P2Y receptor subtypes evoke different Ca2+ signals in cultured aortic smooth muscle cells

Adenine and uridine nucleotides evoke Ca(2+) signals via four subtypes of P2Y receptor in cultured aortic smooth muscle cells, but the mechanisms underlying the different patterns of these Ca(2+) signals are unresolved. Cytosolic Ca(2+) signals were recorded from single cells and populations of cultured rat aortic smooth muscle cells, loaded with a fluorescent Ca(2+) indicator and stimulated with agonists that allow subtype-selective activation of P2Y1, P2Y2, P2Y4, or P2Y6 receptors. Activation of P2Y1, P2Y2, and P2Y6 receptors caused homologous desensitisation, while activation of P2Y2 receptors also caused heterologous desensitisation of the other subtypes. The Ca(2+) signals evoked by each P2Y receptor subtype required activation of phospholipase C and release of Ca(2+) from intracellular stores via inositol 1,4,5-trisphosphate (IP(3)) receptors, but they were unaffected by inhibition of ryanodine or nicotinic acid adenine dinucleotide phosphate (NAADP) receptors. Sustained Ca(2+) signals were independent of the Na(+)/Ca(2+) exchanger and were probably mediated by store-operated Ca(2+) entry. Analyses of single cells established that most cells express P2Y2 receptors and at least two other P2Y receptor subtypes. We conclude that four P2Y receptor subtypes evoke Ca(2+) signals in cultured aortic smooth muscle cells using the same intracellular (IP(3) receptors) and Ca(2+) entry pathways (store-operated Ca(2+) entry). Different rates of homologous desensitisation and different levels of receptor expression account for the different patterns of Ca(2+) signal evoked by each P2Y receptor subtype.     

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.     

Properties of the apamin-sensitive component of Ca2+-dependent K+ current in smooth muscle cells of the guinea pigtaenia coli

With the help of a standard voltage-clamp technique, we investigated transmembrane ion currents in isolated smooth muscle cells of the guinea pigtaenia coli. In Ca²⁺-dependent K⁺ current, we identified and studied the properties of an apamin-sensitive voltage-independent component carried through the channels of low conductance (in many publications called small conductance,I _SK(Ca)). This component did not show the temporal inactivation;I _SK(Ca) was insensitive to the action of 4 mM tetraethylammonium, but was completely blocked by 500 nM of apamin. It was shown thatI _SK(Ca) is very sensitive to changes in the intracellular Ca²⁺ concentration ([Ca²⁺] _i ): a decrease in [Ca²⁺] _i up to 50 nM resulted in the almost complete blockade of the current. The entry of Ca ions into a cell from the external solution through the voltage-operated Ca²⁺ channels of L-type was not an obligatory condition for activation ofI _SK(Ca). The current-voltage relationship forI _SK(Ca) had a maximum within the voltage range of +20 to +50 mV. Neirofiziologiya/Neurophysiology, Vol. 32, No. 2, pp. 87–94, March–April, 2000.     

Demarcation of Ca2+ transport processes in guinea pig stomach smooth muscle

Summary Microsomal fractions were isolated from gastric antrum and fundus smooth muscle of guinea pigs. Ca²⁺ uptake into and Ca²⁺ release from the membrane vesicles were studied by a rapid filtration method, and Ca²⁺ transport properties of the different regions of the stomach were compared. ATP-dependent Ca²⁺ uptake was similar in microsomes isolated from both regions. This uptake was increased by oxalate and was not affected by NaN₃. Oxalate affected Ca²⁺ permeability of both antrum and fundus microsome vesicles similarly. Fundus microsome vesicles preincubated in 100mm NaCl and then diluted to 1/20 concentration with Na⁺-free medium had significantly higher ATP-independent Ca²⁺ uptake than vesicles preincubated in 100mm KCl and treated the same way. This was not true for antrum vesicles. Monensin abolished Na⁺-dependent Ca²⁺ uptake, and NaCl enhanced Ca²⁺ efflux from fundus microsome vesicles. The halflife values of Ca²⁺ loss from fundus vesicles in the presence of NaCl were significantly smaller than those in the presence of KCl. The release of Ca²⁺ from the vesicles within the first 3 min was accelerated by NaCl to three times that by KCl. However, NaCl had ro effect on Ca²⁺ release from antrum microsome vesicles.Results suggest two distinct mechanisms of stomach membrane Ca²⁺ transport: (1) ATP-dependent Ca²⁺ uptake and (2) Na⁺–Ca²⁺ exchange; the latter in the fundus only.     

Properties of the charibdotoxin-sensitive component of Ca2+-dependent K+ current in smooth muscle cells of the guinea pigTaenia Coli

Using the voltage-clamp technique, we investigated transmembrane ion currents in isolated smooth muscle cells of the guinea pigtaenia coli. In our study, we identified and studied a charibdotoxin-sensitive component of Ca²⁺-dependent K⁺ current carried through the channels of high conductance (in most publications called “big conductance,”I _BK(Ca)). This component was completely blocked by 100 nM charibdotoxin and by tetraethylammonium in concentrations as low as 1 mM.I _BK(Ca) demonstrated fast kinetics of inactivation, which nearly coincided with that of Ca²⁺ current. In addition to the dependence on Ca²⁺ concentration, this current also showed voltage-dependent properties: with a rise in the level of depolarization its amplitude increased. In many cells, depolarizing shifts in the membrane potential evoke spontaneous outward currents. Such currents probably represent the secondary effect of cyclic Ca²⁺ release from the caffeine-sensitive intracellular stores that result in short-term activation of charibdotoxin-sensitive Ca²⁺-dependent K⁺ channels.     

Evidence for capacitative and non-capacitative Ca2+ entry pathways coexist in A10 vascular smooth muscle cells

It is generally thought that receptor-operated Ca2+ entry is related to store-operated or capacitative Ca2+ entry mechanism. Recent evidence suggests that non-capacitative Ca2+ entry pathways are also involved in receptor activated Ca2+ influx in many different kinds of cells. In this study, we studied whether alpha1-adrenoreceptor (alpha1-AR)-activated Ca2+ entry is coupled to both capacitative and non-capacitative pathways in A10 vascular smooth muscle cells by fura-2 fluorescence probe and conventional whole-cell patch clamp techniques. We found that both thapsigargin (TG) and phenylephrine (Phe) induced transient increase in cytoplasmic Ca2+ concentration ([Ca2+]i) in Ca2+-free medium, and subsequent addition of Ca2+ evoked a sustained [Ca2+]i rise. When the membrane potential was held at -60 mV, both TG and Phe activated inward currents, which were inhibited by GdCl3(Gd3+), 0Na+/0Ca2+ solution and 1-{beta[3-(4-mehtoxyphenyl)propoxy]-4-methoxypheneth-yl}-1H- imidazole hydro-chloride (SK&F96365), but not by nifedipine. When Ca2+ store was depleted by TG in Ca2+-free solution, Phe failed to further evoke [Ca2+]i rise. However, when capacitative Ca2+ entry was activated by TG in the medium containing Ca2+, 10 microM Phe further increased [Ca2+]i. At the same concentration, TG activated an inward cation current, subsequent addition of Phe also further induced an inward cation current. Furthermore, the amplitudes of [Ca2+]i increase and current density induced by Phe in the presence of TG were less than that induced by Phe alone. Our results suggest that both capacitative and non-capacitative Ca2+ entry pathways are involved in Ca2+ influx induced by activation of alpha1-AR in A10 vascular smooth muscle cells.     

Blockade of insulin sensitive steady-state R-type Ca2+ channel by PN 200-110 in heart and vascular smooth muscle

The effect of high K^– concentration, insulin and the L-type Ca^2– channel blocker PN 200-110 on cytosolic intracellular free calcium ([Ca²⁺]_i) was studied in single ventricular myocytes of 10-day-old embryonic chick heart, 20-week-old human fetus and rabbit aorta (VSM) single cells using the Ca²⁺-sensitive fluorescent dye, Fura-2 microfluorometry and digital imaging technique. Depolarization of the cell membrane of both heart and VSM cells with continuous superfusion of 30 mM [K⁺]_o induced a rapid transient increase of [Ca²⁺]_i that was followed by a sustained component. The early transient increase of [Ca²⁺]_i by high [⁺]_o was blocked by the L-type calcium channel antagonist nifedipine. However, the sustained component was found to be insensitive to this drug. PN 200-110 another L-type Ca²⁺ blocker was found to decrease both the early transient and the sustained increase of [Ca²⁺]_i induced by depolarization of the cell membrane with high [K⁺]_o. Insulin at a concentration of 40 to 80 U/ml only produced a sustained increase of [Ca²⁺]_i that was blocked by PN 200-110 or by lowering the extracellular Ca²⁺ concentration with EGTA. The sustained increase of [Ca²⁺], induced by high [K⁺]_o or insulin was insensitive to metabolic inhibitors such as KCN and ouabain as well to the fast Na⁺ channel blocker, tetrodotoxin and to the increase of intracellular concentrations of cyclic nucleotides. Using the patch clamp technique, insulin did not affect the L-type Ca²⁺ current and the delayed outward K⁺ current. These results suggest that the early increase of (Ca²⁺]_i during depolarization of the cell membrane of heart and VSM cells with high [K⁺]_o is due to the opening and decay of an L-type Ca ²⁺ channel. However, the sustained increase of [Ca²⁺]_i during a sustained depolarization is due to the activation of a resting (R) Ca ²⁺ channel that is insensitive to lowering [ATP]_i and sensitive to insulin.     

Ca microdomains in smooth muscle

In smooth muscle, Ca²⁺ controls diverse activities including cell division, contraction and cell death. Of particular significance in enabling Ca²⁺ to perform these multiple functions is the cell's ability to localize Ca²⁺ signals to certain regions by creating high local concentrations of Ca²⁺ (microdomains), which differ from the cytoplasmic average. Microdomains arise from Ca²⁺ influx across the plasma membrane or release from the sarcoplasmic reticulum (SR) Ca²⁺ store. A single Ca²⁺ channel can create a microdomain of several micromolar near (200 nm) the channel. This concentration declines quickly with peak rates of several thousand micromolar per second when influx ends. The high [Ca²⁺] and the rapid rates of decline target Ca²⁺ signals to effectors in the microdomain with rapid kinetics and enable the selective activation of cellular processes. Several elements within the cell combine to enable microdomains to develop. These include the brief open time of ion channels, localization of Ca²⁺ by buffering, the clustering of ion channels to certain regions of the cell and the presence of membrane barriers, which restrict the free diffusion of Ca²⁺. In this review, the generation of microdomains arising from Ca²⁺ influx across the plasma membrane and the release of the ion from the SR Ca²⁺ store will be discussed and the contribution of mitochondria and the Golgi apparatus as well as endogenous modulators (e.g. cADPR and channel binding proteins) will be considered.     

The importance of Rho-associated kinase-induced Ca2+ sensitization as a component of electromechanical and pharmacomechanical coupling in rat ureteric smooth muscle

Ureteric peristalsis, which occurs via alternating contraction and relaxation of ureteric smooth muscle, ensures the unidirectional flow of urine from the kidney to the bladder. Understanding of the molecular mechanisms underlying ureteric excitation–contraction coupling, however, is limited. To address these knowledge deficits, and in particular to test the hypothesis that Ca²⁺ sensitization via activation of the RhoA/Rho-associated kinase (ROK) pathway plays an important role in ureteric smooth muscle contraction, we carried out a thorough characterization of the electrical activity, Ca²⁺ signaling, MYPT1 (myosin targeting subunit of myosin light chain phosphatase, MLCP) and myosin regulatory light chain (LC₂₀) phosphorylation, and force responses to membrane depolarization induced by KCl (electromechanical coupling) and carbachol (CCh) (pharmacomechanical coupling). The effects of ROK inhibition on these parameters were investigated. We conclude that the tonic, but not the phasic component of KCl- or CCh-induced ureteric smooth muscle contraction is highly dependent on ROK-catalyzed phosphorylation of MYPT1 at T855, leading to inhibition of MLCP and increased LC₂₀ phosphorylation.     

Variable luminal sarcoplasmic reticulum Ca buffer capacity in smooth muscle cells

Sarcoplasmic reticulum contains the internal Ca²⁺ store in smooth muscle cells and its lumen appears to be a continuum that lacks diffusion barriers. Accordingly, the free luminal Ca²⁺ level is the same all throughout the SR; however, whether the Ca²⁺ buffer capacity is the same in all the SR is unknown. We have estimated indirectly the luminal Ca²⁺ buffer capacity of the SR by comparing the reduction in SR Ca²⁺ levels with the corresponding increase in [Ca²⁺]_i during activation of either IP₃Rs with carbachol or RyRs with caffeine, in smooth muscle cells from guinea pig urinary bladder. We have determined that carbachol-sensitive SR has a 2.4 times larger Ca²⁺ buffer capacity than caffeine-sensitive SR. Rapid inhibition of SERCA pumps with thapsigargin revealed that this pump activity accounts for 80% and 60% of the Ca²⁺ buffer capacities of carbachol- and caffeine-sensitive SR, respectively. Moreover, the Ca²⁺ buffer capacity of carbachol-sensitive SR was similar to caffeine-sensitive SR when SERCA pumps were inhibited. Similar rates of Ca²⁺ replenishments suggest similar levels of SERCA pump activities for either carbachol- or caffeine-sensitive SR. Paired pulses of caffeine, in conditions of low Ca²⁺ influx, indicate the relevance of luminal SR Ca²⁺ buffer capacity in the [Ca²⁺]_i response. To further study the importance of luminal SR Ca²⁺ buffer capacity in the release process we used low levels of heparin to partially inhibit IP₃Rs. This condition revealed carbachol-induced transient increase of luminal SR Ca²⁺ levels provided that SERCA pumps were active. It thus appears that SERCA pump activity keeps the luminal SR Ca²⁺-binding proteins in the high-capacity, low-affinity conformation, particularly for IP₃R-mediated Ca²⁺ release.     

Enhanced proliferation and altered calcium handling in RGS2-deficient vascular smooth muscle cells

Abstract

Context: Regulator of G-protein signaling-2 (RGS2) inhibits Gq-mediated regulation of Ca²⁺ signalling in vascular smooth muscle cells (VSMC). Objective: RGS2 knockout (RGS2KO) mice are hypertensive and show arteriolar remodeling. VSMC proliferation modulates intracellular Ca²⁺ concentration [Ca²⁺]i. RGS2 involvement in VSMC proliferation had not been examined. Methods: Thymidine incorporation and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) conversion assays measured cell proliferation. Fura-2 ratiometric imaging quantified [Ca²⁺]i before and after UTP and thapsigargin. [³H]-labeled inositol was used for phosphoinositide hydrolysis. Quantitative RT-PCR and confocal immunofluorescence of select Ca²⁺ transporters was performed in primary aortic VSMC. Results and discussion: Platelet-derived growth factor (PDGF) increased S-phase entry and proliferation in VSMC from RGS2KO mice to a greater extent than in VSMC from wild-type (WT) controls. Consistent with differential PDGF-induced changes in Ca²⁺ homeostasis, RGS2KO VSMC showed lower resting [Ca²⁺]i but higher thapsigargin-induced [Ca²⁺]i as compared with WT. RGS2KO VSMC expressed lower mRNA levels of plasma membrane Ca²⁺ ATPase-4 (PMCA4) and Na⁺ Ca²⁺ Exchanger (NCX), but higher levels of sarco-endoplasmic reticulum Ca²⁺ ATPase-2 (SERCA2). Western blot and immunofluorescence revealed similar differences in PMCA4 and SERCA2 protein, while levels of NCX protein were not reduced in RGS2KO VSMC. Consistent with decreased Ca²⁺ efflux activity, ⁴⁵Ca-extrusion rates were lower in RGS2KO VSMC. These differences were reversed by the PMCA inhibitor La³⁺, but not by replacing extracellular Na⁺ with choline, implicating differences in the activity of PMCA and not NCX. Conclusion: RGS2-deficient VSMC exhibit higher rates of proliferation and coordinate plasticity of Ca²⁺-handling mechanisms in response to PDGF stimulation.     

Na+ entry via TRPC6 causes Ca2+ entry via NCX reversal in ATP stimulated smooth muscle cells

Reversal of the Na+/Ca2+ -exchanger (NCX) has been shown to mediate Ca2+ influx during activation of G-protein linked receptors. Functional coupling between the reverse-mode NCX and the canonical transient receptor potential channels (TRPCs) has been proposed to mediate Ca2+ influx in HEK-293 cells overexpressing TRPC3. In this communication we present evidence for similar functional coupling of NCX to endogenously expressed TRPC6 in rat aorta smooth muscle cells. Selective inhibition of reverse-mode NCX with KB-R7943 and of non-selective cation-channels with SKF-96365 abolished Ca2+ influx in response to agonist stimulation (ATP). Expression of a dominant negative TRPC6 mutant also reduced the Ca2+ influx in proportion to its transfection efficiency. Calyculin A, which is known to disrupt the junctions of the plasma membrane and sarco/endoplasmic reticulum, increased global Na+ elevations and reduced stimulated Ca2+ influx. Together our data provide evidence that localized Na+ elevations are generated by TRPC6 and drive reversal of NCX to mediate Ca2+ influx.     

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.     

Ca2+ Modulation of sarcoplasmic reticulum Ca2+ release in rat skeletal muscle fibers

Ca²⁺ transients and the rate of Ca²⁺ release (dCa_REL/dt) from the sarcoplasmic reticulum (SR) in voltage-clamped, fast-twitch skeletal muscle fibers from the rat were studied with the double Vaseline gap technique and using mag-fura-2 and fura-2 as Ca²⁺ indicators. Single pulse experiments with different returning potentials showed that Ca²⁺ removal from the myoplasm is voltage independent. Thus, the myoplasmic Ca²⁺ removal (dCa_REM/dt) was studied by fitting the decaying phase of the Ca²⁺ transient (Melzer, Ríos & Schneider, 1986) and dCa_REL/dt was calculated as the difference between dCa/dt and dCa_REM/dt. The fast Ca²⁺ release decayed as a consequence of Ca²⁺ inactivation of Ca²⁺ release. Double pulse experiments showed inactivation of the fast Ca²⁺ release depending on the prepulse duration. At constant interpulse interval, long prepulses (200 msec) induced greater inactivation of the fast Ca²⁺ release than shorter depolarizations (20 msec). The correlation (r) between the myoplasmic [Ca²⁺]_i and the inhibited amount of Ca²⁺ release was 0.98. The [Ca²⁺]_i for 50% inactivation of dCa_REL/dt was 0.25 m, and the minimum number of sites occupied by Ca²⁺ to inactivate the Ca²⁺ release channel was 3.0. These data support Ca²⁺ binding and inactivation of SR Ca²⁺ release.This work was supported by Grant-in-Aid from the American Heart Association (National) and Muscular Dystrophy Association (USA). Part of this work was developed in Dr. Stefani's laboratory at Baylor College of Medicine.     

Cytoplasmic Ca2+ does not inhibit the cardiac muscle sarcoplasmic reticulum ryanodine receptor Ca2+ channel,although Ca2+-induced Ca2+ inactivation of Ca2+ release is observed in native vesicles

Single channel properties of cardiac and fast-twitch skeletal muscle sarcoplasmic reticulum (SR) release channels were compared in a planar bilayer by fusing SR membranes in a Cs⁺-conducting medium. We found that the pharmacology, Cs⁺ conductance and selectivity to monovalent and divalent cations of the two channels were similar. The cardiac SR channel exhibited multiple kinetic states. The open and closed lifetimes were not altered from a range of 10^–7 to 10^–3 M Ca²⁺, but the proportion of closed and open states shifted to shorter closings and openings, respectively.However, while the single channel activity of the skeletal SR channel was activated and inactivated by micromolar and millimolar Ca²⁺, respectively, the cardiac SR channel remained activated in the presence of high [Ca²⁺]. In correlation to these studies, [³H]ryanodine binding by the receptors of the two channel receptors was inhibited by high [Ca²⁺] in skeletal but not in cardiac membranes in the presence of adenine nucleotides. There is, however, a minor inhibition of [³H]ryanodine binding of cardiac SR at millimolar Ca²⁺ in the absence of adenine nucleotides.When Ca²⁺-induced Ca²⁺ release was examined from preloaded native SR vesicles, the release rates followed a normal biphasic curve, with Ca²⁺-induced inactivation at high [Ca²⁺] for both cardiac and skeletal SR. Our data suggest that the molecular basis of regulation of the SR Ca²⁺ release channel in cardiac and skeletal muscle is different, and that the cardiac SR channel isoform lacks a Ca²⁺-inactivated site.This work was supported by research grants from the National Institutes of Health HL13870 and AR38970, and the Texas Affiliate of the American Heart Association, 91A-188. M. Fill was the recipient of an NIH fellowship AR01834.     

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.     

Regulation of Ca2+ mobilization by prolactin in mammary gland cells: possible role of secretory pathway Ca2+-ATPase type 2

Regulatory role of prolactin (PRL) on Ca2+ mobilization in human mammary gland cell line MCF-7 was examined. Direct addition of PRL did not affect cytoplasmic Ca2+ concentration ([Ca2+]i); however, treatment with PRL for 24h significantly decreased the peak level and duration time of [Ca2+]i elevation evoked by ATP or thapsigargin (TG). Intracellular Ca2+ release by IP3 or TG in permeablized cells was not decreased after PRL-treatment, indicating that the Ca2+ release was not impaired by PRL treatment. Extracellular Ca2+ entry evoked by ATP or TG was likely to be intact, because entry of extracellular Ba2+ was not affected by PRL treatment. Among Ca2+-ATPases expressed in MCF-7 cells, we found significant increase of secretory pathway Ca2+-ATPase type 2 (SPCA2) mRNA in PRL-treated cells by RT-PCR experiments including quantitative RT-PCR. Knockdown of SPCA2 by siRNA in PRL-treated cells showed similar Ca2+ mobilization to that in PRL-untreated cells. The present results suggest that PRL facilitates Ca2+ transport into Golgi apparatus and may contribute the supply of Ca2+ to milk.     

Functional role of stromal interaction molecule 1 (STIM1) in vascular smooth muscle cells

We investigated the functional role of STIM1, a Ca(2+) sensor in the endoplasmic reticulum (ER) that regulates store-operated Ca(2+) entry (SOCE), in vascular smooth muscle cells (VSMCs). STIM1 was mainly localized at the ER and plasma membrane. The knockdown of STIM1 expression by small interfering (si) RNA drastically decreased SOCE. In contrast, an EF-hand mutant of STIM1, STIM1(E87A), produced a marked increase in SOCE, which was abolished by co-transfection with siRNA to transient receptor potential canonical 1 (TRPC1). In addition, transfection with siRNA against STIM1 suppressed phosphorylation of cAMP-responsive element binding protein (CREB) and cell growth. These results suggest that STIM1 is an essential component of SOCE and that it is involved in VSMC proliferation.