaling in smooth muscle has been inferred, direct information on the biochemical and functional properties of the receptor has been largely lacking. Studies were thus carried out to purify and characterize the RyR in stomach smooth muscle cells from the toad Bufo marinus. Intracellular Ca2+ measurements with the Ca(2+)-sensitive fluorescent indicator fura-2 under voltage clamp indicated the presence of a caffeine- and ryanodine-sensitive internal store for Ca2+ in these cells. The (CHAPS)-solubilized, [3H]ryanodine-labeled RyR of toad smooth muscle was partially purified from microsomal membranes by rate density centrifugation as a 30-S protein complex. SDS/PAGE indicated the comigration of a high molecular weight polypeptide with the peak attributed to 30-S RyR, which had a mobility similar to the cardiac RyR and on immunoblots cross-reacted with a monoclonal antibody to the canine cardiac RyR. Following planar lipid bilayer reconstitution of 30-S stomach muscle RyR fractions, single-channel currents (830 pS with 250 mM K+ as the permeant ion) were observed that were activated by Ca2+ and modified by ryanodine. In vesicle-45Ca2+ efflux measurements, the toad channel was activated to a greater extent at 100-1000 microM than 1-10 microM Ca2+. These results suggest that toad stomach muscle contains a ryanodine-sensitive Ca2+ release channel with properties similar but not identical to those of the mammalian skeletal and cardiac Ca(2+)-release channels.     

Acetylcholine increases voltage-activated Ca2+ current in freshly dissociated smooth muscle cells.

The regulation of voltage-activated Ca2+ current by acetylcholine was studied in single freshly dissociated smooth muscle cells from the stomach of the toad Bufo marinus by using the tight-seal whole-cell recording technique. Ca2+ currents were elicited by positive-going command pulses from a holding level near -80 mV in the presence of internal Cs+ to block outward K+ currents. Ca2+ current was greatest in magnitude at command potentials near 10 mV. At such command potentials, acetylcholine increased the magnitude of the inward current and slowed its decay. The effects of acetylcholine were seen in the absence of external Na+ or with low Cl- (aspartate replacement) in the bathing solution and could be mimicked by muscarine. The peak of the current-voltage relationship for the Ca2+ current was not discernibly shifted along the voltage axis by acetylcholine. These results demonstrate that activation of muscarinic receptors not only suppresses a K+ current (M-current), as we have previously demonstrated [Sims, S. M., Singer, J. J. & Walsh, J. V., Jr. (1985) J. Physiol. (London) 367, 503-529], but also increases the magnitude and slows the decay of Ca2+ current.     

Salt-sensitive hypertension, Na+/Ca2+ exchanger, and vascular smooth muscle

Hypertension is the most common chronic disease and is the leading risk factor for death caused by stroke, myocardial infarction, and end-stage renal failure. The critical importance of excess salt intake in the pathogenesis of hypertension is widely recognized. However, the molecular mechanisms underlying salt-sensitive hypertension remain obscure. Recent studies using selective Na(+)/Ca(2+) exchanger (NCX) inhibitors and genetically engineered mice provide compelling evidence that salt-sensitive hypertension is triggered by Ca(2+) entry through NCX type 1 (NCX1) in arterial smooth muscle. Cardiotonic steroids, such as endogenous ouabain, which may contribute to the pathogenesis of salt-sensitive hypertension, seem to be necessary for NCX1-mediated hypertension. These findings have enabled us to explain how high salt intake leads to hypertension and further to describe the potential of vascular NCX1 as a new therapeutic or diagnostic target for salt-sensitive hypertension.