Ca2+-dependent and Ca2+-independent regulation of smooth muscle contraction

An increase in the cytosolic Ca²⁺ concentration is a prerequisite in activation of contractile activity of smooth muscle. The shape of the Ca²⁺-signal is determined by spatial distribution and kinetics of Ca²⁺-binding sites in the cell. The increase in cytosolic Ca²⁺ activates myosin light chain kinase (MLCK) which in turn phosphorylates the regulatory light chains of myosin II. This Ca²⁺-dependent MLC₂₀ phosphorylation is modulated in a Ca²⁺-independent manner by inhibiting the constitutive active myosin light chain phosphatase mediated by the monomeric GTPase Rho and the Rho-associated kinase as well as protein kinase C or by increasing its activity through cGMP. Furthermore, the activity of MLCK may be decreased due to phosphorylation by CaM kinase II and perhaps p21 activated protein kinase. Hence, smooth muscle tone appears to be regulated by a network of activating and inactivating intracellular signaling cascades which not only show a temporal but also a spatial activation pattern.     

Bradykinin elevates cytosolic Ca2+ concentration in smooth muscle cells isolated from rat duodenum

The effect of bradykinin on the cytosolic Ca2+ concentration were measured in single, Fura-2 loaded, smooth muscle cells isolated from rat duodenum. All cells responded with a Ca2+ signal when exposed to bradykinin. The bradykinin response consisted of an initial Ca2+ spike followed by a plateau. Pre-treatment of single muscle cells with either the phospholipase C blocker U-73122 or thapsigargin, which is a potent inhibitor of the endoplasmic reticulum Ca2+-ATPase, inhibited the response to bradykinin. Pre-treatment of the cells with EGTA or La3+ to inhibit the Ca2+ influx, abolished the response induced by bradykinin. We conclude that bradykinin applied to single smooth muscle cells from rat duodenum, increases cytosolic Ca2+ by emptying intracellular Ca2+ stores, and by contribution from extracellular Ca2+. In contrast to bradykinin-induced response in isolated rat duodenum (a relaxation followed by a contraction), we did not observe a biphasic effect of bradykinin on cytosolic Ca2+ in single muscle cells. Bradykinin may thus cause relaxation of duodenal smooth muscle indirectly through an effect on neighbouring cells as dilatation is brought about by this agent in blood vessels.     

Role of Ca2+ and myosin light chain phosphorylation in regulation of smooth muscle

The time course of phosphorylation of the 20,000-dalton myosin light chain (LC 20) was determined during contraction and relaxation in K+- and histamine-stimulated medial strips of swine carotid arteries. Resting LC 20 phosphorylation levels of 0.15 mol P/mol LC 20 rapidly increased to peak values of 0.6-0.7 mol P/mol LC 20 after stimulation and then declined significantly, although stress continued to rise to a stable steady-state maximum. LC 20 dephosphorylation after agonist washout preceded the decline in isometric stress. Over the entire contraction-relaxation cycle, phosphorylation was correlated with shortening velocity and not with developed stress. The maximum shortening velocity with no external load (Vo) was directly proportional to LC 20 phosphorylation (r = 0.986). The data indicate that LC 20 phosphorylation is necessary for cross-bridge cycling leading to shortening or stress development but that stress can be maintained by additional mechanisms. We suggest that dephosphorylation of an attached cross bridge in the presence of Ca2+ arrests the cycle, forming an attached, noncycling cross bridge.     

Regulation of the mitochondrial proton gradient by cytosolic Ca2+ signals

Mitochondria convert the energy stored in carbohydrate and fat into ATP molecules that power enzymatic reactions within cells, and this process influences cellular calcium signals in several ways. By providing ATP to calcium pumps at the plasma and intracellular membranes, mitochondria power the calcium gradients that drive the release of Ca²⁺ from stores and the entry of Ca²⁺ across plasma membrane channels. By taking up and subsequently releasing calcium ions, mitochondria determine the spatiotemporal profile of cellular Ca²⁺ signals and the activity of Ca²⁺-regulated proteins, including Ca²⁺ entry channels that are themselves part of the Ca²⁺ circuitry. Ca²⁺ elevations in the mitochondrial matrix, in turn, activate Ca²⁺-dependent enzymes that boost the respiratory chain, increasing the ability of mitochondria to buffer calcium ions. Mitochondria are able to encode and decode Ca²⁺ signals because the respiratory chain generates an electrochemical gradient for protons across the inner mitochondrial membrane. This proton motive force (??p) drives the activity of the ATP synthase and has both an electrical component, the mitochondrial membrane potential (???? _m ), and a chemical component, the mitochondrial proton gradient (??pH _m ). ???? _m contributes about 190?mV to ??p and drives the entry of Ca²⁺ across a recently identified Ca²⁺-selective channel known as the mitochondrial Ca²⁺ uniporter. ??pH _m contributes ~30?mV to ??p and is usually ignored or considered a minor component of mitochondria respiratory state. However, the mitochondrial proton gradient is an essential component of the chemiosmotic theory formulated by Peter Mitchell in 1961 as ??pH _m sustains the entry of substrates and metabolites required for the activity of the respiratory chain and drives the activity of electroneutral ion exchangers that allow mitochondria to maintain their osmolarity and volume. In this review, we summarize the mechanisms that regulate the mitochondrial proton gradient and discuss how thermodynamic concepts derived from measurements in purified mitochondria can be reconciled with our recent findings that mitochondria have high proton permeability in situ and that ??pH _m decreases during mitochondrial Ca²⁺ elevations.     

Temperature- and Mg-ATP-dependent regulation of Ca2+ sensitivity of smooth muscle actomyosin ATPase.

Many smooth muscles on metabolic depletion undergo a contraction that is insensitive to EGTA [ethylene glycol-bis (beta-aminoethylether)N,N-tetraacetic acid]. Chicken gizzard actomyosin shows a progressive loss of Ca sensitivity accompanied by activation of EGTA-Mg-ATPase at temperatures near 37 degrees C with decreasing ATP concentrations. Ca2+-dependent phosphorylation still occurs under these conditions when the ATPase is Ca insensitive. Activation of EGTA-Mg-ATPase at low ATP concentration is not due to a pseudo-ATPase, or due to denautration of the actomyosin at 37 degrees C. Magnesium concentrations above 1 mM are required for observing the enhanced EGTA-Mg-ATPase activity and the Ca sensitivity is very markedly influenced by the magnesium concentrations of medium at low ATP. When the Mg-to-ATP ratio (5:1) was kept constant for varying ATP concentrations, activation of EGTA-ATPase was not observed. This activation was not due to the characteristics of the ATP regenerating system (phosphoenolpyruvate and pyruvate kinase) because with phosphocreatine and creatine phosphokinase similar results were obtained. Thus the EGTA-insensitive rise in tension during metabolic depletion is due to activation of Mg-ATPase and loss of Ca sensitivity at 37 degrees C, a temperature at which mammalian smooth muscles normally function.     

Influence of extracellular Na+ removal on cytosolic Ca2+ concentration in smooth muscle cells of rabbit cerebral artery.

There are some controversies over the contribution of Na+/Ca2+ exchanger (NCX) to the regulation of cytosolic Ca2+ concentration ([Ca2+]c) in smooth muscle. To prove the functional role of Na+/Ca2+ exchanger, we examined whether the removal of extracelluar Na+ could affect [Ca2+]c of rabbit cerebral arterial smooth muscle. The fluorescence ratio of fura-2 (R(340/380)) was measured in the single myocyte of rabbit middle cerebral artery and Na+ was substituted with the same concentration of NMDG+ or Li+. In 21 out of 230 cells tested, Na+ removal increased R(340,380) (deltaR(340/380)) by 115 +/- 16.5% of the deltaR(340/380) induced by 10 mM caffeine in the same cell. The Na+ removal-induced deltaR(340/380) was blocked by a selective inhibitor of cardiac type NCX exchanger (KB-R7943, (2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea, 10 microM). In those cells where the Na+ removal by itself did not increase R(340/380), the caffeine-induced deltaR(340/380) was increased by Na+-removal (130 +/- 9.8% of control response, n=30). Under the whole-cell patch clamp condition, short application of caffeine induced transient increase of outward current (I(K,Ca)-transient) which reflect the change of subsarcolemmal [Ca2+]. The application of KB-R7943 increased the amplitude of I(K,Ca)-transient (n=4). These results suggest the functional existence of NCX in rabbit cerebral artery smooth muscle.     

The regulation of cytosolic Ca2+ concentration in a mammalian cell

The assumptions governing the regulation of free Ca2+ in a mammalian cell are presented. There is a possibility that mitochondria, microsomes and plasma membrane play a major role, by regulation of the very low concentration of free Ca2+ in cytosol, as the signal to biochemical activities. The possibility of the loss of very high-affinity Ca2(+)-binding and the number of its sites by preparation has not been taken into consideration in previous hypotheses.     

Inositol 1,4,5-trisphosphate-induced Ca2+ release is regulated by cytosolic Ca2+ in intact skeletal muscle

Microinjection of inositol 1,4,5-trisphosphate (InsP ₃) into intact skeletal muscle fibers isolated from frogs (Rana temporaria) increased resting cytosolic Ca²⁺ concentration ([Ca²⁺]_i) as measured by double-barreled Ca²⁺-selective microelectrodes. In contrast, microinjection of inositol 1-phosphate, inositol 1,4-biphosphate, and inositol 1,4,5,6-tetrakisphosphate did not induce changes in [Ca²⁺]_i. Incubation in low-Ca²⁺ solution, or in the presence of L-type Ca²⁺ channel blockers did not affect InsP ₃-induced release of cytosolic Ca²⁺. Neither ruthenium red, a blocker of ryanodine receptor Ca²⁺-release channels, nor cytosolic Mg²⁺, a known inhibitor of the Ca²⁺-induced Ca²⁺-release process, modified the InsP ₃-induced release of cytosolic Ca²⁺. However, heparin, a blocker of InsP ₃ receptors, inhibited InsP ₃-induced release of cytosolic Ca²⁺. Also, pretreatment with dantrolene or azumulene, two inhibitors of cytosolic Ca²⁺ release, reduced [Ca²⁺]_i, and prevented InsP ₃ from inducing release of cytosolic Ca²⁺. Incubation in caffeine or lengthening of the muscle increased [Ca²⁺]_i and enhanced the ability of InsP ₃ to induce release of cytosolic Ca²⁺. These results indicate that InsP ₃, at physiological concentrations, induces Ca²⁺ release in intact muscle fibers, and suggest that the InsP ₃-induced Ca²⁺ release is regulated by [Ca²⁺]_i. A Ca²⁺-dependent effect of InsP ₃ on cytosolic Ca²⁺ release could be of importance under physiological or pathophysiological conditions associated with alterations in cytosolic Ca²⁺ homeostasis. Received: 15 December 1995/Received after revision and accepted: 10 May 1996     

Changes in cytosolic Ca2+ and contraction induced by various stimulants and relaxants in canine tracheal smooth muscle

Effects of stimulants and relaxants on the cytosolic Ca²⁺ level ([Ca²⁺]_cyt) and contraction were examined in isolated canine tracheal smooth muscle. High K⁺ and carbachol induced a sustained increase in [Ca²⁺]_cyt and muscle tension. Cumulative addition of KCl induced a graded increase in [Ca²⁺]_cyt and muscle tension. Cumulative addition of carbachol induced greater contraction than high K⁺ at a given [Ca²⁺]_cyt. 12-Deoxyphorbol 13-isobutyrate (DPB) (50 nmol/l) induced a small sustained contraction with little effect on [Ca²⁺]_cyt. A higher concentration (1 mol/l) of DPB induced a larger sustained contraction with a decrease in [Ca²⁺]_cyt. DPB (50 nmol/l) potentiated the KCl-induced contraction without or with only a small additional increase in [Ca²⁺]_cyt. By contrast, 1 mol/l DPB potentiated the high-K⁺-induced contraction with a decrease in [Ca²⁺]_cyt. Addition of 50 nmol/l or 1 mol/l DPB in the presence of carbachol inhibited both [Ca²⁺]_cyt and muscle tension. Verapamil, isoprenaline and forskolin did not change or slightly decreased [Ca²⁺]_cyt and muscle tension in resting trachea. Verapamil inhibited the contrction and [Ca²⁺]_cyt stimulated by high K⁺ and carbachol. Isoprenaline and forskolin inhibited the high-K⁺-induced contraction without changing [Ca²⁺]_cyt, whercas these inhibitors inhibited carbachol-induced contraction with a relatively small decrease in [Ca²⁺]_cyt. These results suggest that (a) sustained contractions induced by high K⁺ and carbachol are due to the sustained increase in [Ca²⁺]_cyt, (b) carbachol increases the sensitivity of contractile elements to Ca²⁺, and (c) isoprenaline and forskolin inhibit the contraction by the decrease in [Ca²⁺]_cyt and also by the decrease in the sensitivity of contractile elements to Ca²⁺. The carbachol-induced Ca²⁺ sensitization might be attributable to the activation of protein kinase C following the stimulation of the phosphatidylinositol turnover.     

Mechanism of the ATP-induced rise in cytosolic Ca2+ in freshly isolated smooth muscle cells from human saphenous vein

Effects of exogenous adenosine 5-triphosphate (ATP) were studied by measurements of intracellular Ca²⁺ concentration ([Ca²⁺]_i) and membrane currents in myocytes freshly isolated from the human saphenous vein. At a holding potential of –60 mV, ATP (10 M) elicited a transient inward current and increased [Ca²⁺]_i. These effects of ATP were inhibited by ,-methylene adenosine 5-triphosphate (AMPCPP, 10 M). The ATP-gated current corresponded to a non-selective cation conductance allowing Ca²⁺ entry. The ATP-induced [Ca²⁺]_i rise was abolished in Ca²⁺-free solution and was reduced to 30.1±5.5% (n=14) of the control response when ATP was applied immediately after caffeine, and to 23.7±3.8% (n=11) in the presence of thapsigargin. The Ca²⁺-induced Ca²⁺ release blocker tetracaine inhibited the rise in [Ca²⁺]_i induced by both caffeine and ATP, with apparent inhibitory constants of 70 M and 100 M, respectively. Of the ATP-induced increase in [Ca²⁺]_i 29.3±3.9% (n=8) was tetracaine resistant. It is concluded that the effects of ATP in human saphenous vein myocytes are only mediated by activation of P_2x receptor channels. The ATP-induced [Ca²⁺]_i rise is due to both Ca²⁺ entry and Ca²⁺ release activated by Ca²⁺ ions that enter the cell through P_2x receptor channels.     

Mitochondria contribute to Ca2+ removal in smooth muscle cells

Recent evidence, from a variety of cell types, suggests that mitochondria play an important role in shaping the change in intracellular calcium concentration ([Ca²⁺]_i,) that occurs during physiological stimulation. In the present study, using a range of inhibitors of mitochondrial Ca²⁺ uptake, we have examined the contribution of mitochondria to Ca²⁺ removal from the cytosol of smooth muscle cells following stimulation. In voltage-clamped single smooth muscle cells, we found that following a 8-s train of depolarizing pulses, the rate of Ca²⁺ extrusion from the cytosol was reduced by more than 50% by inhibitors of cytochrome oxidase or exposure of cells to the protonophore carbonyl cyanideP-trifluoromethoxy-phenylhydrazone. Using the potential-sensitive indicator tetramethyl rhodamine ethyl ester, we confirmed that the effect of these agents was associated with depolarization of the mitochondrial membrane. Since, the primary function of the mitochondria is to provide the cell's ATP, it could be argued that it is the ATP supply to the ion pumps which is limiting the rate of Ca²⁺ removal. However, experiments carried out with the mitochondrial Ca²⁺ uniporter inhibitor ruthenium red produced similar results, while the ATP synthetase inhibitor oligomycin had no effect, suggesting that the effect was not due to ATP insufficiency. These results establish that mitochondria in smooth muscle cells play a significant role in removing Ca²⁺ from the cytosol following stimulation. The uptake of Ca²⁺ into mitochondria is proposed to stimulate mitochondrial ATP production, thereby providing a means for matching increased energy demand, following the cell's rise in [Ca²⁺]_i;, with increased cellular ATP production.     

Multiple types of Ca2+ channels in visceral smooth muscle cells

Single-channel currents were recorded from two classes of Ca²⁺ channels in visceral smooth muscle cells isolated from the stomach of the toad, Bufo marinus: a class of small-conductance channels (approximately 11 pS) and a class of large-conductance channels (approximately 26 pS). Small-conductance channels were present in a majority of patches and gave rise to a slowly inactivating current (t_1/2 250 ms at 0 mV). Openings of large-conductance channels could be unequivocally resolved only in the presence of the dihydropyridine Ca²⁺ agonist Bay K 8644. Two subtypes of the large-conductance channels were found — those with a very slow rate of decay (> 500 ms) and those with a faster one (< 100ms). Large-conductance channels resemble L-type Ca²⁺ channels of other preparations. Small-conductance channels do not fit unambiguously into the other existing categories (i.e., N or T). Correspondence between single-channel and macroscopic Ca²⁺ currents is discussed.     

OX-LDL及辛伐他汀对平滑肌细胞PKC活性和胞内Ca2+的影响

目的:探讨OX-LDL是否对大鼠主动脉平滑肌细胞(ASMC)蛋白激酶C(PKC)活性和胞浆内游离钙([Ca²⁺]i)水平有影响。方法:应用γ-[³²P]-ATP磷酸转移法和Fluo-3/Am荧光负载、流式细胞术分别检测PKC活性和胞内([Ca²⁺]i)水平。结果:OX-LDL呈剂量依赖方式促进ASMCPKC总活性增加,并可引起ASMC中PKC发生浆膜的转移。胞浆内[Ca²⁺]i以2个时相升高,即快速相和持续相。而辛伐他汀能明显抑制OX-LDL引起的ASMC中PKC活性的浆膜转移,并显著降低持续相胞浆内钙水平,而对快速相无影响。结论:OX-LDL能引起ASMC内信号通路PKC及[Ca²⁺]i的动态变化,二者密切相关。     

Vascular smooth muscle: The first recorded Ca2+ transients

The bioluminescent calcium indicator aequor in was successfully loaded into vascular smooth muscle cells ofAmphiuma tridactylum by either microinjection or a new method which makes the cells reversibly hyperpermeable. Both gave similar results; however, the latter method produced larger signals. Vasoconstrictors produced a sustained contraction and a light (calcium) response consisting of two component: a large transient followed by a smaller, sustained response. Electrical stimulation produced a light transient that was much briefer than the contraction. These results suggest that tension can be maintained in smooth muscle in the presence of lower calcium levels than those present during force development.     

Functional characterization of the Ca2+-gated Ca2+ release channel of vascular smooth muscle sarcoplasmic reticulum

The Ca²⁺-gated Ca²⁺ release channel of aortic sarcoplasmic reticulum (SR) was partially purified and reconstituted into planar lipid bilayers. Canine and porcine aorta microsomal protein fractions were solubilized in the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulphonate (CHAPS) in the presence and absence of ³[H]-ryanodine and centrifuged through linear sucrose gradients. A single ³[H]-ryanodine receptor peak with an apparent sedimentation coefficient of 30 s was obtained. Upon reconstitution into planar lipid bilayers, the unlabelled 30 s protein fraction induced the formation of a Ca²⁺- and monovalent-ion-conducting channel (110 pS in 100 mM Ca²⁺, 360 pS in 250 mM K⁺). The channel was activated by micromolar Ca²⁺, modulated by millimolar adenosine triphosphate, Mg²⁺ and the Ca²⁺-releasing drug caffeine, and inhibited by micromolar ruthenium red. Micro- to millimolar concentrations of the plant alkaloid ryanodine induced a permanently closed state of the channel. Our results suggest that smooth muscle SR contains a Ca²⁺-gated Ca²⁺ release pathway, with properties similar to those observed for the skeletal and cardiac ryanodine receptor/Ca²⁺ release channel complexes.     

Magnesium regulation of Ca2+ channels in smooth muscle and endothelial cells of human allantochorial placental vessels.

In human allantochorial placental vessels, the vascular tone is regulated by membrane potential controlled by the ion flux through K+ and Ca2+ channels. The effects of MgCl2 and MgSO4 were studied on the membrane potential of vascular smooth muscle cells (VSMCs) and of endothelial cells (VECs). The membrane potential was the main factor of the excitation-contraction coupling of placental vessels. The VSMCs and VECs were predepolarized by high external K+, which blocked voltage-sensitive K+ channels, and depolarized by serotonin addition which activated Ca2+ influx through voltage-gated Ca2+ channels. Addition of MgCl2 or MgSO4 in the external medium induced a depolarization level lower than the previous level, as nifedipine a Ca2+ blocker, corresponding to a Ca2+ influx reduction in VSMCs and VECs and inducing relaxation of the cells. The effect of MgCl2 and MgSO4 was the same on VSMCs and VECs, but the depolarization level reduction was more important with MgCl2 than MgSO4 on VSMCs. These data suggested that Mg salts regulated the Ca2+ influx through voltage-gated Ca2+ channels in VSMCs and VECs and consequently the tonus of human allantochorial placental vessels and that there was a difference between Mg salts.     

Agonist-dependent modulation of Ca2+ sensitivity in rabbit pulmonary artery smooth muscle

The effects of the stable thromboxane analogue U46619, the ₁-adrenergic agent phenylephrine and depolarization with high K⁺ on cytoplasmic Ca²⁺ ([Ca²⁺]_i) and force development were determined in rabbit pulmonary artery smooth muscle. Following stimulation with each of the excitatory agents, the time course of the [Ca²⁺]_i/force relationship described counter-clockwise hysteresis loops with the rise and fall in [Ca²⁺]_i leading, respectively, contraction and relaxation. The rank order of the force/[Ca²⁺]_i ratios evoked by the different methods of stimulation was: U46619 > phenylephrine high K⁺. The difference between the actions of U46619 and phenylephrine was due to the lesser Ca²⁺-releasing and greater Ca²⁺-sensitizing action of U46619. Both U46619 and phenylephrine also released intracellular Ca²⁺ in intact (non-permeabilized) preparations. The effects of the two agonists on force, at constant free cytoplasmic [Ca²⁺] maintained with EGTA, were also determined in preparations permeabilized with staphylococcal -toxin, in which intracellularly stored Ca²⁺ was eliminated with A23187. Sensitization of the contractile response to Ca²⁺ by agonists was indicated by the contractile responses of permeabilized muscles to U46619 and to phenylephrine, in the presence of constant, highly buffered [Ca²⁺]_i. These contractions were inhibited by GDP[S] and could also be elicited by GTP. We conclude that, in addition to changing [Ca²⁺]_i, pharmacomechanical coupling can also modulate contraction by altering the sensitivity of the regulatory/contractile apparatus of smooth muscle to [Ca²⁺]_i, through a G-protein-coupled mechanism.     

Endogenous polyamines modulate Ca2+ channel activity in guinea-pig intestinal smooth muscle

The polyamines spermine and spermidine inhibit L-type Ca2+ channels in whole-cell recordings from guinea-pig ileum cells (Gomez and Hellstrand, Pflügers Arch, 430:501-507, 1995 [4]). To study whether they modulate channel activity under physiological conditions, we further investigated their actions on Ca2+ channels and the effects of altered cellular polyamine contents. In inside-out patches, spermine (0.1-1 mM) inhibited channel activity without affecting the amplitude of unitary currents. In cell-attached recordings, addition of spermine to the bath did not influence channel activity in the patch, indicating that its extracellular action is direct and not mediated via passage of the polyamine through the cell membrane. Cellular contents of spermidine and spermine were decreased by about 50% by organ culture of ileum strips for 5 days with the adenosylmethionine decarboxylase inhibitor CGP 48664 (10 microM). This caused enhanced channel activity in cell-attached recordings, suggesting a reduced level of channel block by endogenous polyamines compared with control cells. Whole-cell recordings in the perforated patch mode showed increased current in polyamine-depleted cells, while this was not seen when cells were dialysed with the pipette solution. We conclude that polyamines block Ca2+ channels from the inside as well as the outside of the cell membrane, and that endogenous polyamines in smooth muscle modulate Ca2+ channel activity.