Regulation of store-operated Ca entry in pulmonary artery smooth muscle cells

Store-operated Ca²⁺ entry (SOCE) is an important mechanism for Ca²⁺ influx in smooth muscle cells; however the activation and regulation of this influx pathway are incompletely understood. In the present study we have examined the effect of several protein kinases in regulating SOCE in pulmonary artery smooth muscle cells (PASMCs) of the rat. Inhibition of protein kinase C with chelerythrine (3 μM) potentiated SOCE by 47 ± 2%, while the tyrosine kinase inhibitors genistein (100 μM) and tyrphostin 23 (100 μM) caused a significant reduction in SOCE of 55 ± 9% and 43 ± 7%, respectively. It has been proposed that Ca²⁺-insensitive phospholipase A₂ (iPLA₂) is involved in the activation of SOCE in many different cell types. The iPLA₂ inhibitor, bromoenol lactone had no effect on SOCE, suggesting that this mechanism was not involved in the activation of the pathway. The calmodulin antagonists, calmidazolium (CMZ) (10 μM) and W-7 (10 μM) appeared to potentiate SOCE in PASMCs. Further investigation established that CMZ was actually activating a Ca²⁺ influx pathway that was independent of the filling state of the sarcoplasmic reticulum. The CMZ-activated Ca²⁺ influx was blocked by Gd³⁺ (10 μM), but unaffected by 2-APB (75 μM), indicating a pharmacological profile distinct from the classical SOCE pathway.     

Role of phospholemman and the 70 kDa inhibitor protein in regulating Na/K ATPase activity in pulmonary artery smooth muscle cells under U46619 stimulation

Treatment of bovine pulmonary smooth muscle cells with U46619 inhibited the Na⁺/K⁺ ATPase activity in two parallel pathways: one of which is mediated via glutathionylation of the pump and the other by augmenting the inhibitory activity of the 70 kDa inhibitor protein of Na⁺/K⁺ ATPase. Although phospholemman deglutathionylates the pump leading to its activation, the inhibitor is responsible for irreversible inhibition of Na⁺/K⁺ ATPase in an isoform specific manner during treatment of the cells with U46619.     

Extracellular alkalosis activates ERK mitogen-activated protein kinase of vascular smooth muscle cells through NADPH-mediated formation of reactive oxygen species

Extracellular alkalosis induced phosphorylation of extracellular signal-regulated kinase (ERK) and enhanced serum-induced ERK phosphorylation in cultured rat aortic smooth muscle cells. While extracellular alkalinization increased verapamil-sensitive (45)Ca(2+) uptake into the cells, ERK phosphorylation induced by extracellular alkalosis was not affected by verapamil. On the other hand, probes for oxidant signaling, such as superoxide dismutase, 4,5-dihydroxy-1,3-benzene-disulfonic acid, a cell-permeable antioxidant, and diphenyliodonium, a NADPH oxidase inhibitor, inhibited extracellular alkalosis-induced phosphorylation of ERK. These results suggest that activation of ERK induced by extracellular alkalosis is not dependent on transplasmalemmal Ca(2+) entry but is caused by reactive oxygen species derived from an activation of NADPH oxidase.     

Mitochondrial ROS-PKCepsilon signaling axis is uniquely involved in hypoxic increase in [Ca2+]i in pulmonary artery smooth muscle cells

The molecular mechanisms underlying hypoxic responses in pulmonary and systemic arteries remain obscure. Here we for the first time report that acute hypoxia significantly increased total PKC and PKCepsilon activity in pulmonary, but not mesenteric arteries, while these two tissues showed comparable PKCepsilon protein expression and activation by the PKC activator phorbol 12-myristate 13-acetate. Hypoxia induced an increase in intracellular reactive oxygen species (ROS) generation in isolated pulmonary artery smooth muscle cells (PASMCs), but not in mesenteric artery SMCs. Inhibition of mitochondrial ROS generation with rotenone, myxothiazol, or glutathione peroxidase-1 overexpression prevented hypoxia-induced increases in total PKC and PKCepsilon activity in pulmonary arteries. The inhibitory effects of rotenone were reversed by exogenous hydrogen peroxide. A PKCepsilon translocation peptide inhibitor or PKCepsilon gene deletion decreased hypoxic increase in [Ca(2+)](i) in PASMCs, whereas the conventional PKC inhibitor GO6976 had no effect. These data suggest that acute hypoxia may specifically increase mitochondrial ROS generation, which subsequently activates PKC, particularly PKCepsilon, contributing to hypoxia-induced increase in [Ca(2+)](i) and contraction in PASMCs.     

The protein kinase inhibitor, staurosporine, inhibits L-type Ca2+ current in rabbit atrial myocytes

A whole-cell patch recording was used to determine the effects of staurosporine (ST), a potent protein kinase C (PKC) inhibitor, on L-type Ca(2+) channel (LTCC) activity in rabbit atrial myocytes. Bath application of ST (300 nM) caused a significant reduction in peak I-V relationship of LTCC (from -16.8+/-2.55 to -3.74+/-1.22pApF(-1) at 0 mV). The level of L-type Ca(2+) current (I(Ca,L)) inhibition produced by ST was independent of the voltage at which the effect was measured. ST inhibited the I(Ca,L) in a dose-dependent manner with a K(d) value of 61.98+/-6.802 nM. ST shifted the activation curve to more positive potentials, but did not have any significant effect on the voltage dependence of the inactivation curve. Other PKC inhibitors, GF 109203X (1 microM) and chelerythrine (3 microM), and PKA inhibitor, PKA-IP (5 microM), did not show any inhibitory effect on I(Ca,L). Additional application of ST in the presence of isoproterenol (1 microM), a selective beta-adrenoreceptor agonist, reduced peak I(Ca,L) (78.2%) approximately to the same level with single application of ST (77.8%). In conclusion, our results indicate that ST directly blocks the LTCC in a PKC or PKA-independent manner on LTCC and it should be taken into consideration when ST is used in functional studies of ion channel modulation by protein phosphorylation.     

IP3 decreases coronary artery tone via activating the BKCa channel of coronary artery smooth muscle cells in pigs

Large conductance Ca²⁺-activated K⁺ channel (BK_Ca) is a potential target for coronary artery-relaxing medication, but its functional regulation is largely unknown. Here, we report that inositol trisphosphate (IP3) activated BK_Ca channels in isolated porcine coronary artery smooth muscle cells and by which decreased the coronary artery tone. Both endogenous and exogenous IP3 increased the spontaneous transient outward K⁺ currents (STOC, a component pattern of BK_Ca currents) in perforated and regular whole-cell recordings, which was dependent on the activity of IP3 receptors. IP3 also increased the macroscopic currents (MC, another component pattern of BK_Ca currents) via an IP3 receptor- and sarcoplasmic Ca²⁺ mobilization-independent pathway. In inside-out patch recordings, direct application of IP3 to the cytosolic side increased the open probability of single BK_Ca channel in an IP3 receptor-independent manner. We conclude that IP3 is an activator of BK_Ca channels in porcine coronary smooth muscle cells and exerts a coronary artery-relaxing effect. The activation of BK_Ca channels by IP3 involves the enhancement of STOCs via IP3 receptors and stimulation of MC by increasing the Ca²⁺ sensitivity of the channels.     

Ca2+-induced redistribution of Ca2+/calmodulin-dependent protein kinase II associated with an endoplasmic reticulum stress response in vascular smooth muscle

The relation between CaM kinase II activity and high Ca²⁺-mediated stress responses was studied in cultured vascular smooth muscle cells. Treatment with ionomycin (1 M) for 5 min caused a significant loss of CaM kinase II activity in whole cell homegenates and prominent vesiculation of the endoplasmic reticulum (ER). Similar losses of CaM kinase II activity were observed in the soluble lysate as assessed by activity measurements and Western blotting. Examination of the post-lysate particulate fraction showed that the loss of CaM kinase II from the soluble lysate was accompanied by a redistribution of CaM kinase II to this fraction. The ionomycin-mediated response was limited to this concentration (1 M); lower concentrations of ionomycin as well as stimulation with angiotensin II (1 M) or ATP (100 M) did not cause a shift in CaM kinase II distribution. Treatment with neither the CaM kinase II inhibitor KN-93 nor the phosphatase inhibitor okadaic acid altered the ionomycin-induced redistribution indicating that CaM kinase II activation and/or phosphorylation was not part of the mechanism. The response, however, was eliminated when the cells were treated in Ca²⁺-free medium. Washout of ionomycin led to only a partial restoration of the kinase activity in the soluble fraction after 10 min. Immunofluorescence microscopy of resting cells indicated colocalization of antibodies to CaM kinase II and an ER protein marker. ER vesiculation induced by ionomycin coincided with a parallel redistribution of CaM kinase II and ER marker proteins. These data link ionomycin-induced ER restructuring to a progressive redistribution of CaM kinase II protein to an insoluble particulate fraction and loss of cellular CaM kinase II activity. We propose that redistribution of CaM kinase II and loss of cellular activity are components of a common Ca²⁺-overload induced cellular stress response in cells.     

Calcineurin activation by slow calcium release from intracellular stores suppresses protein kinase C regulation of L-type calcium channels in L6 cells

L-type Ca²⁺ channel activity was assayed in L6 cells as the rate of nifedipine-sensitive Ba²⁺ influx in a depolarizing medium. In the absence of extracellular Ca²⁺, activation of protein kinase C (PKC) with phorbol-12-myristate-13-acetate (PMA) or thymeleatoxin (TMX) inhibited Ba²⁺ influx by 38%. Thapsigargin (Tg), a selective inhibitor of the Ca²⁺-ATPase in the sarcoplasmic reticulum, evoked a rise in the cytosolic Ca²⁺ concentration ([Ca²⁺]_i) in a Ca²⁺-free medium from 30 to 80 nM. This [Ca²⁺]_i increase declined slowly, giving rise to a modest elevation of [Ca²⁺]_i that persisted for >5 min. The inhibitory effects of PMA and TMX on channel activity were abolished when tested in Tg-treated cells in a Ca²⁺-free medium. However, when the Ca²⁺ ionophore, ionomycin, was applied with Tg, PMA and TMX retained their inhibitory effect on L-type Ca²⁺ channel activity, suggesting that a lower amplitude and prolonged release of Ca²⁺ stores is necessary for abrogating PKC-mediated inhibition of LCC. Cyclosporin A (5 μM) and ascomycin (5 μM), inhibitors of the Ca²⁺/calmodulin-dependent protein phosphatase, calcineurin, fully restored the inhibitory effect of PMA and TMX on channel activity. Addition of 1 mM CaCl₂ to the Tg-treated cells increased [Ca²⁺]_i to 165 nM and also restored the inhibitory effects of PMA and TMX. These results indicate that a small, relatively prolonged [Ca²⁺]_i increase elicited by passive depletion of internal Ca²⁺ stores led to activation of calcineurin, giving rise to an increase in protein phosphatase activity that counteracted the inhibitory effects of PKC on channel activity. A larger increase in [Ca²⁺]_i via store-dependent Ca²⁺ entry enhanced the activity of PKC sufficiently to overcome the protein phosphatase activity of calcineurin. This study is the first to demonstrate that the regulation of L-type Ca²⁺ channels in a myocyte model involves a balance between the differential Ca²⁺ sensitivities and opposing actions of PKC and calcineurin.     

Endothelin-1 induces intracellular [Ca2+] increase via Ca2+ influx through the L-type Ca2+ channel, Ca2+-induced Ca2+ release and a pathway involving ETA receptors, PKC, PKA and AT1 receptors in cardiomyocytes

Using fura-2-acetoxymethyl ester (AM) fluorescence imaging and patch clamp techniques, we found that endothelin-1 (ET-1) significantly elevated the intracellular calcium level ([Ca²⁺]_i) in a dose-dependent manner and activated the L-type Ca²⁺ channel in cardiomyocytes isolated from rats. The effect of ET-1 on [Ca²⁺]_i elevation was abolished in the presence of the ET_A receptor blocker BQ123, but was not affected by the ET_B receptor blocker BQ788. ET-1-induced an increase in [Ca²⁺]_i, which was inhibited 46.7% by pretreatment with a high concentration of ryanodine (10 μmol/L), a blocker of the ryanodine receptor. The ET-1-induced [Ca²⁺]_i increase was also inhibited by the inhibitors of protein kinase A (PKA), protein kinase C (PKC) and angiotensin type 1 receptor (AT1 receptor). We found that ET-1 induced an enhancement of the amplitude of the whole cell L-type Ca²⁺ channel current and an increase of open-state probability (NPo) of an L-type single Ca²⁺ channel. BQ123 completely blocked the ET-1-induced increase in calcium channel open-state probability. In this study we demonstrated that ET-1 regulates calcium overload through a series of mechanisms that include L-type Ca²⁺ channel activation and Ca²⁺-induced Ca²⁺ release (CICR). ETA receptors, PKC, PKA and AT1 receptors may also contribute to this pathway. Supported by the National Natural Science Foundation of China (Grant No. 200830870910).     

Phospholipase A2-derived lysophosphatidylcholine triggers Ca entry in dystrophic skeletal muscle fibers

Duchenne muscular dystrophy is an inherited disease caused by the absence of dystrophin, a structural protein normally located under the sarcolemma of skeletal muscle fibers. Muscle degeneration occurring in this disease is thought to be partly caused by increased Ca²⁺ entry through sarcolemmal cationic channels. Using the Mn²⁺ quench method, we show here that Mn²⁺ entry triggered by Ca²⁺ store depletion but not basal Mn²⁺ entry relies on Ca²⁺-independent PLA₂ (iPLA₂) activity in dystrophic fibers isolated from a murine model of Duchenne muscular dystrophy, the mdx^5cv mouse. iPLA₂ was found to be localized in the vicinity of the sarcolemma and consistently, the iPLA₂ lipid product lysophosphatidylcholine was found to trigger Ca²⁺ entry through sarcolemmal channels, suggesting that it acts as an intracellular messenger responsible for store-operated channels opening in dystrophic fibers. Our results suggest that inhibition of iPLA₂ and lysophospholipid production may be of interest to reduce Ca²⁺ entry and subsequent degeneration of dystrophic muscle.     

Airway smooth muscle relaxation induced by 5-HT(2A) receptors: role of Na(+)/K(+)-ATPase pump and Ca(2+)-activated K(+) channels

AIMS: Although 5-hydroxytryptamine (5-HT) contracts airway smooth muscle in many mammalian species, in guinea pig and human airways 5-HT causes a contraction followed by relaxation. This study explored potential mechanisms involved in the relaxation induced by 5-HT. MAIN METHODS: Using organ baths, patch clamp, and intracellular Ca(2+) measurement techniques, the effect of 5-HT on guinea pig airway smooth muscle was studied. KEY FINDINGS: A wide range of 5-HT concentrations caused a biphasic response of tracheal rings. Response to 32 muM 5-HT was notably reduced by either tropisetron or methiothepin, and almost abolished by their combination. Incubation with 10 nM ketanserin significantly prevented the relaxing phase. Likewise, incubation with 100 nM charybdotoxin or 320 nM iberiotoxin and at less extent with 10 muM ouabain caused a significant reduction of the relaxing phase induced by 5-HT. Propranolol, L-NAME and 5-HT(1A), 5-HT(1B)/5-HT(1D) and 5-HT(2B) receptors antagonist did not modify this relaxation. Tracheas from sensitized animals displayed reduced relaxation as compared with controls. In tracheas precontracted with histamine, a concentration response curve to 5-HT (32, 100 and 320 muM) induced relaxation and this effect was abolished by charybdotoxin, iberiotoxin or ketanserin. In single myocytes, 5-HT in the presence of 3 mM 4-AP notably increased the K(+) currents (I(K(Ca))), and they were completely abolished by charybdotoxin, iberiotoxin or ketanserin. SIGNIFICANCE: During the relaxation induced by 5-HT two major mechanisms seem to be involved: stimulation of the Na(+)/K(+)-ATPase pump, and increasing activity of the high-conductance Ca(2+)-activated K(+) channels, probably via 5-HT(2A) receptors.     

Big-conductance Ca2+-activated K+ channels in physiological and pathophysiological urinary bladder smooth muscle cells

Contraction and relaxation of urinary bladder smooth muscle cells (UBSMCs) represent the important physiological functions of the bladder. Contractile responses in UBSMCs are regulated by a number of ion channels including big-conductance Ca²⁺- activated K⁺ (BK) channels. Great progress has been made in studies of BK channels in UBSMCs. The intent of this review is to summarize recent exciting findings with respect to the functional interactions of BK channels with muscarinic receptors, ryanodine receptors (RyRs) and inositol triphosphate receptors (IP₃Rs) as well as their functional importance under normal and pathophysiological conditions. BK channels are highly expressed in UBSMCs. Activation of muscarinic M₃ receptors inhibits the BK channel activity, facilitates opening of voltage-dependent Ca²⁺ (Ca_V) channels, and thereby enhances excitability and contractility of UBSMCs. Signaling molecules and regulatory mechanisms involving RyRs and IP₃Rs have a significant effect on functions of BK channels and thereby regulate cellular responses in UBSMCs under normal and pathophysiological conditions including overactive bladders. Moreover, BK channels may represent a novel target for the treatment of bladder dysfunctions.     

A model for the generation of localized transient [Na] elevations in vascular smooth muscle

We present a stochastic computational model to study the mechanism of signaling between a source and a target ionic transporter, both localized on the plasma membrane (PM). In general this requires a nanometer-scale cytoplasmic space, or nanodomain, between the PM and a peripheral organelle to reflect ions back towards the PM. Specifically we investigate the coupling between Na⁺ entry via the transient receptor potential canonical channel 6 (TRPC6) and the Na⁺/Ca²⁺ exchanger (NCX), a process which is essential for reloading the sarcoplasmic reticulum (SR) via the sarco/endoplasmic reticulum Ca²⁺ATPase (SERCA) and maintaining Ca²⁺ oscillations in activated vascular smooth muscle. Having previously modeled the flow of Ca²⁺ between reverse NCX and SERCA during SR refilling, this quantitative approach now allows us to model the upstream linkage of Na⁺ entry through TRPC6 to reversal of NCX. We have implemented a random walk (RW) Monte Carlo (MC) model with simulations mimicking a diffusion process originating at the TRPC6 within PM-SR junctions. The model calculates the average Na⁺ in the nanospace and also produces profiles as a function of distance from the source. Our results highlight the necessity of a strategic juxtaposition of the relevant ion translocators as well as other physical structures within the nanospaces to permit adequate Na⁺ build-up to initiate NCX reversal and Ca²⁺ influx to refill the SR.     

Age-dependent changes in diastolic Ca and Na concentrations in dystrophic cardiomyopathy: Role of Ca entry and IP3

Duchenne muscular dystrophy (DMD) is a lethal X-inherited disease caused by dystrophin deficiency. Besides the relatively well characterized skeletal muscle degenerative processes, DMD is also associated with a dilated cardiomyopathy that leads to progressive heart failure at the end of the second decade. The aim of the present study was to characterize the diastolic Ca²⁺ concentration ([Ca²⁺]_d) and diastolic Na⁺ concentration ([Na⁺]_d) abnormalities in cardiomyocytes isolated from 3-, 6-, 9-, and 12-month old mdx mice using ion-selective microelectrodes. In addition, the contributions of gadolinium (Gd³⁺)-sensitive Ca²⁺ entry and inositol triphosphate (IP₃) signaling pathways in abnormal [Ca²⁺]_d and [Na⁺]_d were investigated. Our results showed an age-dependent increase in both [Ca²⁺]_d and [Na⁺]_d in dystrophic cardiomyocytes compared to those isolated from age-matched wt mice. Gd³⁺ treatment significantly reduced both [Ca²⁺]_d and [Na⁺]_d at all ages. In addition, blockade of the IP₃-pathway with either U-73122 or xestospongin C significantly reduced ion concentrations in dystrophic cardiomyocytes. Co-treatment with U-73122 and Gd³⁺ normalized both [Ca²⁺]_d and [Na⁺]_d at all ages in dystrophic cardiomyocytes. These data showed that loss of dystrophin in mdx cardiomyocytes produced an age-dependent intracellular Ca²⁺ and Na⁺ overload mediated at least in part by enhanced Ca²⁺ entry through Gd³⁺ sensitive transient receptor potential channels (TRPC), and by IP₃ receptors.     

Role of MMP-2 in inhibiting Na+ dependent Ca2+ uptake by H2O2 in microsomes isolated from pulmonary smooth muscle

Treatment of microsomes (preferentially enriched with endoplasmic reticulum) isolated from bovine pulmonary artery smooth muscle tissue with H₂O₂ (1 mM) markedly stimulated matrix metalloproteinase activity and also inhibited Na⁺ dependent Ca²⁺ uptake. Electron micrograph revealed that H₂O₂ (1 mM) does not cause any damage to the microsomes. MMP-2 and TIMP-2 were determined to be the ambient protease and corresponding antiprotease of the microsomes. Pretreatment with vitamin E (1 mM) and TIMP-2 (50 g/ml) reversed the effect produced by H₂O₂ (1 mM) on Na⁺ dependent Ca²⁺ uptake in the microsomes. However, H₂O₂ (1 mM) caused changes in MMP-2 activity and Na⁺ dependent Ca²⁺ uptake were not reversed upon pretreatment of the microsomes with a low concentration of 5 g/ml of TIMP-2 which otherwise reversed MMP-2 (1 g/ml) mediated increase in ¹⁴C-gelatin degradation and inhibition of Na⁺ dependent Ca²⁺ uptake. Combined treatment of the microsomes with a low dose of MMP-2 (0.5 g/ml) and H₂O₂ (0.5 mM) inhibited Na⁺ dependent Ca²⁺ uptake in the microsomes compared to the respective low dose of either of them. Direct treatment of TIMP-2 (5 g/ml) with H₂O₂ (1 mM) abolished the inhibitory effect of the inhibitor on ¹⁴C-gelatinolytic activity elicited by 1 g/ml of MMP-2. Thus, one of the mechanisms by which H₂O₂ activates MMP-2 could be due to inactivation of TIMP-2 by the oxidant. The resulting activation of MMP-2 subsequently inhibits Na⁺ dependent Ca²⁺ uptake in the microsomes. (Mol Cell Biochem 270: 79–87, 2005)     

Leukotriene D4 induced calcium changes in U937 cells may utilize mechanisms additional to inositol phosphate production that are pertussis toxin insensitive but are blocked by phorbol myristate acetate

DMSO differentiated U937 cells responded to 10⁻⁶ M LTD₄, LTB₄ and FMLP with an increase in both InsP formation and [Ca²⁺]_i. FMLP caused a greater rise in InsPs than either LTD₄ or LTB₄, which were equivalent. LTD₄, however, caused a greater increase in [Ca²⁺]_i than LTB₄ (4-fold) or FMLP. The FMLP [Ca²⁺]_i and InsP responses were abolished by pertussis toxin (100 ng/ml for 4 h) but were unaffected by PMA (10⁻⁷ M for 3 min). In contrast, the LTD₄ [Ca²⁺]_i and InsP responses were reduced by only 50% by pertussis toxin, whilst PMA reduced the [Ca²⁺]_i and InsP responses to LTD₄ by 75 and 30%, respectively. These results suggest that mechanisms additional to InsP formation exist for mediating LTD₄ evoked increases in [Ca²⁺]_i.     

Activation of choline kinase by extracellular Ca is Ca-sensing receptor, Gα12 and Rho-dependent in breast cancer cells

Breast cancer cell metastases to bone result in osteolysis and release of large quantities of Ca²⁺ into the bone microenviroment. Extracellular Ca²⁺ (Ca_o²⁺) acting through the Ca²⁺-sensing receptor (CaR), a member of G protein-coupled receptor superfamily, plays an important role in the regulation of multiple signaling pathways. Here, we find that expression of the CaR and Gα₁₂ is significantly up-regulated in breast cancer cells (MDA-MB-231 and MCF-7) compared with nonmalignant breast cells (Hs 578Bst and MCF-10A). Ca_o²⁺ induces a significant increase in extracellular [³H]phosphocholine (P-cho) production in breast cancer cells. Using an anti-CaR antibody to block Ca_o²⁺ binding to the CaR and small interfering RNA (siRNA) to silence CaR gene expression, our data demonstrate that [³H]P-cho production in response to Ca_o²⁺-stimulation is CaR-dependent. By analyzing cellular lipid profiles and using siRNA to silence choline kinase (ChoK) expression, we determine that the production of [³H]P-cho is primarily related to CaR-induced ChoK activation, and not degradation of choline phospholipids. Finally, by pretreatment of the cells with either pertussis toxin or C₃ exoenzyme, co-immunoprecipiation of Gα_i, Gα_q or Gα₁₂ with the CaR, and RhoA translocation, we found that the enhancement of ChoK activation and P-cho production in breast cancer cells occurs via a CaR-Gα₁₂-Rho signaling pathway.     

Role of protein kinase C in mediating alpha-1-adrenoceptor-induced negative inotropic response in rat ventricles

The aim of this study was to determine the effect of protein kinase C (PKC) activation on intracellular Ca(2+) transient and its relation to alpha(1)-adrenoceptor (alpha(1)-AR)-stimulated negative inotropic response in rat ventricles. The electromechanical responses to phenylephrine (PE) in rat ventricular muscles were concomitantly examined using the conventional microelectrode method. The responses of intracellular Ca(2+) transient and cell contractions to PE in the absence of certain pharmacological interventions were ascertained in fura-2-loaded myocytes. The influence of PE on L-type Ca(2+) current (I(Ca,L)) was also examined using a voltage clamp in a whole-cell configuration. PE did not alter the action potential parameters during the negative inotropic phase. The negative inotropic effect (NIE) was inhibited by prazosin, chloroethylclonidine (CEC) and staurosporine, but was insensitive to pertussis toxin. Desensitization of PKC after prolonged pretreatment of rat ventricles with PDBu also abolished the NIE of PE. Caffeine modulated the NIE, but thapsigargin did not. The evoked intracellular Ca(2+) transient and cell contraction were initially decreased by PE, while I(Ca,L) was not altered. Prazosin and staurosporine significantly inhibited the responses. Our data indicated that alpha(1)AR-mediated NIE in rat ventricular muscles was due to the decrease of intracellular Ca(2+) transients by the modulation of PKC on Ca(2+)-releasing channels signaling through a CEC-sensitive alpha(1)AR subtype.     

Developmental regulation of expression of the α1 and α2 subunits mRNAs of the voltage-dependent calcium channel in a differentiating myogenic cell line

The voltage-dependent calcium channel (VDCC) in skeletal muscle probably plays a key role in transducing membrane charge movement to the calcium release channel. We report here that the expression of VDCC α₁ and α₂ mRNAs is developmentally regulated in differentiating C2Cl2 myogenic cells. The α₁ mRNA is not detectable in the myoblast form of C2Cl2 cells while its expression is induced 20-fold in differentiated myotubes. In contrast, the α₂ mRNA is weakly expressed in myoblasts but is also induced upon myogenic differentiation.     

Large conductance, Ca-activated K channels (BKCa) and arteriolar myogenic signaling

Myogenic, or pressure-induced, vasoconstriction is critical for local blood flow autoregulation. Underlying this vascular smooth muscle (VSM) response are events including membrane depolarization, Ca²⁺ entry and mobilization, and activation of contractile proteins. Large conductance, Ca²⁺-activated K⁺ channel (BK_Ca) has been implicated in several of these steps including, (1) channel closure causing membrane depolarization, and (2) channel opening causing hyperpolarization to oppose excessive pressure-induced vasoconstriction. As multiple mechanisms regulate BK_Ca activity (subunit composition, membrane potential (Em) and Ca²⁺ levels, post-translational modification) tissue level diversity is predicted. Importantly, heterogeneity in BK_Ca channel activity may contribute to tissue-specific differences in regulation of myogenic vasoconstriction, allowing local hemodynamics to be matched to metabolic requirements. Knowledge of such variability will be important to exploiting the BK_Ca channel as a therapeutic target and understanding systemic effects of its pharmacological manipulation.