Functional relevance of the de novo coupling between hTRPC1 and type II IP3 receptor in store-operated Ca2+ entry in human platelets

Store-operated Ca2+ entry (SOCE), a major mechanism for Ca2+ entry in non-excitable cells, is regulated by the filling state of the intracellular Ca2+ stores. We have previously reported that a de novo conformational coupling between the type II IP3 receptor (IP3RII) and hTRPC1 channel occurs after depletion of the intracellular Ca2+ stores in human platelets, which might be involved in the activation of SOCE in these cells. Here we present for the first time direct evidence for the functional relevance of the coupling between hTRPC1 and IP3RII in SOCE in human platelets. Our data suggest that at least two pathways may contribute to SOCE in these cells. An early component, insensitive to cytochalasin D (Cyt D), is followed by a late component which is sensitive to Cyt D. Introduction of a peptide corresponding to IP3RII(317-334) (IP3BD-peptide(317-334)) in the cells by electrotransjection impairs the coupling between hTRPC1 and IP3RII but not the interaction between hTRPC1 and STIM1 induced by store depletion. Coimmunoprecipitation experiments indicated that endogenously expressed hTRPC1 interacts with the IP3BD-peptide(317-334). Electrotransjection of cells with IP3BD-peptide(317-334), significantly attenuated the late stage of Ca2+ and Mn2+ entry induced by 10 nM thapsigargin (TG) or 20 microM 2,5-di-(tert-butyl)-1,4-hydroquinone (TBHQ), providing evidence for a functional role of the de novo coupling between hTRPC1 and IP3RII in the activation of SOCE in human platelets.     

Intracellular Ca2+ store depletion induces the formation of macromolecular complexes involving hTRPC1, hTRPC6, the type II IP3 receptor and SERCA3 in human platelets

Endogenously expressed human canonical transient receptor potential 1 (hTRPC1) and human canonical transient receptor potential 6 (hTRPC6) have been shown to play a role in store-operated Ca2+ entry (SOCE) in human platelets, where two mechanisms for SOCE, regulated by the dense tubular system (DTS) or the acidic granules, have been identified. In cells preincubated for 1 min with 100 microM flufenamic acid we show that hTRPC6 is involved in SOCE activated by both mechanisms, as demonstrated by selective depletion of the DTS or the acidic stores, using thapsigargin (TG) (10 nM) or 2,5-di-(tert-butyl)-1,4-hydroquinone (TBHQ) (20 microM), respectively, although it is more relevant after acidic store depletion. Co-immunoprecipitation experiments indicated that depletion of both stores separately results in time-dependent interaction between hTRPC1 and hTRPC6, and also between both hTRPCs and the type II IP3 receptor (IP3RII). The latter was greater after treatment with TG. TBHQ-induced coupling between hTRPC1 and 6 was transient and decreased after 30s of treatment, while that induced by TG increased for at least 3 min. TBHQ induced association between SERCA3, located in the acidic stores, hTRPC1, hTRPC6 and Orai1. TBHQ also evoked coupling between SERCA3 and IP3RII, presumably located in the DTS, thus suggesting interplay between both Ca2+ stores. Similarly, TG induces the interaction of SERCA2b with hTRPC1 and 6 and the IP3RII. The interactions between hTRPC1, hTRPC6, IP3RII and SERCA3 were impaired by disruption of the microtubules, supporting a role for microtubules in Ca2+ homeostasis. In conclusion, the present data demonstrate for the first time that hTRPC1, hTRPC6, IP3RII and SERCA3 are parts of a macromolecular protein complex activated by depletion of the intracellular Ca2+ stores in human platelets.     

The inositol trisphosphate receptor antagonist 2-aminoethoxydiphenylborate (2-APB) blocks Ca2+ entry channels in human platelets: cautions for its use in studying Ca2+ influx.

It has been reported that store-mediated Ca2+ entry (SMCE) in human platelets is likely to be mediated by a secretion-like coupling mechanism. Recently, 2-aminoethoxydiphenylborate (2-APB) has been used in the investigation of SMCE. Here, the mechanism of action of 2-APB is investigated in human platelets. In a Ca2+-free medium (EGTA added), addition of 0.1 U/ml thrombin caused an elevation in [Ca2+]i. Preincubation with 100 microM 2-APB for 170s abolished the release of internal Ca2+. In platelets whose internal Ca2+ stores had been depleted by treatment with 200 nM thapsigargin, addition of extracellular Ca2+ caused an elevation in [Ca2+]i indicative of SMCE. Preincubation with 2-APB decreased SMCE by 95.5+/-1.1%. After activation of SMCE, addition of 2-APB rapidly decreased [Ca2+]i to basal levels; in contrast, the coupling between Trp1 and IP3RII, which has been shown to play an important role in SMCE in platelets, remained intact at the same time points. The rate of decrease of [Ca2+]i and the absence of measurable latency in the effect of 2-APB were comparable to the effects of La3+ (a cation channel blocker). These data suggest that 2-APB may act as a blocker of Ca2+ permeable plasma membrane channels. These data provide further information regarding the mechanism and site of action of 2-APB and highlight the necessity of careful interpretation of work performed using this molecule.     

A key role for reverse Na+/Ca2+ exchange influenced by the actin cytoskeleton in store-operated Ca2+ entry in human platelets: evidence against the de novo conformational coupling hypothesis

We have previously demonstrated a role for the reorganization of the actin cytoskeleton in store-operated calcium entry (SOCE) in human platelets and interpreted this as evidence for a de novo conformational coupling step in SOCE activation involving the type II IP(3) receptor and the platelet hTRPC1-containing store-operated channel (SOC). Here, we present evidence challenging this model. The actin polymerization inhibitors cytochalasin D or latrunculin A significantly reduced Ca2+ but not Mn2+ or Na+ entry into thapsigargin (TG)-treated platelets. Jasplakinolide, which induces actin polymerization, also inhibited Ca2+ but not Mn2+ or Na+ entry. However, an anti-hTRPC1 antibody inhibited TG-evoked entry of all three cations, indicating that they all permeate an hTRPC1-containing store-operated channel (SOC). These results indicate that the reorganization of the actin cytoskeleton is not involved in SOC activation. The inhibitors of the Na+/Ca2+ exchanger (NCX), KB-R7943 or SN-6, caused a dose-dependent inhibition of Ca2+ but not Mn2+ or Na+ entry into TG-treated platelets. The effects of the NCX inhibitors were not additive with those of actin polymerization inhibitors, suggesting a common point of action. These results indicate a role for two Ca2+ permeable pathways activated following Ca2+ store depletion in human platelets: A Ca2+-permeable, hTRPC1-containing SOC and reverse Na+/Ca2+ exchange, which is activated following Na+ entry through the SOC and requires a functional actin cytoskeleton.     

Dual role of tubulin-cytoskeleton in store-operated calcium entry in human platelets

Two mechanisms for store-operated Ca(2+) entry (SOCE) regulated by two independent Ca(2+) stores, the dense tubular system (DTS) and the acidic stores, have been described in platelets. We have previously suggested that coupling between the type II IP(3) receptor (IP(3)RII) and hTRPC1, involving reorganization of the actin microfilaments, play an important role in SOCE. However, the involvement of the tubulin microtubules, located beneath the plasma membrane, remains unclear. Here we show that the microtubule disrupting agent colchicine reduced Ca(2+) entry stimulated by low concentrations (0.1 U/mL) of thrombin, which activates SOCE mostly by depleting acidic Ca(2+)-store. Consistently, colchicine reduced SOCE activated by 2,5 di-(tertbutyl)-1,4-hydroquinone (TBHQ), which selectively depletes the acidic Ca(2+) stores. In contrast, colchicine enhanced SOCE mediated by depletion of the DTS, induced by high concentrations of thapsigargin (TG), which depletes both the acidic Ca(2+) stores and the DTS, the major releasable Ca(2+) store in platelets. These findings were confirmed by using Sr(2+) as a surrogate for Ca(2+) entry. Colchicine attenuated the coupling between IP(3)RII and hTRPC1 stimulated by thrombin while it enhanced that evoked by TG. Paclitaxel, which induces microtubular stabilization and polymerization, exerted the opposite effects on thrombin- and TG-evoked SOCE and coupling between IP(3)RII and hTRPC1 compared with colchicine. Neither colchicine nor paclitaxel altered the ability of platelets to extrude Ca(2+). These findings suggest that tubulin microtubules play a dual role in SOCE, acting as a barrier that prevents constitutive SOCE regulated by DTS, but also supporting SOCE mediated by the acidic Ca(2+) stores.     

Overexpression of TRPC1 enhances pulmonary vasoconstriction induced by capacitative Ca2+ entry

Transient receptor potential (TRP) cation channels are a critical pathway for Ca2+ entry during pulmonary artery (PA) smooth muscle contraction. However, whether canonical TRP (TRPC) subunits and which TRP channel isoforms are involved in store depletion-induced pulmonary vasoconstriction in vivo remain unclear. This study was designed to test whether overexpression of the human TRPC1 gene (hTRPC1) in rat PA enhances pulmonary vasoconstriction due to store depletion-mediated Ca2+ influx. The hTRPC1 was infected into rat PA rings with an adenoviral vector. RT-PCR and Western blot analyses confirmed the mRNA and protein expression of hTRPC1 in the arterial rings. The amplitude of active tension induced by 40 mM K+ (40K) in PA rings infected with an empty adenoviral vector (647 +/- 88 mg/mg) was similar to that in PA rings infected with hTRPC1 (703 +/- 123 mg/mg, P = 0.3). However, the active tension due to capacitative Ca2+ entry (CCE) induced by cyclopiazonic acid was significantly enhanced in PA rings overexpressing hTRPC1 (91 +/- 13% of 40K-induced contraction) compared with rings infected with an empty adenoviral vector (61 +/- 14%, P < 0.001). Endothelial expression of hTRPC1 was not involved since the CCE-induced vasoconstriction was also enhanced in endothelium-denuded PA rings infected with the adenoviral vector carrying hTRPC1. These observations demonstrate that hTRPC1 is an important Ca(2+)-permeable channel that mediates pulmonary vasoconstriction when PA smooth muscle cell intracellular Ca2+ stores are depleted.     

Hydrogen peroxide generation induces pp60src activation in human platelets: evidence for the involvement of this pathway in store-mediated calcium entry

Reactive oxygen species, such as H2O2, have been recognized as intracellular messengers involved in several cell functions. Here we report the activation of the tyrosine kinase pp60(src) by H2O2, a mechanism required for the activation of store-mediated Ca2+ entry (SMCE) in human platelets. Treatment of platelets with H2O2 resulted in a time- and concentration-dependent activation of pp60(src). Incubation with GF 109203X, a protein kinase C (PKC) inhibitor, prevented H2O2-induced pp60(src) activation. In contrast, dimethyl-BAPTA loading did not affect this response, suggesting that activation of pp60(src) by H2O2 is independent of increases in [Ca2+](i). Cytochalasin D, an inhibitor of actin polymerization, significantly reduced H2O2-induced pp60(src) activation. We found that platelet stimulation with thapsigargin (TG) plus ionomycin (Iono) or thrombin induced rapid H2O2 production, a mechanism independent of elevations in [Ca2+](i). Treatment of platelets with catalase attenuated TG plus Iono- and thrombin-induced activation of pp60(src). In addition, catalase as well as the pp60(src) inhibitor, PP1, reduced both the activation of SMCE and the coupling between the hTrp1 and the IP(3)R type II without having any effect on the maintenance of SMCE. Consistent with the role of PKC in the activation of pp60(src) by H2O2, the PKC inhibitors GF 109202X and Ro-31-8220 were found to reduced SMCE in platelets. This study suggests that platelet activation with TG plus Iono or thrombin is associated with H2O2 production, which acts as a second messenger by stimulating pp60(src) by a PKC-dependent pathway and is involved in the activation of SMCE in these cells.     

Phosphatidylinositol 4,5-bisphosphate enhances store-operated calcium entry through hTRPC6 channel in human platelets

Phosphatidylinositol 4,5-bisphosphate (PIP2) is a versatile regulator of TRP channels. We report that inclusion of a PIP2 analogue, PIP2 1,2-dioctanoyl, does not induce non-capacitative Ca2+ entry per se but enhanced Ca2+ entry stimulated either by thrombin or by selective depletion of the Ca2+ stores in platelets, the dense tubular system, using 10 nM TG, and the acidic stores, using 20 microM 2,5-di-(tert-butyl)-1,4-hydroquinone (TBHQ). Reduction of PIP2 levels by blocking PIP2 resynthesis with Li+ or introducing a monoclonal anti-PIP2 antibody, or sequestering PIP2 using poly-lysine, attenuated Ca2+ entry induced by thrombin, TG and TBHQ, and reduced thrombin-evoked, but not TG- or TBHQ-induced, Ca2+ release from the stores. Incubation with the anti-hTRPC1 antibody did not alter the stimulation of Ca2+ entry by PIP2, whilst introduction of anti-hTRPC6 antibody directed towards the C-terminus of hTRPC6 reduced Ca2+ and Mn2+ entry induced by thrombin, TG or TBHQ, and abolished the stimulation of Ca2+ entry by PIP2. The anti-hTRPC6 antibody, but not the anti-hTRPC1 antibody or PIP2, reduced non-capacitative Ca2+ entry by the DAG analogue 1-oleoyl-2-acetyl-sn-glycerol. In summary, hTRPC6 plays a role both in store-operated and in non-capacitative Ca2+ entry. PIP2 enhances store-operated Ca2+ entry in human platelets, most probably by stimulation of hTRPC6 channels.     

Two pathways for store-mediated calcium entry differentially dependent on the actin cytoskeleton in human platelets

A major pathway for stimulated Ca(2+) entry in non-excitable cells is activated following depletion of intracellular Ca(2+) stores. Secretion-like coupling between elements in the plasma membrane (PM) and Ca(2+) stores has been proposed as the most likely mechanism to activate this store-mediated Ca(2+) entry (SMCE) in several cell types. Here we identify two mechanisms for SMCE in human platelets activated by depletion of two independent Ca(2+) pools, which are differentially modulated by the actin cytoskeleton. Ca(2+) entry induced by depletion of a 2,5-di-(tert-butyl)-1,4-hydroquinone (TBHQ)-sensitive pool is increased by disassembly of the actin cytoskeleton and that induced by a TBHQ-insensitive pool is reduced. Stabilization of the actin cytoskeleton prevented Ca(2+) entry by both mechanisms. We propose that the membrane-associated actin network prevents constitutive Ca(2+) entry via both pathways. Reorganization of the actin cytoskeleton permits the activation of Ca(2+) entry via both mechanisms, but only SMCE activated by the TBHQ-insensitive pool requires new actin polymerization, which may support membrane trafficking toward the PM.     

Interaction of STIM1 with endogenously expressed human canonical TRP1 upon depletion of intracellular Ca2+ stores

STIM1 (stromal interaction molecule 1) has recently been proposed to communicate the intracellular Ca(2+) stores with the plasma membrane to mediate store-operated Ca(2+) entry. Here we describe for the first time that Ca(2+) store depletion stimulates rapid STIM1 surface expression and association with endogenously expressed human canonical TRP1 (hTRPC1) independently of rises in cytosolic free Ca(2+) concentration. These events require the support of the actin cytoskeleton in human platelets, as reported for the coupling between type II inositol 1,4,5-trisphosphate receptor in the Ca(2+) stores and hTRPC1 in the plasma membrane, which has been suggested to underlie the activation of store-operated Ca(2+) entry in these cells. Electrotransjection of cells with anti-STIM1 antibody, directed toward the N-terminal sequence that includes the Ca(2+)-binding region, prevented the migration of STIM1 toward the plasma membrane, the interaction between STIM1 and hTRPC1, the coupling between hTRPC1 and type II inositol 1,4,5-trisphosphate receptor, and reduced store-operated Ca(2+) entry. These findings provide evidence for a role of STIM1 in the activation of store-operated Ca(2+) entry probably acting as a Ca(2+) sensor.     

Role of lipid rafts in the interaction between hTRPC1, Orai1 and STIM1

Store-operated Ca2+ entry (SOCE) is a mechanism regulated by the filling state of the intracellular Ca2+ stores that requires the participation of the Ca2+ sensor STIM1, which communicates the Ca2+ content of the stores to the plasma membrane Ca2+-permeable channels. We have recently reported that Orai1 mediates the communication between STIM1 and the Ca2+ channel hTRPC1. This event is important to confer hTRPC1 store depletion sensitivity, thus supporting the functional role of the STIM1-Orai1-hTRPC1 complex in the activation of SOCE. Here we have explored the relevance of lipid rafts in the formation of the STIM1-Orai1-hTRPC1 complex and the activation of SOCE. Disturbance of lipid raft domains, using methyl-beta-cyclodextrin, reduces the interaction between endogenously expressed Orai1 and both STIM1 and hTRPC1 upon depletion of the intracellular Ca2+ stores and attenuates thapsigargin-evoked Ca2+ entry. These findings suggest that TRPC1, Orai1 and STIM1 form a heteromultimer associated with lipid raft domains and regulated by the intracellular Ca2+ stores.     

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.     

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.     

A key role for reverse Na+/Ca2+ exchange influenced by the actin cytoskeleton in store-operated Ca2+ entry in human platelets: Evidence against the de novo conformational coupling hypothesis

We have previously demonstrated a role for the reorganization of the actin cytoskeleton in store-operated calcium entry (SOCE) in human platelets and interpreted this as evidence for a de novo conformational coupling step in SOCE activation involving the type II IP₃ receptor and the platelet hTRPC1-containing store-operated channel (SOC). Here, we present evidence challenging this model. The actin polymerization inhibitors cytochalasin D or latrunculin A significantly reduced Ca²⁺ but not Mn²⁺ or Na⁺ entry into thapsigargin (TG)-treated platelets. Jasplakinolide, which induces actin polymerization, also inhibited Ca²⁺ but not Mn²⁺ or Na⁺ entry. However, an anti-hTRPC1 antibody inhibited TG-evoked entry of all three cations, indicating that they all permeate an hTRPC1-containing store-operated channel (SOC). These results indicate that the reorganization of the actin cytoskeleton is not involved in SOC activation. The inhibitors of the Na⁺/Ca²⁺ exchanger (NCX), KB-R7943 or SN-6, caused a dose-dependent inhibition of Ca²⁺ but not Mn²⁺ or Na⁺ entry into TG-treated platelets. The effects of the NCX inhibitors were not additive with those of actin polymerization inhibitors, suggesting a common point of action. These results indicate a role for two Ca²⁺ permeable pathways activated following Ca²⁺ store depletion in human platelets: A Ca²⁺-permeable, hTRPC1-containing SOC and reverse Na⁺/Ca²⁺ exchange, which is activated following Na⁺ entry through the SOC and requires a functional actin cytoskeleton.     

STIM1 and STIM2 are located in the acidic Ca2+ stores and associates with Orai1 upon depletion of the acidic stores in human platelets

Mammalian cells accumulate Ca2+ into agonist-sensitive acidic organelles, vesicles that possess a vacuolar proton-ATPase. Acidic Ca2+ stores include secretory granules and lysosome-related organelles. Current evidence clearly indicates that acidic Ca2+ stores participate in cell signaling and function, including the activation of store-operated Ca2+ entry in human platelets upon depletion of the acidic stores, although the mechanism underlying the activation of store-operated Ca2+ entry controlled by the acidic stores remains unclear. STIM1 has been presented as the endoplasmic reticulum Ca2+ sensor, but its role sensing intraluminal Ca2+ concentration in the acidic stores has not been investigated. Here we report that STIM1 and STIM2 are expressed in the lysosome-related organelles and dense granules in human platelets isolated by immunomagnetic sorting. Depletion of the acidic Ca2+ stores using the specific vacuolar proton-ATPase inhibitor, bafilomycin A1, enhanced the association between STIM1 and STIM2 as well as between these proteins and the plasma membrane channel Orai1. Depletion of the acidic Ca2+ stores also induces time-dependent co-immunoprecipitation of STIM1 with the TRPC proteins hTRPC1 and hTRPC6, as well as between Orai1 and both TRPC proteins. In addition, bafilomycin A1 enhanced the association between STIM2 and SERCA3. These findings demonstrate the location of STIM1 and STIM2 in the acidic Ca2+ stores and their association with Ca2+ channels and ATPases upon acidic stores discharge.     

Homers regulate calcium entry and aggregation in human platelets: a role for Homers in the association between STIM1 and Orai1

Homer is a family of cytoplasmic adaptor proteins that play different roles in cell function, including the regulation of G-protein-coupled receptors. These proteins contain an Ena (Enabled)/VASP (vasodilator-stimulated phosphoprotein) homology 1 domain that binds to the PPXXF sequence motif, which is present in different Ca2?-handling proteins such as IP3 (inositol 1,4,5-trisphosphate) receptors and TRPC (transient receptor potential canonical) channels. In the present study we show evidence for a role of Homer proteins in the STIM1 (stromal interaction molecule 1)-Orai1 association, as well as in the TRPC1-IP3RII (type II IP3 receptor) interaction, which might be of relevance in platelet function. Treatment of human platelets with thapsigargin or thrombin results in a Ca2?-independent association of Homer1 with TRPC1 and IP3RII. In addition, thapsigargin and thrombin enhanced the association of Homer1 with STIM1 and Orai1 in a Ca2?-dependent manner. Interference with Homer function by introduction of the synthetic PPKKFR peptide into cells, which emulates the proline-rich sequences of the PPXXF motif, reduced STIM1-Orai1 and TRPC1- IP3RII associations, as compared with the introduction of the inactive PPKKRR peptide. The PPKKFR peptide attenuates thrombin-evoked Ca2? entry and the maintenance of thapsigargin-induced store-operated Ca2? entry. Finally, the PPKKFR peptide attenuated thrombin-induced platelet aggregation. The findings of the present study support an important role for Homer proteins in thrombin-stimulated platelet function, which is likely to be mediated by the support of agonist-induced Ca2? entry.     

Capacitative calcium entry in guinea pig gallbladder smooth muscle in vitro

This study investigates the involvement of capacitative Ca2+ entry in excitation-contraction coupling in guinea pig gallbladder smooth muscle. Thapsigargin (0.1 nM-1 microM, a sarcoplasmic reticulum Ca(2+)-ATPase inhibitor) produced slowly developing sustained tonic contractions in guinea pig isolated gallbladder strips. All contractions approached 50% of the response to carbachol (10 microM) after 55 min. Contractile responses to thapsigargin (1 microM) were abolished in a Ca(2+)-free medium. Subsequent re-addition of Ca2+ (2.5 mM) produced a sustained tonic contraction (99 +/- 6% of the carbachol response). The contractile response to Ca2+ re-addition following incubation of tissues in a Ca(2+)-free bathing solution in the absence of thapsigargin was significantly less than in its presence (79 +/- 4 % vs 100 +/- 7 % of carbachol; p < 0.05). Contractile responses to Ca2+ re-addition following treatment with thapsigargin were attenuated by (a) the L-type voltage-operated Ca2+ channel antagonist, nifedipine (10 microM) and (b) the general inhibitor of Ca2+ entry channels including store-operated channels, SK&F96365 (50 microM and 100 microM). In separate experiments, responses to Ca2+ re-addition were essentially abolished by the tyrosine kinase inhibitor, genistein (100 microM). These results suggest that capacitative Ca2+ entry provides a source of activator Ca2+ for guinea pig gallbladder smooth muscle contraction. Contractile responses to Ca2+ re-addition following depletion of sarcoplasmic reticulum Ca2+ stores with thapsigargin, are mediated in part by Ca2+ entry through voltage-operated Ca2+ channels and by capacitative Ca2+ entry through store-operated Ca2+ channels which can be blocked by SK&F96365. Furthermore, capacitative Ca2+ entry in this tissue may be modulated by tyrosine kinase.     

Involvement of inositol 1,4,5-trisphosphate in Ca2+ influx in thrombin stimulated human platelets

By incubating platelets at low temperature (10 degrees C), the relationship between Ca2+ mobilization and formation of inositol 1,4,5-trisphosphate (IP3) in thrombin stimulated platelets could be precisely investigated. In the presence of 1 mM EGTA, time dependent changes in the intracellular free calcium concentration [( Ca2+]i) were closely related to those in IP3 formation. Time course of the influx of external Ca2+, estimated by delta [Ca2+]i obtained by subtracting [Ca2+]i in the presence of 1 mM EGTA from that in the presence of 1 mM CaCl2 was also very similar to that of IP3 formed. Furthermore, the increase in delta [Ca2+]i was extremely well correlated with the amount of IP3 formed (Y = 49X - 34, r = 0.99). Thus, these data indicate that IP3 might be involved not only in intracellular Ca2+ mobilization but in Ca2+ influx of human platelets stimulated by thrombin.     

Effects of methyl-beta-cyclodextrin-mediated cholesterol depletion in porcine sperm compared to somatic cells

In this study, the use of methyl-beta-cyclodextrin (MBCD) to support capacitation of sperm cells was studied. Sperm were incubated with MBCD or alternatively capacitated in an in vitro fertilization medium. The effects of these incubations on phospholipid scrambling (using merocyanin), cholesterol depletion, GM-1 localization (using cholera-toxin B (CTX)), and membrane deterioration were assessed. For comparison, this was also tested in MBCD-treated MDCK cells. In MDCK cells, upto 71% of cholesterol was depleted, which coincided with a more diffuse CTX staining without any obvious effects on cell viability. In sperm, a similar depletion of 53% cholesterol was found after a 10 mM MBCD treatment. However, no merocyanin response was observed in viable sperm after MBCD treatments (indicating a lack of membrane changes associated with sperm capacitation). In contrast to MDCK, cells >1 mM MBCD caused plasma membrane disintegration and rendered sperm immotile. At higher concentrations also acrosome disruption was noted. CTX staining was absent at < 0.1 mM MBCD incubations but appeared at higher MBCD levels and was found to be specific for deteriorated cells that showed morphological signs of acrosome disruption. No significant plasma membrane deterioration, acrosome disruption, and sperm immotility nor CTX staining and only a modest (< 15%) cholesterol depletion were observed in conventionally capacitated sperm, where 40% of the intact sperm showed merocyanin staining. Taken together, the results indicate that membranes of sperm are more sensitive to MBCD-mediated cholesterol depletion than MDCK cells and that the use of MBCD to support sperm capacitation cannot be recommended due to its spermicidal effects.