Contribution of both Ca2+ entry and Ca2+ sensitization to the alpha1-adrenergic vasoconstriction of rat penile small arteries

Sympathetic adrenergic nerves maintain the flaccid state of the penis through the tonic release of norepinephrine that contracts trabecular and arterial smooth muscle. Simultaneous measurements of intracellular Ca(2+) concentration ([Ca(2+)](i)) and tension and experiments with alpha-toxin-permeabilized arteries were performed in branches of the rat dorsal penile artery to investigate the intracellular Ca(2+) signaling pathways underlying alpha(1)-adrenergic vasoconstriction. Phenylephrine increased both [Ca(2+)](i) and tension, these increases being abolished by extracellular Ca(2+) removal and reduced by about 50% by the L-type Ca(2+) channel blocker nifedipine (0.3 microM). Non-L-type Ca(2+) entry through store-operated channels was studied by inhibiting the sarcoplasmic reticulum Ca(2+)-ATPase with cyclopiazonic acid (CPA). CPA (30 microM) induced variable phasic contractions that were abolished by extracellular Ca(2+) removal and by the store-operated channels antagonist 2-aminoethoxydiphenyl borate (2-APB, 50 microM) and largely inhibited by nifedipine (0.3 microM). CPA induced a sustained increase in [Ca(2+)](i) that was reduced in a Ca(2+)-free medium. Under conditions of L-type channels blockade, Ca(2+) readmission after store depletion with CPA evoked a sustained and marked elevation in [Ca(2+)](i) not coupled to contraction. 2-APB (50 microM) inhibited the rise in [Ca(2+)](i) evoked by CPA and the nifedipine-insensitive increases in both [Ca(2+)](i) and contraction elicited by phenylephrine. In alpha-toxin-permeabilized penile arteries, activation of G proteins with guanosine 5'-O-(3-thiotriphosphate) and of the alpha(1)-adrenoceptor with phenylephrine both enhanced the myofilament sensitivity to Ca(2+). This Ca(2+) sensitization was reduced by selective inhibitors of PKC, tyrosine kinase (TK), and Rho kinase (RhoK) by 43%, 67%, and 82%, respectively. As a whole, the present data suggest the alpha(1)-adrenergic vasoconstriction in penile small arteries involves Ca(2+) entry through both L-type and 2-APB-sensitive receptor-operated channels, as well as Ca(2+) sensitization mechanisms mediated by PKC, TK, and RhoK. A capacitative Ca(2+) entry coupled to noncontractile functions of the smooth muscle cell is also demonstrated.     

Impaired Ca2+ handling in penile arteries from prediabetic Zucker rats: involvement of Rho kinase

Diabetes is associated with an increased vascular tone usually involved in the pathogenesis of diabetic cardiovascular complications such as hypertension, stroke, coronary artery disease, or erectile dysfunction (ED). Enhanced contractility of penile erectile tissue has been associated with augmented activity of the RhoA/Rho kinase (RhoK) pathway in models of diabetes-associated ED. The present study assessed whether abnormal vasoconstriction in penile arteries from prediabetic obese Zucker rats (OZRs) is due to changes in the intracellular Ca(2+) concentration ([Ca(2+)](i)) and/or in myofilament Ca(2+) sensitivity. Penile arteries from OZRs and lean Zucker rats (LZRs) were mounted on microvascular myographs for simultaneous measurements of [Ca(2+)](i) and tension. The relationships between [Ca(2+)](i) and contraction for the α(1)-adrenergic vasoconstrictor phenylephrine (PE) were left shifted and steeper in OZRs compared with LZRs, although the magnitude of the contraction was similar in both groups. In contrast, the vasoconstriction induced by the thromboxane A(2) receptor agonist U-46619 was augmented in arteries from OZRs, and this increase was associated with an increase in both the sensitivity and maximum responses to Ca(2+). The RhoK inhibitor Y-27632 (10 μM) reduced the vasoconstriction induced by PE to a greater extent in OZRs than in LZRs, without altering Ca(2+). Y-27632 inhibited with a greater potency the contraction elicited by high KCl in arteries from OZRs compared with LZRs without changing [Ca(2+)](i). RhoK-II expression was augmented in arteries from OZRs. These results suggest receptor-specific changes in the Ca(2+) handling of penile arteries under conditions of metabolic syndrome. Whereas augmented vasoconstriction upon activation of the thromboxane A(2) receptor is coupled to enhanced Ca(2+) entry, a RhoK-mediated enhancement of myofilament Ca(2+) sensitivity is coupled with the α(1)-adrenergic vasoconstriction in penile arteries from OZRs.     

Rho kinase and Ca2+ entry mediate increased pulmonary and systemic vascular resistance in L-NAME-treated rats

The small GTP-binding protein and its downstream effector Rho kinase play an important role in the regulation of vasoconstrictor tone. Rho kinase activation maintains increased pulmonary vascular tone and mediates the vasoconstrictor response to nitric oxide (NO) synthesis inhibition in chronically hypoxic rats and in the ovine fetal lung. However, the role of Rho kinase in mediating pulmonary vasoconstriction after NO synthesis inhibition has not been examined in the intact rat. To address this question, cardiovascular responses to the Rho kinase inhibitor fasudil were studied at baseline and after administration of an NO synthesis inhibitor. In the intact rat, intravenous injections of fasudil cause dose-dependent decreases in systemic arterial pressure, small decreases in pulmonary arterial pressure, and increases in cardiac output. L-NAME caused a significant increase in pulmonary and systemic arterial pressures and a decrease in cardiac output. The intravenous injections of fasudil after L-NAME caused dose-dependent decreases in pulmonary and systemic arterial pressure and increases in cardiac output, and the percent decreases in pulmonary arterial pressure in response to the lower doses of fasudil were greater than decreases in systemic arterial pressure. The Ca(++) entry blocker isradipine also decreased pulmonary and systemic arterial pressure in L-NAME-treated rats. Infusion of sodium nitroprusside restored pulmonary arterial pressure to baseline values after administration of L-NAME. These data provide evidence in support of the hypothesis that increases in pulmonary and systemic vascular resistance following L-NAME treatment are mediated by Rho kinase and Ca(++) entry through L-type channels, and that responses to L-NAME can be reversed by an NO donor.     

Arachidonic acid-induced Ca2+ entry is involved in early steps of tumor angiogenesis

Growth factor-induced intracellular calcium signals in endothelial cells regulate cytosolic and nuclear events involved in the angiogenic process. Among the intracellular messengers released after proangiogenic stimulation, arachidonic acid (AA) plays a key role and its effects are strictly related to calcium homeostasis and cell proliferation. Here, we studied AA-induced intracellular calcium signals in endothelial cells derived from human breast carcinomas (B-TEC). AA promotes B-TEC proliferation and organization of vessel-like structures in vitro. The effect is directly mediated by the fatty acid without a significant contribution of its metabolites. AA induces Ca(2+)(i) signals in the entire capillary-like structure during the early phases of tubulogenesis in vitro. No such responses are detectable in B-TECs organized in more structured tubules. In B-TECs growing in monolayer, AA induces two different signals: a Ca(2+)(i) increase due to Ca(2+) entry and an inhibition of store-dependent Ca(2+) entry induced by thapsigargin or ATP. An inhibitor of Ca(2+) entry and angiogenesis, carboxyamidotriazole, significantly and specifically decreases AA-induced B-TEC tubulogenesis, as well as AA-induced Ca(2+) signals in B-TECs. We conclude that (a) AA-activated Ca(2+) entry is associated with the progression through the early phases of angiogenesis, mainly involving proliferation and tubulogenesis, and it is down-regulated during the reorganization of tumor-derived endothelial cells in capillary-like structures; and (b) inhibition of AA-induced Ca(2+) entry may contribute to the antiangiogenic action of carboxyamidotriazole.     

Capacitative Ca2+ entry is involved in cAMP synthesis in mouse parotid acini

Muscarinicreceptor interaction leading to augmentation ofisoproterenol-stimulated cAMP accumulation in mouse parotid acini involves Ca²⁺ (28). Theeffectiveness of capacitative Ca²⁺entry and intracellular Ca²⁺release on this response was determined in time course studies by usingthree independent tools to manipulate the free intracellular Ca²⁺ concentration: the muscarinicagonist carbachol, thapsigargin, and ionomycin. Time course studiesrevealed that Ca²⁺ release fromintracellular stores by carbachol produced an early rapid increase(0.25-0.5 min) in stimulated cAMP levels, whereas capacitativeCa²⁺ entry resulted in a sustainedincrease in stimulated cAMP levels that was blocked byLa³⁺. CapacitativeCa²⁺ entry, alone, was involved inthapsigargin and ionomycin augmentation of stimulated cAMPaccumulation. The inability of phosphodiesterase inhibitors,3-isobutyl-1-methylxanthine and milrinone, to prevent agonistaugmentation of cAMP levels, as well as the finding that the type VIIIadenylyl cyclase (ACVIII) is expressed in parotid acini, suggests thatcapacitative Ca²⁺ entry augmentsstimulated cAMP accumulation, at least in part, via activation of thisadenylyl cyclase isoenzyme.

     

Ca2+-independent activation of Bruton's tyrosine kinase is required for store-mediated Ca2+ entry in human platelets

Store-mediated Ca(2+) entry (SMCE), which is rapidly activated by depletion of the intracellular Ca(2+) stores, is a major mechanism for Ca(2+) influx. Several studies have involved tyrosine kinases in the activation of SMCE, such as pp60(src), although at present those involved in the early activation steps are unknown. Here we report the involvement of Bruton's tyrosine kinase (Btk) in the early stages of SMCE in human platelets. Cell treatment with thrombin or thapsigargin (TG) plus ionomycin (Iono) results in rapid activation of Btk, which was independent of rise in intracellular Ca(2+) concentration ([Ca(2+)](i)) but dependent on H(2)O(2) generation. Platelet treatment with Btk inhibitors, LFM-A13 or terreic acid, significantly reduced TG+Iono- and thrombin-evoked SMCE. Btk was rapidly activated by addition of low concentrations of H(2)O(2), whose effect on Ca(2+) entry was prevented by Btk inhibitors. Our results indicate that pp60(src) and Btk co-immunoprecipitate after platelet stimulation with TG+Iono, thrombin or H(2)O(2). In addition, we have found that LFM-A13 impaired actin filament reorganization after store depletion and agonist-induced activation of pp60(src), while the inhibitor of pp60(src), a protein that requires actin reorganization for its activation, did not modify Btk activation, suggesting that Btk is upstream of pp60(src). We propose a role for Btk in the early steps of activation of SMCE in human platelets.     

Involvement of protein kinase C, tyrosine kinases, and Rho kinase in Ca(2+) handling of human small arteries

The mechanisms of Ca(2+) handling and sensitization were investigated in human small omental arteries exposed to norepinephrine (NE) and to the thromboxane A(2) analog U-46619. Contractions elicited by NE and U-46619 were associated with an increase in intracellular Ca(2+) concentration ([Ca(2+)](i)), an increase in Ca(2+)-independent signaling pathways, or an enhancement of the sensitivity of the myofilaments to Ca(2+). The two latter pathways were abolished by protein kinase C (PKC), tyrosine kinase (TK), and Rho-associated protein kinase (ROK) inhibitors. In Ca(2+)-free medium, both NE and U-46619 elicited an increase in tension that was greatly reduced by PKC inhibitors and abolished by caffeine or ryanodine. After depletion of Ca(2+) stores with NE and U-46619 in Ca(2+)-free medium, addition of CaCl(2) in the continuous presence of the agonists produced increases in [Ca(2+)](i) and contractions that were inhibited by nitrendipine and TK inhibitors but not affected by PKC inhibitors. NE and U-46619 induced tyrosine phosphorylation of a 42- or a 58-kDa protein, respectively. These results indicate that the mechanisms leading to contraction elicited by NE and U-46619 in human small omental arteries are composed of Ca(2+) release from ryanodine-sensitive stores, Ca(2+) influx through nitrendipine-sensitive channels, and Ca(2+) sensitization and/or Ca(2+)-independent pathways. They also show that the TK pathway is involved in the tonic contraction associated with Ca(2+) entry, whereas TK, PKC, and ROK mechanisms regulate Ca(2+)-independent signaling pathways or Ca(2+) sensitization.     

Rho kinase is an effector underlying Ca2+-desensitizing hypoxic relaxation in porcine coronary artery

Acute hypoxia dilates most systemic arteries leading to increased tissue perfusion. We have previously shown that at high-stimulus conditions, porcine coronary artery was relaxed by hypoxia without a change in intracellular [Ca(2+)] (27). This Ca(2+)-desensitizing hypoxic relaxation (CDHR) was validated in permeabilized porcine coronary artery smooth muscle (PCASM) in which hypoxia decreased force and myosin regulatory light chain phosphorylation (p-MRLC) despite fixed [Ca(2+)] (10). Rho kinase-dependent phosphorylation of myosin phosphatase-targeting subunit 1 (p-MYPT1) is associated with decreased MRLC phosphatase activity and increased Ca(2+) sensitivity of both p-MRLC and force. We recently reported that p-MYPT1 dephosphorylation was a key effector in CDHR (33). In the current study, we tested the hypothesis that Rho kinase and not p-MYPT1 phosphatase is the regulated enzyme involved in CDHR. We used alpha-toxin to permeabilize deendothelialized PCASM. CDHR was attenuated in contractions attributable to myosin light chain kinase (MLCK, in the presence of the Rho kinase inhibitor Y-27632). In contrast, hypoxia relaxed contractions attributable to Rho kinase phosphorylation of MYPT1 and MRLC or MRLC alone (in the presence of the MLCK inhibitor ML7). Using an in situ assay, we showed that Rho kinase activity, measured as thiophosphorylation of MYPT1 and MRLC, was nearly abolished by hypoxia. The in vitro activity of the catalytically active fragment of Rho kinase was not affected by hypoxia. Our evidence strongly implicates that hypoxia directly inhibits Rho kinase-dependent phosphorylation of MYPT1. This underlies the decreases in both p-MYPT1 and p-MRLC and thereby leads to the Ca(2+)-desensitizing hypoxic relaxation.     

Rho kinase inhibition partly weakens myogenic reactivity in rat small arteries by changing calcium sensitivity

The hypothesis that Rho kinase is involved in myogenic reactivity was investigated in pressurized rat tail small arteries using videomicroscopic diameter determination and calcium fluorimetry. The potent Rho kinase inhibitor Y-27632 reversibly increased vessel diameter at 80 mmHg without changing the intracellular calcium concentration ([Ca](i)) shifting the relationship between diameter change and [Ca](i) to higher calcium levels. Neither endothelium removal nor inhibition of neural transmission affected the Y-27632-induced effect. Y-27632 at 3 x 10(-6) mol/l attenuated the myogenic response in the pressure range from 10 to 120 mmHg, shifting the relationship between vessel tone and [Ca](i) to higher calcium levels. In addition, the Y-27632-induced shift of the relationship between vessel tone and [Ca](i) was larger at 80 than at 10 mmHg. These results suggest that smooth muscle cell Rho kinase in rat tail small arteries 1) is in an active state partly determining the level of the myogenic tone, and 2) alters the strength of the myogenic response by changing calcium sensitivity, probably caused by the pressure-induced activation of the kinase.     

The Ca2+ release-activated Ca2+ current (ICRAC) mediates store-operated Ca2+ entry in rat microglia

Ca²⁺ signaling plays a central role in microglial activation, and several studies have demonstrated a store-operated Ca²⁺ entry (SOCE) pathway to supply this ion. Due to the rapid pace of discovery of novel Ca²⁺ permeable channels, and limited electrophysiological analyses of Ca²⁺ currents in microglia, characterization of the SOCE channels remains incomplete. At present, the prime candidates are ‘transient receptor potential’ (TRP) channels and the recently cloned Orai1, which produces a Ca²⁺-release-activated Ca²⁺ (CRAC) current. We used cultured rat microglia and real-time RT-PCR to compare expression levels of Orai1, Orai2, Orai3, TRPM2, TRPM7, TRPC1, TRPC2, TRPC3, TRPC4, TRPC5, TRPC6 and TRPC7 channel genes. Next, we used Fura-2 imaging to identify a store-operated Ca²⁺ entry (SOCE) pathway that was reduced by depolarization and blocked by Gd3+, SKF-96365, diethylstilbestrol (DES), and a high concentration of 2-aminoethoxydiphenyl borate (50 μM 2-APB). The Fura-2 signal was increased by hyperpolarization, and by a low concentration of 2-APB (5 μM), and exhibited Ca²⁺-dependent potentiation. These properties are entirely consistent with Orai1/CRAC, rather than any known TRP channel and this conclusion was supported by patch-clamp electrophysiological analysis. We identified a store-operated Ca²⁺ current with the same properties, including high selectivity for Ca²⁺ over monovalent cations, pronounced inward rectification and a very positive reversal potential, Ca²⁺-dependent current potentiation, and block by SKF-96365, DES and 50 μM 2-APB. Determining the contribution of Orai1/CRAC in different cell types is crucial to future mechanistic and therapeutic studies; this comprehensive multi-strategy analysis demonstrates that Orai1/CRAC channels are responsible for SOCE in primary microglia.     

Neuronal galvanotropism is independent of external Ca2+ entry or internal Ca2+ gradients

The mechanism by which growing neurites sense and respond to small applied electrical fields is not known, but there is some evidence that the entry of Ca²⁺ from the external medium, with the subsequent formation of intracellular Ca²⁺ gradients, is important in this process. We have employed two approaches to test this idea. Xenopus spinal neurites were exposed to electrical fields in a culture medium in which Ca²⁺ was chelated to very low levels compared to the normal extracellular concentration of 2 mM. In other experiments, loading the neurites with the calcium buffer, 1,2‐bis(o‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid (BAPTA), disrupted the putative internal Ca²⁺ gradients, and the effects on the electrical response were determined. Fields of 100 mV/mm were applied for 12 h, and no difference was detected in the cathodal turning response between the treated neurites and the untreated controls. Using the Differential Growth Index (DGI), an asymmetry index, to quantitate the turning response, we recorded DGIs of −0.64, −0.65, and −0.62 for control cells, cells in Ca²⁺‐free medium, and cells preloaded with BAPTA, respectively. Furthermore, we detected an increase in neurite length for those neurons cultured in Ca²⁺‐free medium; they were 1.5–1.7 times as long as neurites from neurons cultured in normal Ca²⁺ medium. Likewise, we found that BAPTA‐loaded neurites were longer than control neurites. Our data indicate that neuronal galvanotropism is independent of the entry of external Ca²⁺ or of internal Ca²⁺ gradients. Both cell‐permeant agonistic and antagonistic analogs of cyclic 3′,5′‐adenosine monophosphate (cAMP) increased the response to applied electrical fields. © 2000 John Wiley & Sons, Inc. J Neurobiol 45: 30–38, 2000     

Ca2+-independent phospholipase A2 is a novel determinant of store-operated Ca2+ entry

Store-operated cation (SOC) channels and capacitative Ca(2+) entry (CCE) play very important role in cellular function, but the mechanism of their activation remains one of the most intriguing and long lasting mysteries in the field of Ca(2+) signaling. Here, we present the first evidence that Ca(2+)-independent phospholipase A(2) (iPLA(2)) is a crucial molecular determinant in activation of SOC channels and store-operated Ca(2+) entry pathway. Using molecular, imaging, and electrophysiological techniques, we show that directed molecular or pharmacological impairment of the functional activity of iPLA(2) leads to irreversible inhibition of CCE mediated by nonselective SOC channels and by Ca(2+)-release-activated Ca(2+) (CRAC) channels. Transfection of vascular smooth muscle cells (SMC) with antisense, but not sense, oligonucleotides for iPLA(2) impaired thapsigargin (TG)-induced activation of iPLA(2) and TG-induced Ca(2+) and Mn(2+) influx. Identical inhibition of TG-induced Ca(2+) and Mn(2+) influx (but not Ca(2+) release) was observed in SMC, human platelets, and Jurkat T-lymphocytes when functional activity of iPLA(2) was inhibited by its mechanism-based suicidal substrate, bromoenol lactone (BEL). Moreover, irreversible inhibition of iPLA(2) impaired TG-induced activation of single nonselective SOC channels in SMC and BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid)-induced activation of whole-cell CRAC current in rat basophilic leukemia cells. Thus, functional iPLA(2) is required for activation of store-operated channels and capacitative Ca(2+) influx in wide variety of cell types.     

Store-operated Ca2+ entry is not required for fertilization-induced Ca2+ signaling in mouse eggs

Repetitive oscillations in cytoplasmic Ca²⁺ due to periodic Ca²⁺ release from the endoplasmic reticulum (ER) drive mammalian embryo development following fertilization. Influx of extracellular Ca²⁺ to support the refilling of ER stores is required for sustained Ca²⁺ oscillations, but the mechanisms underlying this Ca²⁺ influx are controversial. Although store-operated Ca²⁺ entry (SOCE) is an appealing candidate mechanism, several groups have arrived at contradictory conclusions regarding the importance of SOCE in oocytes and eggs. To definitively address this question, Ca²⁺ influx was assessed in oocytes and eggs lacking the major components of SOCE, the ER Ca²⁺ sensor STIM proteins, and the plasma membrane Ca²⁺ channel ORAI1. We generated oocyte-specific conditional knockout (cKO) mice for Stim1 and Stim2, and also generated Stim1/2 double cKO mice. Females lacking one or both STIM proteins were fertile and their ovulated eggs displayed normal patterns of Ca²⁺ oscillations following fertilization. In addition, no impairment was observed in ER Ca²⁺ stores or Ca²⁺ influx following store depletion. Similar studies were performed on eggs from mice globally lacking ORAI1; no abnormalities were observed. Furthermore, spontaneous Ca²⁺ influx was normal in oocytes from Stim1/2 cKO and ORAI1-null mice. Finally, we tested if TRPM7-like channels could support spontaneous Ca²⁺ influx, and found that it was largely prevented by NS8593, a TRPM7-specific inhibitor. Fertilization-induced Ca²⁺ oscillations were also impaired by NS8593. Combined, these data robustly show that SOCE is not required to support appropriate Ca²⁺ signaling in mouse oocytes and eggs, and that TRPM7-like channels may contribute to Ca²⁺ influx that was previously attributed to SOCE.     

Endogenously expressed Trp1 is involved in store-mediated Ca2+ entry by conformational coupling in human platelets

Physical interaction between transient receptor potential (Trp) channels and inositol 1,4,5-trisphosphate receptors (IP(3)Rs) has been presented as a candidate mechanism for the activation of store-mediated Ca(2+) entry. The role of a human homologue of Drosophila transient receptor potential channel, hTrp1, in the conduction of store-mediated Ca(2+) entry was examined in human platelets. Incubation of platelets with a specific antibody, which recognizes the extracellular amino acid sequence 557-571 of hTrp1, inhibited both store depletion-induced Ca(2+) and Mn(2+) entry in a concentration-dependent manner. Stimulation of platelets with the physiological agonist thrombin activated coupling between the IP(3) receptor type II and endogenously expressed hTrp1. This event was reversed by refilling of the internal Ca(2+) stores but maintained after removal of the agonist if the stores were not allowed to refill. Inhibition of IP(3) recycling using Li(+) or inhibition of IP(3)Rs with xestospongin C or treatment with jasplakinolide, to stabilize the cortical actin filament network, abolished thrombin-induced coupling between hTrp1 and IP(3)R type II. Incubation with the anti-hTrp1 antibody inhibited thrombin-evoked Ca(2+) entry without affecting Ca(2+) release from intracellular stores. These results provide evidence for the involvement of hTrp1 in the activation of store-mediated Ca(2+) entry by coupling to IP(3)R type II in normal human cells.     

O-linked N-acetylglucosaminylation is involved in the Ca2+ activation properties of rat skeletal muscle

O-Linked N-acetylglucosaminylation termed O-GlcNAc is a dynamic cytosolic and nuclear glycosylation that is dependent both on glucose flow through the hexosamine biosynthesis pathway and on phosphorylation because of the existence of a balance between phosphorylation and O-GlcNAc. This glycosylation is a ubiquitous post-translational modification, which probably plays an important role in many aspects of protein functions. We have previously reported that, in skeletal muscle, proteins of the glycolytic pathway, energetic metabolism, and contractile proteins were O-GlcNAc-modified and that O-Glc-NAc variations could control the muscle protein homeostasis and be implicated in the regulation of muscular atrophy. In this paper, we report O-N-acetylglucosaminylation of a number of key contractile proteins (i.e. myosin heavy and light chains and actin), which suggests that this glycosylation could be involved in skeletal muscle contraction. Moreover, our results showed that incubation of skeletal muscle skinned fibers in N-acetyl-d-glucosamine, in a concentration solution known to inhibit O-GlcNAc-dependent interactions, induced a decrease in calcium sensitivity and affinity of muscular fibers, whereas the cooperativity of the thin filament proteins was not modified. Thus, our results suggest that O-GlcNAc is involved in contractile protein interactions and could thereby modulate muscle contraction.     

Phospholipase C-gamma is required for agonist-induced Ca2+ entry

We report here that PLC-gamma isoforms are required for agonist-induced Ca2+ entry (ACE). Overexpressed wild-type PLC-gamma1 or a lipase-inactive mutant PLC-gamma1 each augmented ACE in PC12 cells, while a deletion mutant lacking the region containing the SH3 domain of PLC-gamma1 was ineffective. RNA interference to deplete either PLC-gamma1 or PLC-gamma2 in PC12 and A7r5 cells inhibited ACE. In DT40 B lymphocytes expressing only PLC-gamma2, overexpressed muscarinic M5 receptors (M5R) activated ACE. Using DT40 PLC-gamma2 knockout cells, M5R stimulation of ER Ca2+ store release was unaffected, but ACE was abolished. Normal ACE was restored by transient expression of PLC-gamma2 or a lipase-inactive PLC-gamma2 mutant. The results indicate a lipase-independent role of PLC-gamma in the physiological agonist-induced activation of Ca2+ entry.     

Lysosomal cysteine protease cathepsin S is involved in cancer cell motility by regulating store-operated Ca2+ entry

Cathepsin S (CTSS), a lysosomal cysteine protease, has been reported to be associated with extracellular matrix (ECM) degradation, thus promoting cell migration and invasion, but whether CTSS regulates other intracellular mechanisms during metastasis remains unknown. The expression of CTSS was knocked down using siRNA transfection, and enzymatic activity was inhibited by the highly-selective CTSS inhibitor RJW-58. The results of in vitro functional assays, western blot analysis, and an in vivo colonization model demonstrated that CTSS was positively related to cellular adhesive ability. Moreover, both CTSS knockdown and inhibition significantly decreased Ca²⁺ influx via store-operated Ca²⁺ entry (SOCE) without changing STIM1 and Orai1 expression levels, while RJW-58 dose-dependently reduced the activation of the Ca²⁺-dependent downstream effectors, NFAT1 and Rac1. The results of immunoprecipitation assays demonstrated that CTSS could bind to STIM1, which was reversed by CTSS inhibition. In addition, confocal microscopy and super-resolution imaging showed that CTSS inhibition led to STIM1 puncta accumulation in the endoplasmic reticulum and reduced the interaction between active STIM1 and EB1. In conclusion, we have demonstrated for the first time that the lysosomal cysteine protease, CTSS, plays an important role in mediating Ca²⁺ homeostasis by regulating STIM1 trafficking, which leads to the suppression of cell migration and invasion.     

Capacitative Ca2+ entry involves Ca2+ influx factor in rat glioma C6 cells.

Capacitative Ca2+ entry exists in rat glioma C6 cells; however, how the information of depletion of Ca2+ in intracellular stores transmits to the plasma membrane is unknown. In the present study, we examined whether Ca2+ influx factor (CIF) causes capacitative Ca2+ entry in C6 cells. CIF was extracted from non-treated (Non-CIF), bombesin-treated (BBS-CIF) and thapsigargin-treated (TG-CIF) C6 cells by a reverse-phase silica cartridge. The addition of BBS-CIF and TG-CIF gradually increased cytoplasmic Ca2+ concentration ([Ca2+]i) but Non-CIF did not increase [Ca2+]i. Neither BBS-CIF nor TG-CIF elevated [Ca2+]i in the absence of extracellular Ca2+. Gd3+ inhibited the increase in [Ca2+]i induced by BBS-CIF and TG-CIF. Genistein abolished an elevation of [Ca2+]i induced by BBS-CIF and TG-CIF. BBS-CIF and TG-CIF did not increase inositol 1,4,5-trisphosphate accumulation. The results suggest that capacitative Ca2+ entry is caused by CIF in rat glioma C6 cells.     

Norepinephrine activates store-operated Ca2+ entry coupled to large-conductance Ca2+-activated K+ channels in rat pinealocytes

Norepinephrine (NE) is one of the major neurotransmitters that determine melatonin production in the pineal gland. Although a substantial amount of Ca²⁺ influx is triggered by NE, the Ca²⁺ entry pathway and its physiological relevance have not been elucidated adequately. Herein we report that the Ca²⁺ influx triggered by NE significantly regulates the protein level of serotonin N-acetyltransferase, or arylalkylamine N-acetyltransferase (AANAT), a critical enzyme in melatonin production, and is responsible for maintaining the Ca²⁺ response after repetitive stimulation. Ca²⁺ entry evoked by NE was dependent on PLC activation. NE evoked a substantial amount of Ca²⁺ entry even after cells were treated with 1-oleoyl-2-acetyl-sn-glycerol (OAG), an analog of diacylglycerol. To the contrary, further OAG treatment after cells had been exposed to OAG did not evoke additional Ca²⁺ entry. Moreover, NE failed to induce further Ca²⁺ entry after the development of Ca²⁺ entry induced by thapsigargin (Tg), suggesting that the pathway of Ca²⁺ entry induced by NE might be identical to that of Tg. Interestingly, Ca²⁺ entry evoked by NE or Tg induced membrane hyperpolarization that was reversed by iberiotoxin (IBTX), a specific inhibitor of large-conductance Ca²⁺-activated K⁺ (BK) channels. Moreover, IBTX-sensitive BK current was observed during application of NE, suggesting that activation of the BK channels was responsible for the hyperpolarization. Furthermore, the activation of BK channels triggered by NE contributed to regulation of the protein level of AANAT. Collectively, these results suggest that NE triggers Ca²⁺ entry coupled to BK channels and that NE-induced Ca²⁺ entry is important in the regulation of AANAT. serotonin N-acetyltransferase; pineal gland