The effects of isoproterenol on intracellular calcium concentration

beta-Adrenergic agonist, isoproterenol (ISO), is a potent relaxant of tracheal smooth muscle and inhibits carbachol-induced contraction. The effect of ISO on intracellular free Ca2+ concentration ([Ca2+]i) was examined in bovine tracheal smooth muscle strips, employing aequorin as Ca2+ indicator. Surprisingly, 10 microM ISO induces a 5-fold increase in [Ca2+]i which then gradually declines but still remains higher than basal after 1 h of stimulation. The ISO-induced increase in [Ca2+]i is dose-dependent, and the ED50 is approximately 50 nM. The ISO-induced increase in [Ca2+]i is inhibited by a beta-receptor blocker, propranolol, not by an alpha-blocker, phentolamine. The ISO-induced rise in [Ca2+]i is dependent on extracellular Ca2+. Forskolin, an adenylate cyclase activator, and vasoactive intestinal peptide, which is known to stimulate adenylate cyclase via a specific receptor in this tissue, have similar effects on [Ca2+]i, suggesting that a rise in cyclic AMP concentration mediates this effect of ISO on [Ca2+]i. Pretreatment of muscle with 10 microM ISO inhibits both the initial Ca2+ transient and the contractile response induced by 0.3 microM carbachol. Conversely, in carbachol-pretreated muscle strips, addition of ISO causes a fall rather than a rise in [Ca2+]i, and an inhibition of contraction. These results indicate that ISO has effects on cellular Ca2+ metabolism at more than a single site in bovine tracheal smooth muscle, that these effects are different in control and carbachol-pretreated muscle, and that the relaxing effect of ISO is not due solely to its effect on Ca2+ metabolism.     

Genistein inhibits contractile force, intracellular Ca2+ increase and Ca2+ oscillations induced by serotonin in rat aortic smooth muscle

The soy-derived isoflavones genistein and daidzein affect the contractile state of different kinds of smooth muscle. We describe acute effects of genistein and daidzein on contractile force and intracellular Ca2+ concentration ([Ca2+]i) in in situ smooth muscle of rat aorta. Serotonin (5-HT) (2 microM) or a depolarizing high K+ solution produced the contraction of aortic rings, which were immediately relaxed by 20 microM genistein and by 20 microM daidzein. Accordingly, both 5-HT and a high K+ solution increased the [Ca2+]i in in situ smooth muscle cells. Genistein strongly inhibited the [Ca2+]i increase evoked by 5-HT (74.0 +/- 7.3%, n = 11, p < 0.05), and had a smaller effect on high K+ induced [Ca2+]i increase (19.9 +/- 4.0%, n = 7, p < 0.05). The K+ channels blocker tetraethylammonium (TEA) (0.5 mM) diminished genistein effects on 5-HT-induced [Ca2+]i increase. Interestingly, during prolonged application of 5-HT, the [Ca2+]i oscillated and a short (90 s) preincubation with genistein (20 microM) significantly diminished the frequency of the oscillations. This effect was totally abolished by TEA. In conclusion, in rat aortic smooth muscle, genistein is capable of diminishing the increase in [Ca2+]i and in force evoked by 5-HT and high K+ solution, and of decreasing the frequency of [Ca2+]i oscillations induced by 5-HT. The short time required by genistein, and the relaxing effect of daidzein suggest that tyrosine kinases inhibition is not involved. The small inhibiting effect of genistein on the [Ca2+]i increase evoked by high K+ and the effect of TEA point to the activation by genistein of calcium-activated K+ channels.     

Clearance of large Ca2+ loads in a single smooth muscle cell: examination of the role of mitochondrial Ca2+ uptake and intracellular pH

Redistribution of cytosolic free Ca2+ following Ca2+ influx into the cytoplasm was studied in single smooth muscle cells isolated from guinea-pig urinary bladder. Voltage-clamped cells were loaded with a low-affinity fluorophore Indo-1FF. A decay of free intracellular Ca2+ ([Ca2+]i) after the termination of the depolarizing pulse (1 s from -50 mV to +20 mV) was fitted with a single exponential and the effect of various substances on the time constant was compared. At a holding potential of +80 mV the [Ca2+]i decay was 1.56 times slower compared to that at -50 mV suggesting the presence of a voltage-dependent process redistributing Ca2+. In the presence of cyclopiazonic acid (CPA, 10 microM), an inhibitor of sarco(endo)plasmatic Ca2+ pump (SERCa), the [Ca2+]i decay was 3.93 times slower than that in the absence of the inhibitor. Introduction of a polycation Ruthenium Red (RR) (20 microM), an inhibitor of the mitochondrial Ca2+ uniporter, into a cell or collapsing a transmitochondrial H+ gradient with the protonophore CCCP (2 microM) slowed down the [Ca2+]i decay 6.05-fold and 9.78-fold, respectively. The apparent amplitude of [Ca2+]i increments was also increased by CCCP. Increasing H+ buffering power in the intracellular solution from 10 mM to 40 mM of HEPES greatly reduced the effect of CCCP on [Ca2+]i decay. A further increase in HEPES concentration to 100 mM eliminated the effects of CCCP both on the time course of [Ca2+]i decay and on the amplitude of [Ca2+]i increment. Perfusion of RR together with 100 mM HEPES into the cytoplasm was without effect on the decay time course of [Ca2+]i. The effect of CPA on [Ca2+]i decay was also reduced in cells loaded with 100 mM HEPES; the time constant in the presence of CPA was slowed down by a factor of 2.18. Application of 10 mM Na(+)-butyrate to the cells loaded with 10 mM HEPES resulted in a slowing down of [Ca2+]i decay: the time constant was increased by a factor of 5.84. Measurement of intracellular pH with SNARF-1 confirmed cytoplasmic acidification during application of Na(+)-butyrate and CCCP. It is concluded that the contribution of mitochondrial Ca2+ uptake to the rapid [Ca2+]i decay is much less than could be extrapolated from action of protonophores in these smooth muscle cells. The results also demonstrate the importance of intracellular pH for Ca2+ handling in the cytoplasm of smooth muscle cells.     

Ca2+]i changes in guinea pig tracheal smooth muscle cells in culture: effects of Na+ and ouabain

The objective of this work was to confirm that the contractile effects of ouabain and Na(+)-free solutions in guinea pig tracheal rings are associated with increments in the cytosolic free Ca2+ concentration ([Ca2+]i) in cultured tracheal smooth muscle (TSM) cells. Cultured cells were alpha-actin positive. Histamine (50 microM) and Na(+)-free solution elicited a transient increase in [Ca2+]i, while the responses to thapsigargin (1 microM) and ouabain (1 mM) were long lasting. However, carbachol (10, 200, and 500 mM) and high K(+)-solution produced no effect on [Ca2+]i, suggesting that cultured guinea pig TSM cells display a phenotype change but maintain some of the tracheal rings physiological properties. The transient rise in [Ca2+]i in response to the absence of extracellular Na+ and the effect of ouabain may indicate the participation of the Na(+)/Ca2+ exchanger (NCX) in the regulation of [Ca2+]i.     

Magnesium regulates intracellular free ionized calcium concentration and cell geometry in vascular smooth muscle cells.

Regulatory effects of extracellular magnesium ions ([Mg2+]o) on intracellular free ionized calcium ([Ca2+]i) were studied in cultured vascular smooth muscle cells (VSMCs) from rat aorta by use of the fluorescent indicator fura-2 and digital imaging microscopy. With normal Mg2+ (1.2 mM)-containing incubation media, [Ca2+]i in VSMCs was 93.6 +/- 7.93 nM with a heterogeneous cellular distribution. Lowering [Mg2+]o to 0 mM or 0.3 mM (the lowest physiological range) resulted in 5.8-fold (579.5 +/- 39.99 nM) and 3.5-fold (348.0 +/- 31.52 nM) increments of [Ca2+]i, respectively, without influencing the cellular distribution of [Ca2+]i. Surprisingly, [Mg2+]o withdrawal induced changes of cell geometry in many VSMCs, i.e., the cells rounded up. However, elevation of [Mg2+]o up to 4.8 mM only induced slight decrements of [Ca2+]i (mean = 72.0 +/- 4.55 nM). The large increment of [Ca2+]i induced by [Mg2+]o withdrawal was totally inhibited when [Ca2+]o was removed. The data suggest that: (1) [Mg2+]o regulates the level of [Ca2+]i in rat aortic smooth muscle cells, and (2) [Mg2+] acts as an important regulatory ion by modulating cell shapes in cultured VSMc and their metabolism to control vascular contractile activities.     

Decreasing extracellular Na+ concentration triggers inositol polyphosphate production and Ca2+ mobilization

Removing extracellular Na+ (Na+o) evoked a large increase in cytosolic free Ca2+ concentration ([Ca2+]i in human skin fibroblasts. Decreasing [Na+]o from 120 to 14 mM caused the half-maximal peak increase in [Ca2+]i. Removing Na+o strongly stimulated 45Ca2+ efflux and decreased total cell Ca2+ by about 40%. Bradykinin caused changes in [Ca2+]i, total Ca2+, and 45Ca2+ fluxes similar to those evoked by removing Na+o. Prior stimulation of the cells with bradykinin prevented Na+o removal from increasing [Ca2+]i and vice versa. Na+o removal rapidly increased [3H]inositol polyphosphate production. Loading the cells with Na+ had no effect on the increase in 45Ca2+ efflux produced by Na+o removal. Therefore, decreasing [Na+]o probably stimulates a "receptor(s)" which is sensitive to extracellular, not intracellular, Na+. Removing Na+o also mobilized intracellular Ca2+ in smooth muscle and endothelial cells cultured from human umbilical and dog coronary arteries, respectively.     

Fendiline-Induced Ca2+ movement in A10 smooth muscle cells

The effect of fendiline, an anti-anginal drug, on cytosolic free Ca2+ levels ([Ca2+]i) in A10 smooth muscle cells was explored by using fura-2 as a Ca2+ indicator. Fendiline at concentrations between 10-50 microM increased [Ca2+]i in a concentration-dependent manner with an EC50 of 20 microM. External Ca2+ removal reduced the Ca2+ signal by 75%. Addition of 3 mM Ca2+ increased [Ca2+]i in cells pretreated with fendiline in Ca2+-free medium. The 50 microM fendiline-induced [Ca2+]i increase in Ca2+-containing medium was inhibited by 10 microM of La3+, nifedipine, or verapamil. In Ca2+-free medium, pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor) to deplete the endoplasmic reticulum Ca2+ store partly inhibited 50 microM fendiline-induced Ca2+ release; whereas pretreatment with 50 microM fendiline abolished 1 microM thapsigargin-induced Ca2+ release. Inhibition of phospholipase C activity with 2 microM U73122 did not alter 50 microM fendiline-induced Ca2+ release. Incubation with 50 microM fendiline for 10-30 min decreased cell viability by 10-20%. Together, the findings indicate that in smooth muscle cells fendiline induced [Ca2+]i increases. Fendiline acted by activating Ca2+ influx via L-type Ca2+ channels, and by releasing internal Ca2+ in a phospholipase C-independent manner. Prolonged exposure of cells to fendiline induced cell death.     

Cellular mechanisms involved in iso-osmotic high K+ solutions-induced contraction of the estrogen-primed rat myometrium

The aim of the present study was to investigate the mechanisms involved in the contraction evoked by iso-osmotic high K+ solutions in the estrogen-primed rat uterus. In Ca2+-containing solution, iso-osmotic addition of KCl (30, 60 or 90 mM K+) induced a rapid, phasic contraction followed by a prolonged sustained plateau (tonic component) of smaller amplitude. The KCl (60 mM)-induced contraction was unaffected by tetrodotoxin (3 microM), omega-conotoxin MVIIC (1 microM), GF 109203X (1 microM) or calphostin C (3 microM) but was markedly reduced by tissue treatment with neomycin (1 mM), mepacrine (10 microM) or U-73122 (10 microM). Nifedipine (0.01-0.1 microM) was significantly more effective as an inhibitor of the tonic component than of the phasic component. After 60 min incubation in Ca2+-free solution containing 3 mM EGTA, iso-osmotic KCl did not cause any increase in tension but potentiated contractions evoked by oxytocin (1 microM), sodium orthovanadate (160 micrM) or okadaic acid (20 microM) in these experimental conditions. In freshly dispersed myometrial cells maintained in Ca2+-containing solution and loaded with indo 1, iso-osmotic KCl (60 mM) caused a biphasic increase in the intracellular Ca2+ concentration ([Ca2+]i). In cells superfused for 60 min in Ca2+-free solution containing EGTA (1 mM), KCl did not increase [Ca2+]i. In Ca2+-containing solution, KCl (60 mM) produced a 76.0 +/- 16.2% increase in total [3H]inositol phosphates above basal levels and increased the intracellular levels of free arachidonic acid. These results suggest that, in the estrogen-primed rat uterus, iso-osmotic high K+ solutions, in addition to their well known effect on Ca2+ influx, activate other cellular processes leading to an increase in the Ca2+ sensitivity of the contractile machinery by a mechanism independent of extracellular Ca2+.     

Slow changes in cytosolic free Ca2+ in Escherichia coli highlight two putative influx mechanisms in response to changes in extracellular calcium.

Free intracellular Ca2+ ([Ca2+]i) in Escherichia coli was measured using the bioluminescent protein aequorin. Overall, the bacteria maintained a tight control on their free [Ca2+]i. The results indicated a slow Ca2+ influx, the magnitude of the initial rise in free [Ca2+]i being dependent upon the concentrations of external Ca2+. This was followed by the slow removal of free Ca2+ until normal levels were restored. Specifically, addition of external Ca2+ (0.25-10 mM) resulted in a gradual rise in intracellular free Ca2+ from a basal level of approximately 272 nM, maximally reaching a peak of 0.85-5.4 microM within 30-40 min. This was followed by a slow fall over the next 30 min, culminating in an oscillatory pattern of free [Ca2+]i (range 0.3-0.7 microM for 0.25 mM external Ca2+). In the presence of EGTA, free [Ca2+]i was dramatically reduced. Neither the influx of Ca2+ nor restoration of intracellular free Ca2+ required protein synthesis. Moreover, preincubation with Ca2+ increased the rising phase of intracellular Ca2+ in response to further exposure to external Ca2+. This was further evidence against a specific adaptation process such as the synthesis of calcium exporters. A putative Ca2+ influx channel was demonstrated in stationary phase cells in particular, which could be blocked by La3+. This channel was consistent with the voltage-activated poly-3-hydroxybutyrate/polyphosphate Ca2+ channels previously detailed by Reusch et al. [23] Even in the presence of La3+, however, the free [Ca2+]i of log phase and stationary phase bacteria still increased two-fold over resting values in response to external Ca2+. This suggested the presence of at least two Ca2+ influx processes, one inhibited by La3+ and the other not.     

Novel role for K+-dependent Na+/Ca2+ exchangers in regulation of cytoplasmic free Ca2+ and contractility in arterial smooth muscle

Cytoplasmic free Ca2+ ([Ca2+]cyt) is essential for the contraction and relaxation of blood vessels. The role of plasma membrane Na+/Ca2+ exchange (NCX) activity in the regulation of vascular Ca2+ homeostasis was previously ascribed to the NCX1 protein. However, recent studies suggest that a relatively newly discovered K+-dependent Na+/Ca2+ exchanger, NCKX (gene family SLC24), is also present in vascular smooth muscle. The purpose of the present study was to identify the expression and function of NCKX in arteries. mRNA encoding NCKX3 and NCKX4 was demonstrated by RT-PCR and Northern blot in both rat mesenteric and aortic smooth muscle. NCXK3 and NCKX4 proteins were also demonstrated by immunoblot and immunofluorescence. After voltage-gated Ca2+ channels, store-operated Ca2+ channels, and Na+ pump were pharmacologically blocked, when the extracellular Na+ was replaced with Li+ (0 Na+) to induce reverse mode (Ca2+ entry) activity of Na+/Ca2+ exchangers, a large increase in [Ca2+]cyt signal was observed in primary cultured aortic smooth muscle cells. About one-half of this [Ca2+]cyt signal depended on the extracellular K+. In addition, after the activity of NCX was inhibited by KB-R7943, Na+ replacement-induced Ca2+ entry was absolutely dependent on extracellular K+. In arterial rings denuded of endothelium, a significant fraction of the phenylephrine-induced and nifedipine-resistant aortic or mesenteric contraction could be prevented by removal of extracellular K+. Taken together, these data provide strong evidence for the expression of NCKX proteins in the vascular smooth muscle and their novel role in mediating agonist-stimulated [Ca2+]cyt and thereby vascular tone.     

Ca2+-dependent modulation of intracellular Mg2+ concentration with amiloride and KB-R7943 in pig carotid artery

It has long been recognized that magnesium is associated with several important diseases, including diabetes, hypertension, cardiovascular, and cerebrovascular diseases. In the present study, we measured the intracellular free Mg2+ concentration ([Mg2+]i) using 31P nuclear magnetic resonance (NMR) in pig carotid artery smooth muscle. In normal solution, application of amiloride (1 mm) decreased [Mg2+]i by approximately 12% after 100 min. Subsequent washout tended to further decrease [Mg2+]i. In contrast, application of amiloride significantly increased [Mg2+]i (by approximately 13% after 100 min) under Ca2+-free conditions, where passive Mg2+ influx is facilitated. The treatments had little effect on intracellular ATP and pH (pHi). Essentially the same Ca2+-dependent changes in [Mg2+]i were produced with KB-R7943, a selective blocker of reverse mode Na+-Ca2+ exchange. Application of dimethyl amiloride (0.1 mM) in the presence of Ca2+ did not significantly change [Mg2+]i, although it inhibited Na+-H+ exchange at the same concentration. Removal of extracellular Na+ caused a marginal increase in [Mg2+]i after 100-200 min, as seen in intestinal smooth muscle in which Na+-Mg2+ exchange is known to be the primary mechanism of maintaining a low [Mg2+]i against electrochemical equilibrium. In Na+-free solution (containing Ca2+), neither amiloride nor KB-R7943 decreased [Mg2+]i, but they rather increased it. The results suggest that these inhibitory drugs for Na+-Ca2+ exchange directly modulate Na+-Mg2+ exchange in a Ca2+-dependent manner, and consequently produce the paradoxical decrease in [Mg2+]i in the presence of Ca2+.     

Characterization of oscillations in cytosolic free Ca2+ concentration and measurement of cytosolic Na+ concentration changes evoked by angiotensin II and vasopressin in individual rat aortic smooth muscle cells. Use of microfluorometry and digital imaging

Dual wavelength microfluorometry was used to characterize the changes in cytosolic free Ca2+ concentration [( Ca2+]i) in individual cultured rat aortic vascular smooth muscle cells (VSMC). Angiotensin II (ANG II) at 10(-8) M induced a transient rise in [Ca2+]i from 43 +/- 2 to 245 +/- 23 nM, lasting for approximately 60 s (n = 42). In half of the population, discrete oscillations in [Ca2+]i of smaller amplitude occurred after the initial [Ca2+]i peak, with a period of 58 +/- 8 s and a maximum height of 132 +/- 24 nM. A similar oscillatory pattern was observed with arginine vasopressin (AVP). The oscillations depended upon the presence of extracellular Ca2+. Cytosolic free Na+ concentration ([Na+]i) in VSMC was also measured using the fluorescent Na+ probe sodium-binding benzofuran isophthalate. ANG II induced a gradual and sustained elevation of [Na+]i, from 24.0 +/- 6.2 to 36 +/- 9.7 mM. In response to AVP, [Na+]i rose to 41.0 +/- 11.6 mM. Video imaging of individual VSMC, with on-line ratio calibration of [Ca2+]i, revealed an inhomogeneous distribution of Ca2+ within the cell. [Ca2+] in the nucleus was invariably lower than in the cytoplasm in resting cells. In the cytoplasm, there were small regions in which [Ca2+] was elevated, or "hot spots." In Ca(2+)-containing medium, the initial rise in [Ca2+]i triggered by ANG II and AVP appeared to emanate from the hot spots and to spread evenly throughout the cytoplasm. Between [Ca2+]i oscillations, Ca2+ retreated back to the original hot spots. This study demonstrates the cellular and subcellular heterogeneity of [Ca2+]i both in resting VSMC and during stimulation by ANG II and AVP and reports the direct measurement of [Na+]i in VSMC. The results suggest an action of Ca2+ in both the initial and sustained phases of the response in VSMC and a link between changes in [Ca2+]i and [Na+]i.     

Cytosolic free calcium levels in monolayers of cultured rat aortic smooth muscle cells. Effects of angiotensin II and vasopressin

A rise in cytosolic free calcium ([Ca2+]i) is thought to be the principal mediator in vascular smooth muscle contraction. Quantitative changes of [Ca2+]i in response to two vasoconstrictor peptide hormones, angiotensin II and vasopressin, were directly measured in monolayers of adherent cultured rat aortic smooth muscle cells loaded with the fluorescent calcium indicator Quin 2. Angiotensin II induced rapid, concentration-dependent rises in [Ca2+]i from 1.53 +/- 0.27 X 10(-7) (n = 16) up to 1.2 X 10(-6) M, with ED50 of 0.45 X 10(-9) M, an effect which was blocked by the antagonist analogue [Sar1, Ala8]angiotensin II. Vasopressin also elicited transient rises in [Ca2+]i to peak levels of about 8 X 10(-7) M, with ED50 of 1.05 X 10(-9) M, and this response was completely abolished by a vasopressor antagonist. In calcium-free medium, basal [Ca2+]i levels fell to 0.92 +/- 0.24 X 10(-7) M (n = 4), and both hormones were still able to raise [Ca2+]i, although to a lesser extent. Readdition of extracellular calcium following the [Ca2+]i transient induced a second, slower [Ca2+]i rise. In calcium-containing medium, lanthanum ion (2 X 10(-5) M) reduced peptide-evoked [Ca2+]i rises to the values observed in calcium-free medium. Stimulation with each peptide completely desensitized the smooth muscle cells to a subsequent identical challenge, with little crosstachyphylaxis. Potassium ion (50 mM) only minimally affected [Ca2+]i levels. The calcium channel blocker nifedipine (10(-6) M) did not prevent the [Ca2+]i rises induced by angiotensin II, vasopressin, or potassium. These findings indicate that the two physiologically important vasoconstrictor hormones angiotensin II and vasopressin rapidly raise [Ca2+]i in cultured vascular smooth muscle cells, in part by mobilizing calcium from intracellular pools and in part through activation of receptor-operated calcium channels.     

Properties of the depolarization-activated calcium and barium entry in osteoblast-like cells

Measurements of free cystolic Ca2+ ([Ca2+]i) and Ba2+ ([Ba2+]i) concentrations with Fura 2 were used to identify and characterize the properties of a depolarization-activated Ca2+ and Ba2+ entry in the plasma membrane of osteoblast-like cells. The presence of this pathway was demonstrated in two osteoblastic cell lines, UMR-106 and MC3T3-E1 and osteoblasts isolated from rat long bone and rat neonatal calvariae. Subsequent characterization of the pathway was performed in the osteosarcoma cell line UMR-106. Depolarization of the cells with high medium K+ was followed by an increase in [Ca2+]i which was dependent on medium Ca2+. Ba2+ ions depolarized the cells and were transported by this pathway. Mg2+ ions interfered with Ca2+ and Ba2+ entry. At 140 mM KCl and 1 mM MgCl2, the pathway could be saturated with Ca2+ or Ba2+. The apparent affinity for Ca2+ was 0.78 mM and for Ba2+ 1.82 mM. Ca2+ or Ba2+ entry into the cells was blocked by low concentrations of nicardipine, diltiazem, verapamil, and La3+. In the absence of an increase in [Ca2+]i or [Ba2+]i, the pathway inactivated within about 5 min after depolarization. When [Ca2+]i or [Ba2+]i was allowed to increase, the pathway inactivated within about 20 s. These properties suggest that Ca2+ and Ba2+ entry are mediated by an L-type, depolarization-activated Ca2+ channel in osteoblasts. The activity of these channels changes little with an increase or decrease in cell volume. Thus, it is concluded that these pathways do not provide the Ca2+ entry pathway required for initiation of volume decrease by osteoblasts.     

Contractile response of skeletal muscle to low peroxide concentrations: myofibrillar calcium sensitivity as a likely target for redox-modulation.

Endogenous peroxides and related reactive oxygen species may influence various steps in the contractile process. Single mouse skeletal muscle fibers were used to study the effects of hydrogen peroxide (H2O2) and t-butyl hydroperoxide (t-BOOH) on force and myoplasmic Ca2+ concentration ([Ca2+]i). Both peroxides (1010 to 105 M) decreased tetanic [Ca2+]i and increased force during submaximal tetani. Catalase (1 kU/ml) blocked the effect of H2O2, but not of t-BOOH. The decrease in tetanic [Ca2+]i was constant, while the effect on force was biphasic: A transitory increase was followed by a steady decline to the initial level. Myofibrillar Ca2+ sensitivity remained increased during incubation with either peroxide. Only the highest peroxide concentration (10 mM) increased resting [Ca2+]i and slowed the return of [Ca2+]i to its resting level after a contraction, evidence of impaired sarcoplasmic reticulum Ca2+ re-uptake. The peroxides increased maximal force production and the rate of force redevelopment, and decreased maximum shortening velocity. N-ethylmaleimide (25 mM, thiol-alkylating agent) prevented the response to 1 mM H2O2. These results show that myofibrillar Ca2+ sensitivity and cross-bridge kinetics are influenced by H2O2 and t-BOOH concentrations that approach those found physiologically, and these findings indicate a role for endogenous oxidants in the regulation of skeletal muscle function.     

Extracellular signal-regulated kinase1/2 in contraction of vascular smooth muscle

Smooth muscle contractility is regulated by both intracellular Ca2+ concentration ([Ca2+]i) and Ca2+ sensitivity of the contractile apparatus. Extracellular signal-regulated kinases1/2 (ERK1/2) have been implicated in modulating Ca2+ sensitivity of smooth muscle contraction but mechanisms of action remain elusive. This study investigated the roles of ERK1/2 in modulating [Ca2+]i, calcium sensitivity and the 20-kDa myosin light chain (MLC20) phosphorylation during contraction activated by alpha1-adrenoceptor agonist phenylephrine and thromboxane A2 mimetic U46619 in rat tail artery strips. A specific inhibitor for ERK1/2 activation, U0126, inhibited phenylephrine- and U46619-induced contraction, shifting both concentration-response curves rightward. During phenylephrine-stimulated contraction, U0126 exhibited concentration-dependent inhibition towards force but significant decreases in [Ca2+]i were detected only at higher concentration. Both phenylephrine and U46619 induced a transient activation of ERK1/2 which was abolished by U0126 but unaffected by a general tyrosine kinase inhibitor genistein or Rho kinase inhibitor Y27632 at concentrations inhibiting more than 50% force. Interestingly, U0126 had no effect on steady-state MLC20 phosphorylation levels stimulated by both receptor agonists. These results indicated that during contraction of rat tail artery smooth muscle activated by alpha1-adrenoceptor agonist or thromboxane A2 analogue, ERK1/2 increase Ca2+ sensitivity that does not involve the modulation of MLC20 phosphorylation.     

Endothelin-sensitive intracellular Ca2+ store overlaps with caffeine-sensitive one in rat aortic smooth muscle cells in primary cultures

We made use of quin2 microfluorometry to determine the effects of endothelin (ET) on cytosolic free Ca2+ concentrations [Ca2+]i) in rat aortic smooth muscle cells in primary culture. In Ca2+-containing medium, ET induced a rapid and sustained elevation of [Ca2+]i. In the latter component, in particular, the elevation of [Ca2+]i was inhibited by diltiazem. In Ca2+-free medium, ET induced a rapid and transient [Ca2+]i elevation, which was not inhibited by diltiazem. When the caffeine-sensitive intracellular Ca2+ store was practically depleted by repeated treatment with caffeine in Ca2+-free media, ET did not elevate [Ca2+]i. Thus, it was suggested that ET induces [Ca2+]i elevation not only by extracellular Ca2+-dependent, mechanisms but also by releasing Ca2+ from the intracellular store, and that the ET-sensitive Ca2+ store may overlap with the caffeine-sensitive one, in cultured vascular smooth muscle cells.     

Calcium entry in squid axons during voltage clamp pulses

Squid giant axons were injected with aequorin and tetraethylammonium and were impaled with sodium ion sensitive, current and voltage electrodes. The axons were usually bathed in a solution of varying Ca2+ concentration ([Ca2+]o) containing 150mM each of Na+, K+ and an inert cation such as Li+, Tris or N-methylglucamine and had ionic currents pharmacologically blocked. Voltage clamp pulses were repeatedly delivered to the extent necessary to induce a change in the aequorin light emission, a measure of axoplasmic Ca2+ level, [Ca2+]i. The effect of membrane voltage on [Ca2+]i was found to depend on the concentration of internal Na+ ([Na+]i). Voltage clamp hyperpolarizing pulses were found to cause a reduction of [Ca2+]i. For depolarizing pulses a relationship between [Ca2+]i gain and [Na+]i indicates that Ca2+ entry is sigmoid with a half maximal response at 22 mM Na+. This Ca2+ entry is a steep function of [Na+]i suggesting that 4 Na+ ions are required to promote the influx of 1 Ca2+. There was little change in Ca2+ entry with depolarizing pulses when [Ca2+]o is varied from 1 to 10mM, while at 50mM [Ca2+]o calcium entry clearly increases suggesting an alternate pathway from that of Na+/Ca2+ exchange. This entry of Ca2+ at high [Ca2+]o, however, was not blocked by Cs+o. The results obtained lend further support to the notion that Na+/Ca2+ exchange in squid giant axon is sensitive to membrane voltage no matter whether this is applied as a constant change in membrane potential or as an intermittent one.     

Regulation of metabolism and contraction by cytoplasmic calcium in the intestinal smooth muscle

Reduced pyridine nucleotides (PNred) and oxidized flavoproteins (FPox) were measured fluorometrically in the intestinal smooth muscle strip of guinea pig taenia caeci simultaneously with contractile tension. Cytoplasmic free Ca2+ levels ([Ca2+]cyt) were also measured by a fura-2-Ca2+ fluorescence technique. PNred, FPox, and [Ca2+]cyt increased during spontaneous contraction or upon the addition of high K+ or carbachol and decreased upon the removal of these stimulants. [Ca2+]cyt increased before the increase in muscle tension. PNred increased almost simultaneously with or immediately after the onset of contraction, while FPox increased before the initiation of contraction. Both PNred and FPox decreased a few seconds after the initiation of relaxation. In the K+-depolarized, Ca2+-depleted muscle, graded elevation of external Ca2+ increased PNred, FPox, and muscle tension. The sensitivity to Ca2+ was in the order of FPox greater than PNred greater than muscle tension. Changes in PNred were inhibited when glycolysis was inhibited by substitution of external glucose with oxaloacetate, pyruvate, or beta-hydroxybutylate, but not when oxidative phosphorylation was inhibited by N2 bubbling or by NaCN. In contrast to this, changes in the FPox were inhibited by N2 bubbling or NaCN, but not by the inhibition of glycolysis. These results suggest that an elevation of intracellular Ca2+ activates carbohydrate metabolism and contractile elements independently, resulting in the reduction of cytoplasmic pyridine nucleotides, oxidation of mitochondrial flavoproteins, and development of tension in the intestinal smooth muscle.     

Intracellular alkalinization induces Ca2+ influx via non-voltage-operated Ca2+ channels in rat aortic smooth muscle cells

In smooth muscle, the cytosolic Ca2+ concentration ([Ca2+](i)) is the primary determinant of contraction, and the intracellular pH (pH(i)) modulates contractility. Using fura-2 and 2',7'-biscarboxyethyl-5(6) carboxyfluorescein (BCECF) fluorometry and rat aortic smooth muscle cells in primary culture, we investigated the effect of the increase in pH(i) on [Ca2+](i). The application of the NH(4)Cl induced concentration-dependent increases in both pH(i) and [Ca2+](i). The extent of [Ca2+](i) elevation induced by 20mM NH(4)Cl was approximately 50% of that obtained with 100mM K(+)-depolarization. The NH(4)Cl-induced elevation of [Ca2+](i) was completely abolished by the removal of extracellular Ca2+ or the addition of extracellular Ni2+. The 100mM K(+)-induced [Ca2+](i) elevation was markedly inhibited by a voltage-operated Ca2+ channel blocker, diltiazem, and partly inhibited by a non-voltage-operated Ca2+ channel blocker, SKF96365. On the other hand, the NH(4)Cl-induced [Ca2+](i) elevation was resistant to diltiazem, but was markedly inhibited by SKF96365. It is thus concluded that intracellular alkalinization activates the Ca2+ influx via non-voltage-operated Ca2+ channels and thereby increases [Ca2+](i) in the vascular smooth muscle cells. The alkalinization-induced Ca2+ influx may therefore contribute to the enhancement of contraction.