Characterization of voltage-independent Ca2+ channels activated by endothelin-1

To clarify Ca2+ entry channels involved in the endothelin-1 (ET-1)-induced increase in the intracellular concentration ([Ca2+]i), we performed whole-cell recordings of patch-clamp techniques and monitoring of [Ca2+]i with Ca2+ indicators fura-2 and fluo-3 in A7r5 cells (a cell line derived from rat thoracic aortic smooth muscle cells). With whole-cell recordings, lower concentrations (< or = 1 nM) of ET-1 activated a Ca(2+)-permeable nonselective cation channel (designated NSCC-1). In contrast, higher concentrations (> or = 1 nM) of ET-1 activated two types of Ca(2+)-permeable nonselective cation channel (designated NSCC-1 and NSCC-2) and store-operated Ca2+ channel (SOCC). Importantly, we found that these Ca2+ channels can be pharmacologically discriminated using blockers of the so-called receptor operated Ca2+ influx such as SK&F 96365 and LOE 908. That is, NSCC-1 is resistant to SK&F 96365 but sensitive to LOE 908; NSCC-2 is sensitive to both SK&F 96365 and LOE 908; SOCC is sensitive to SK&F 96365 but resistant to LOE 908. Using these blockers, we analyzed the ET-1-induced increase in [Ca2+]i. The increase in [Ca2+]i induced by lower concentrations of ET-1 was resistant to SK&F 96365 but sensitive to LOE 908. In contrast, the increase in [Ca2+]i induced by higher concentrations of ET-1 was partially suppressed to approximately 30% of controls by either SK&F 96365 or LOE 908 alone, and it was abolished by their combination. These results show that the increase in [Ca2+]i induced by lower concentrations (< or = 1 nM) of ET-1 results from Ca2+ influx through NSCC-1, whereas the increase in [Ca2+]i induced by higher concentrations (> or = 10 nM) of ET-1 results from Ca2+ influx through NSCC-1, NSCC-2 and SOCC.     

Characterization of Ca2+ channels involved in endothelin-1-induced contraction of rabbit basilar artery

This study attempted to characterize Ca2+ channels involved in endothelin-1-induced contraction of rabbit basilar artery using whole-cell patch-clamp and measurement of intracellular free Ca2+ concentration. Endothelin-1 activates two types of Ca2+-permeable nonselective cation channels (NSCC-1 and NSCC-2) and a store-operated Ca2+ channel (SOCC) in addition to the voltage-operated Ca2+ channel (VOCC). These channels can be discriminated using Ca2+ channel blockers, SK&F 96365 and LOE 908. Tension study was conducted to clarify the Ca2+ channels involved in endothelin-1-induced contraction of basilar artery. Endothelin-1-induced basilar artery contraction is fully dependent on extracellular Ca2+ influx. Based on sensitivity to nifedipine, an L-type VOCC blocker, VOCCs have a minor role in endothelin-1-induced contraction. Both LOE 908 and SK&F 96365 inhibit endothelin-1-induced contraction in a concentration-dependent manner, and their combination abolished it. The median inhibitory concentrations of these blockers for endothelin-1-induced contraction correlated well with those of the endothelin-1-induced [Ca2+]i responses. Thus, the inhibitory action of these blockers on endothelin-1-induced contraction may be mediated by blockade of NSCC-1, NSCC-2, and the SOCC. Extracellular Ca2+ influx through NSCC-1, NSCC-2, and SOCC may be essential for endothelin-1-induced basilar artery contraction.     

Involvement of extracellular Ca2+ influx and epidermal growth factor receptor tyrosine kinase transactivation in endothelin-1-induced arachidonic acid release

1. Endothelin-1 (ET-1) activates two types of Ca(2+)-permeable nonselective cation channels (designated NSCC-1 and NSCC-2) and a store-operated Ca(2+) channel (SOCC) in vascular smooth muscle cells (VSMCs). These channels can be distinguished by their sensitivity to Ca(2+)-channel blockers, SK&F 96365 and LOE 908. LOE 908 is sensitive to NSCC-1 and NSCC-2, and SK&F 96365 is sensitive to NSCC-2 and SOCC. Moreover, these channels play essential roles in ET-1-induced epidermal growth factor receptor protein tyrosine kinase (EGFR PTK) transactivation. The main purpose of the present study was to demonstrate the involvement of EGFR PTK transactivation in ET-1-induced arachidonic acid release in VSMCs. 2. Both SK&F 96365 and LOE 908 inhibited ET-1-induced arachidonic acid release with the IC(50) values correlated to those of ET-1-induced Ca(2+) influx. Moreover, combined treatment with these blockers abolished ET-1-induced arachidonic acid release. 3. AG1478, a specific inhibitor of EGFR PTK, inhibited ET-1-induced arachidonic acid release and extracellular signal-regulated kinase 1 and 2 (ERK1/2). The IC(50) values of AG1478 for ET-1-induced arachidonic acid release and ERK1/2 correlated well with those for ET-1-induced EGFR PTK transactivation. 4. Mitogen-activated protein kinase kinase inhibitor, PD 98059, inhibited ET-1-induced arachidonic acid release. The IC(50) values of PD 98059 for ET-1-induced arachidonic acid release were similar to those for ET-1-induced ERK1/2 activity. In contrast, PD 98059 failed to inhibit ET-1-induced EGFR PTK transactivation. 5. These results indicate that (1) extracellular Ca(2+) influx through NSCCs and SOCC plays important roles for ET-1-induced arachidonic acid release, (2) EGFR PTK transactivation/ERK1/2 pathways are involved in ET-1-induced arachidonic acid release.     

Ca2+ entry channels involved in contractions of rat aorta induced by endothelin-1, noradrenaline,and vasopressin

Endothelin-1 (ET-1) has been shown to activate three types of Ca2+ channel, namely two Ca2+-permeable nonselective cation channels (designated NSCC-1 and NSCC-2) and a store-operated Ca2+ channel (SOCC), and that these channels can be discriminated by Ca2+ channel blockers such as LOE 908 (a blocker of NSCC-1 and NSCC-2) and SK&F 96365 (a blocker of NSCC-2 and SOCC). This study pharmacologically compared Ca2+ entry channels involved in contractions of rat thoracic aorta without endothelium induced by ET-1, noradrenaline (NA), or arginine-vasopressin (AVP). These agonists-induced contractions of aortic rings without endothelium and increases in the intracellular free Ca2+ concentration ([Ca2+]i) of cultured aortic smooth muscle cells were abolished by removal of extracellular Ca2+. A blocker of L-type voltage-operated Ca2+ channel (VOCC), nifedipine had no effect on the responses to ET-1, but it suppressed the responses to NA and AVP to 70% and 65% of control responses, respectively. LOE 908 partially suppressed the nifedipine-resistant responses to ET-1 and AVP, but not those to NA. SK&F 96365 also partially suppressed the nifedipine-resistant responses to ET-1 and AVP, whereas it abolished the responses to NA. LOE 908 in combination with SK&F 96365 abolished the nifedipine-resistant responses to either of the agonists. These results show that the contraction of rat aorta involves different Ca2+ entry channel depending on agonists: (a) NSCC-1, NSCC-2, and SOCC for ET-1; (b) VOCC and SOCC for NA; and (c) VOCC, NSCC-1, NSCC-2, and SOCC for AVP.     

Molecular mechanisms for activation of voltage-independent Ca2+ channels by endothelin-1/endothelin-A receptors

Endothelin-1 (ET-1) activates two types of Ca2+- permeable non-selective cation channels (designated NSCC-1 and NSCC-2) and a store-operated Ca2+ channel (SOCC) in Chinese hamster ovary cells expressing endothelin-A receptors (CHOETAR), which couple with Gq, Gs and G12. The purpose of this study was to identify the G proteins involved in the activation of these Ca channels, using mutated ETARs with coupling to either Gq or Gs/G12 (designated ETAR(Delta)385 and SerETAR, respectively) and a dominant negative mutant of G12 (G12G228A). ETAR(Delta)385 is truncated downstream of Cys385 in the C-terminal as palmitoylation sites, whereas SerET(A)R is unpalmitoylated because of substitution of all the cysteine residues to serine (CysCys --> SerSer). ET-1 activated SOCC in CHO-ET(A)R(Delta)385. In CHO-SerET(A)R or CHO-ET(A)R pretreated with U73122, an inhibitor of phospholipase C, ET-1 activated NSCC-1. ET-1 activated SOCC in CHO-ETAR microinjected with G12G228A. Moreover, ET-1 activated NSCC-1 in CHO-ETAR treated with LY 294002, the phosphoinositide 3-kinase inhibitor. These results indicate that NSCC-1 is activated via a G12-dependent pathway, NSCC-2 via Gq/phospholipase C-dependent and G12-dependent pathways, and SOCC via a Gq-phospholipase C-dependent pathway. In addition, NSCC-2 and SOCC are stimulated by ET-1 via a phosphoinositide 3-kinase-dependent cascade, whereas NSCC-1 is stimulated via a phosphoinositide 3-kinase-independent cascade.     

Ca2+ stores regulate ryanodine receptor Ca2+ release channels via luminal and cytosolic Ca2+ sites

1. In muscle, intracellular calcium concentration, hence skeletal muscle force and cardiac output, is regulated by uptake and release of calcium from the sarcoplasmic reticulum. The ryanodine receptor (RyR) forms the calcium release channel in the sarcoplasmic reticulum. 2. The free [Ca2+] in the sarcoplasmic reticulum regulates the excitability of this store by stimulating the Ca2+ release channels in its membrane. This process involves Ca2+-sensing mechanisms on both the luminal and cytoplasmic sides of the RyR. In the cardiac RyR, these have been shown to be a luminal Ca2+ activation site (L-site; 60 micromol/L affinity), a cytoplasmic activation site (A-site; 0.9 micromol/L affinity) and a cytoplasmic Ca2+ inactivation site (I2-site; 1.2 micromol/L affinity). 3. Cardiac RyR activation by luminal Ca2+ occurs by a multistep process dubbed 'luminal-triggered Ca2+ feed-through'. Binding of Ca2+ to the L-site initiates brief (1 msec) openings at a rate of up to 10/s. Once the pore is open, luminal Ca2+ has access to the A-site (producing up to 30-fold prolongation of openings) and to the I2-site (causing inactivation at high levels of Ca2+ feed-through). 4. The present paper reviews the evidence for the principal aspects of the 'luminal-triggered Ca2+ feed-through' model, the properties of the various Ca2+-dependent gating mechanisms and their likely role in controlling sarcoplasmic reticulum (SR) Ca2+ release in cardiac muscle. 5. The model makes the following important predictions: (i) there will be a close link between luminal and cytoplasmic regulation of RyRs and any cofactor that prolongs channel openings triggered by cytoplasmic Ca2+ will also promote RyR activation by luminal Ca2+; (ii) luminal Mg2+ (1 mmol/L) is essential for the control of SR excitability in cardiac muscle by luminal Ca2+; and (iii) the different RyR isoforms in skeletal and cardiac muscle will be controlled quite differently by the luminal milieu. For example, Mg2+ in the SR lumen (approximately 1 mmol/L) can strongly inhibit RyR2 by competing with Ca2+ for the L-site, whereas RyR1 is not affected by luminal Mg2+.     

Ca2+ influx is not involved in acute cytotoxicity of arachidonic acid

Arachidonic acid (AA; 20:4, n-6) has been implicated in cell damage in the brain under ischemia-reperfusion and other pathological conditions. In our experiments, PC12 cells exposed to >10 microM AA died within 1-2 hr, as assessed by the LDH release assay. Since AA is known to induce Ca2+/cation-permeable conductance in the plasma membrane, we investigated whether Ca2+ influx plays a role in this acute cell death. We found that extracellular Ca2+ was not required for the toxic effect of AA. In fact, the removal of extracellular Ca2+ dramatically accelerated its development: the half-time of the toxic effect of 40 microM AA decreased from 70.1 +/- 0.3 min in the presence of 5 mM Ca2+ to 7.4 +/- 0.3 min in the Ca-free solution. The extent of cell killing depended only weakly on AA concentration and ion composition, remaining within the 70-95% range. The AA-induced acute death was not affected by inhibitors of AA metabolism (nordihydroguaiaretic acid, indomethacin, proadifen), whereas some antioxidants tested (deferoxamine and ellagic acid), but not all (melatonin), partially suppressed it. Also, it was not affected by changes in the extracellular ionic strength or mimicked by an acetylenic analog of AA 5,8,11,14-eicosatetraynoic acid. We conclude that lethal injuries sustained by cells during short exposures to AA were caused by the fatty acid itself and were not mediated by the AA-induced influx of Ca2+/cations. Moreover, direct physical effects of AA on the plasma membrane (changes in membrane fluidity or detergent-like action) were also excluded.     

Characterization of G proteins involved in activation of nonselective cation channels by endothelin(B) receptor

1: We recently demonstrated that endothelin-1 (ET-1) activates two types of Ca(2+)-permeable nonselective cation channels (NSCC-1 and NSCC-2) in Chinese hamster ovary cells expressing endothelin(B) receptors (CHO-ET(B)R) that couple with G(q) and G(i). The purpose of the present study was to identify the G proteins involved in the activation of these Ca(2+) channels by ET-1. For this purpose, we constructed CHO cells expressing an unpalmitoylated (Cys(402)Cys(403) Cys(405)-->Ser(402)Ser(403)Ser(405)) ET(B)R (CHO-SerET(B)R) and ET(B)R truncated at the cytoplasmic tail downstream of Cys(403) (CHO-ET(B)RDelta403). 2: Based on the data obtained from actin stress fibre formation, CHO-ET(B)R couple with G(13). Therefore, CHO-ET(B)R couple with G(q), G(i) and G(13). CHO-SerET(B)R and CHO-ET(B)RDelta403 couple with G(13) and G(q), respectively. 3: ET-1 activated NSCC-1 in CHO-ET(B)R preincubated with phospholipase C (PLC) inhibitor, U73122, and in CHO-SerET(B)R. On the other hand, ET-1 failed to activate Ca(2+) channels in CHO-ET(B)RDelta403. Microinjection of dominant negative mutants of G(13) (G(13)G225A) abolished activation of NSCC-1 and NSCC-2 in CHO-ET(B)R and that of NSCC-1 in CHO-SerET(B)R. 4: Y-27632, a specific Rho-associated kinase (ROCK) inhibitor, did not affect the ET-1-induced transient and sustained increase in [Ca(2+)](i) in CHO-ET(B)R. 5: These results indicate that (1) the cytoplasmic tail downstream of the palmitoylation sites of ET(B)R, but not the palmitoylation site itself, is essential for coupling with G(13), (2) the activation mechanism of each Ca(2+) channel by ET-1 is different in CHO-ET(B)R. NSCC-1 activation depends on G(13)-dependent cascade, and NSCC-2 activation depends on both G(q)/PLC- and G(13)-dependent cascades. Moreover, ROCK-dependent cascade is not involved in the activation of these channels.     

Characterization of Ca(2+) channels involved in endothelin-1-induced mitogenic responses in vascular smooth muscle cells.

Ca(2+) channels involved in the endothelin-1-induced mitogenic response of cultured rat thoracic aorta smooth muscle cells, A7r5 cells, were characterized using the Ca(2+) channel blockers, LOE 908 and SK&F 96365. Stimulation of A7r5 cells with endothelin-1 induced a mitogenic response as well as a biphasic increase in the intracellular-free Ca(2+) concentration. Based on the sensitivity to nifedipine, a specific blocker of L-type voltage-operated Ca(2+) channel (VOCC), Ca(2+) influx through VOCC has a minor role in endothelin-1-induced mitogenic responses. On the other hand, Ca(2+) influx through voltage-independent Ca(2+) channels (VICCs) plays an important part in endothelin-1-induced mitogenesis. Moreover, based on their sensitivity to SK&F 96365 and LOE 908, VICCs consist of two types of Ca(2+)-permeable nonselective cation channels (designated NSCC-1 and NSCC-2) and a store-operated Ca(2+) channel (SOCC). Ca(2+) influx through NSCC-1, NSCC-2 and SOCC contributes to 35%, 30% and 35%, respectively, to the nifedipine-resistant component of the endothelin-1 mitogenic response.     

Potentiation by endothelin-1 of Ca2+ sensitivity of contractile elements depends on Ca2+ influx through L-type Ca2+ channels in the canine cerebral artery

• 1.1. Endothelin-1 (ET-1) contracted canine cerebral artery in a concentration-dependent manner with an increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i), and at higher concentrations it produced a greater contraction with a smaller increase in [Ca²⁺]_i.
• 2.2. Ca²⁺ channel antagonist such as d-cis-diltiazem inhibited the tension more effectively than the [Ca²⁺]_i increased by ET-1.
• 3.3. In Ca²⁺-free solution containing 0.2 mM EGTA, ET-1 elicited a transient increase in [Ca²⁺]_i and tension.
• 4.4. In the Staphylococcus aureus α-toxin-permeabilized artery, ET-1 shifted the pCa-tension relationship leftwards in the presence of GTP.
• 5.5. These findings suggest that ET-I contracts the canine cerebral artery by increasing not only the Ca²⁺ influx through L-type Ca²⁺ channels, but also Ca²⁺ release from the intracellular storage sites, and also Ca²⁺ sensitivity of contractile elements. The degree of Ca²⁺ sensitivity is strongly affected by [Ca²⁺]_i which is increased by the Ca²⁺ influx through L-type Ca²⁺ channels.

     

Ca2+ entry channels in rat thoracic aortic smooth muscle cells activated by endothelin-1.

The contraction of the rat aorta induced by endothelin-1 (ET-1) requires entry of extracellular Ca2+, but involvement of voltage-operated Ca2+ channel is minor. Using whole-cell recordings of patch-clamp and monitoring of the intracellular free Ca2+ concentration ([Ca2+]i), we characterized Ca2+ entry channels in A7r5 cells activated by ET-1. ET-1 activates three types of voltage-independent Ca2+ entry channels: two types of Ca2+-permeable nonselective cation channels (designated NSCC-1 and NSCC-2) and a store-operated Ca2+ channel (SOCC). Furthermore, it was found that these channels can be pharmacologically discriminated using Ca2+ channel blockers such as SK&F 96365 and LOE 908. NSCC-1 is resistant to SK&F 96365, but sensitive to LOE 908, whereas NSCC-2 is sensitive to both SK&F 96365 and LOE 908. SOCC is sensitive to SK&F 96365, but resistant to LOE 908. Using these channel blockers, we analyzed Ca2+ entry channels involved in the ET-1-induced contractions of rat thoracic aorta and increases in [Ca2+]i of single smooth muscle cells. The responses to lower concentrations of ET-1 (< or = 0.1 nM) were abolished by either SK&F 96365 or LOE 908 alone. In contrast, the responses to higher concentrations of ET-1 (> or = 1 nM) were suppressed by SK&F 96365 or LOE 908 to about 10% and 35% of controls, respectively, and abolished by combined treatment with SK&F 96365 and LOE 908. These results show that the responses of rat aorta to lower concentrations of ET-1 involve only one Ca2+ channel that is sensitive to SK&F 96365 and LOE 908 (NSCC-2), whereas those to higher concentrations of ET-1 involve NSCC-1, NSCC-2 and SOCC, contributing 10%, 55% and 35%, respectively, to total Ca2+ entry.     

OX1 orexin/hypocretin receptor signaling through arachidonic acid and endocannabinoid release

We showed previously that OX(1) orexin receptor stimulation produced a strong (3)H overflow response from [(3)H]arachidonic acid (AA)-labeled cells. Here we addressed this issue with a novel set of tools and methods, to distinguish the enzyme pathways responsible for this response. CHO-K1 cells heterologously expressing human OX(1) receptors were used as a model system. By using selective pharmacological inhibitors, we showed that, in orexin-A-stimulated cells, the AA-derived radioactivity was released as two distinct components, i.e., free AA and the endocannabinoid 2-arachidonoyl glycerol (2-AG). Two orexin-activated enzymatic cascades are responsible for this response: cytosolic phospholipase A(2) (cPLA(2)) and diacylglycerol lipase; the former cascade is responsible for part of the AA release, whereas the latter is responsible for all of the 2-AG release and part of the AA release. Essentially only diacylglycerol released by phospholipase C but not by phospholipase D was implicated as a substrate for 2-AG production, although both phospholipases were strongly activated. The 2-AG released acted as a potent paracrine messenger through cannabinoid CB(1) receptors in an artificial cell-cell communication assay that was developed. The cPLA(2) cascade, in contrast, was involved in the activation of orexin receptor-operated Ca(2+) influx. 2-AG was also released upon OX(1) receptor stimulation in recombinant HEK-293 and neuro-2a cells. The results directly show, for the first time, that orexin receptors are able to generate potent endocannabinoid signals in addition to arachidonic acid signals, which may explain the proposed orexin-cannabinoid interactions (e.g., in neurons).     

Direct regulation of cardiac Ca2+ channels by G proteins: neither proven nor necessary?

The cardiac L-type Ca2+ channel has served as a model for ion channel regulation for over a decade. The Ca2+ current is increased by beta-adrenoceptor stimulation and this effect is inhibited by muscarinic acetylcholine receptor stimulation. It is well established that beta-adrenoceptor stimulation increases this current largely by cAMP-dependent phosphorylation but recently data have been presented that suggest that this channel may also be regulated directly by G proteins. This review by Criss Hartzell and Rodolphe Fischmeister evaluates evidence for this second regulatory pathway and concludes that, although G proteins affect cardiac Ca2+ channels in bilayers and excised patches, there is little evidence that this pathway is physiologically significant.     

Failure of endothelin-1 to activate store-operated Ca2+ channels by lack of mobilization from intracellular Ca2+ stores in cultured bovine adrenal chromaffin cells

We have recently shown that in addition to L-type voltage-operated Ca2+ channel (VOC), endothelin-1 (ET-1) stimulation opens two types of Ca2+-permeable nonselective cation channels [designated nonselective cation channel-1 (NSCC-1) and NSCC-2]. However, in this Ca2+ entry, the involvement of store-operated Ca2+ channel (SOCC), which is suggested to exist in chromaffin cells, was unclear. Those NSCCs as well as SOCC can be pharmacologically discriminated using Ca2+ channel blockers such as SK&F 96365 and LOE 908. To clarify whether SOCC should actually exist and play a role in Ca2+ entry in chromaffin cells stimulated with ET-1, we examined the effects of removal of extracellular Ca2+, thapsigargin (TG, an inhibitor of endoplasmic reticulum Ca2+-ATPase), LOE 908 and SK&F 96365 on cytosolic free Ca2+ concentrations ([Ca2+]i) in cultured bovine adrenal chromaffin cells. After the cells were exposed to Ca2+-free medium followed by exposure to TG to deplete Ca2+ from the intracellular Ca2+ store, restoration of extracellular Ca2+ caused a gradual increase in [Ca2+]i (to about 200% of control). The increase was unaffected by LOE 908, but completely abolished by SK&F 96365. In the Ca2+-free medium, no increase in [Ca2+]i by ET-1 was observed, but the subsequent restoration of extracellular Ca2+ induced a rapid increase in [Ca2+]i (to the same level of [Ca2+]i as that evoked by ET-1 in the normal medium (1.0 mM Ca2+)). Since SK&F 96365 is also a blocker of SOCC, these results indicate that in bovine adrenal chromaffin cells, Ca2+ entry through SOCC (Ca2+ influx through the capacitative Ca2+ entry system) occurs but is comparably weak, and that it virtually does not work on the stimulation of ET-1.     

Involvements of voltage-independent Ca2+ channels and phosphoinositide 3-kinase in endothelin-1-induced PYK2 tyrosine phosphorylation

We demonstrated recently that endothelin-1 (ET-1) activates two types of Ca(2+)-permeable nonselective cation channels [designated nonselective cation channel (NSCC)-1 and NSCC-2] and a store-operated Ca(2+) channel (SOCC) in rabbit internal carotid artery vascular smooth muscle cells (ICA VSMCs). These channels can be distinguished by their sensitivity to Ca(2+) channel blockers 1-(beta-[3-(4-methoxyphenyl) propoxy]-4-methoxyphenethyl)-1H-imidazole hydrochloride (SK&F 96365) and (R,S)-(3,4-dihydro-6,7-dimethoxy-isochinolin-1-yl)-2-phenyl-N,N-di[2-(2,3,4-trimethoxyphenyl)ethyl]acetamid mesylate (LOE 908). NSCC-1 is sensitive to LOE 908 and resistant to SK&F 96365, NSCC-2 is sensitive to both LOE 908 and SK&F 96365, and SOCC is resistant to LOE 908 and sensitive to SK&F 96365. The purpose of the present study was to identify the Ca(2+) channels involved in the ET-1-induced, proline-rich tyrosine kinase 2 (PYK2) phosphorylation in ICA VSMCs. Based on sensitivity to nifedipine, an L-type voltage-operated Ca(2+) channel (VOCC) blocker, Ca(2+) influx through VOCC seems to play a minor role in the ET-1-induced PYK2 phosphorylation. In the presence of nifedipine, PYK2 phosphorylation was abolished by blocking Ca(2+) influx through NSCC-1, NSCC-2, and SOCC. The phosphoinositide 3-kinase (PI3K) inhibitors wortmannin and 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY 294002), inhibited ET-1-induced Ca(2+) influx through NSCC-2 and SOCC. In addition, these inhibitors blocked PYK2 phosphorylation that depends on Ca(2+) influx through NSCC-2 and SOCC. These results indicate that 1) Ca(2+) influx through NSCC-1, NSCC-2, and SOCC plays essential roles in ET-1-induced PYK2 phosphorylation, 2) NSCC-2 and SOCC are stimulated by ET-1 via a PI3K-dependent cascade, whereas NSCC-1 is stimulated via a PI3K-independent cascade, and 3) PI3K is involved in the PYK2 phosphorylation that depends on Ca(2+) influx through SOCC and NSCC-2.     

Inhibition of endothelial cell Ca2+ entry and transient receptor potential channels by Sigma-1 receptor ligands

Background and Purpose

The Sigma-1 receptor (Sig1R) impacts on calcium ion signalling and has a plethora of ligands. This study investigated Sig1R and its ligands in relation to endogenous calcium events of endothelial cells and transient receptor potential (TRP) channels.

Experimental Approach

Intracellular calcium and patch clamp measurements were made from human saphenous vein endothelial cells and HEK 293 cells expressing exogenous human TRPC5, TRPM2 or TRPM3. Sig1R ligands were applied and short interfering RNA was used to deplete Sig1R. TRP channels tagged with fluorescent proteins were used for subcellular localization studies.

Key Results

In endothelial cells, 10–100 μM of the Sig1R antagonist BD1063 inhibited sustained but not transient calcium responses evoked by histamine. The Sig1R agonist 4-IBP and related antagonist BD1047 were also inhibitory. The Sig1R agonist {"type":"entrez-protein","attrs":{"text":"SKF10047","term_id":"1156210965","term_text":"SKF10047"}}SKF10047 had no effect. Sustained calcium entry evoked by VEGF or hydrogen peroxide was also inhibited by BD1063, BD1047 or 4-IBP, but not {"type":"entrez-protein","attrs":{"text":"SKF10047","term_id":"1156210965","term_text":"SKF10047"}}SKF10047. 4-IBP, BD1047 and BD1063 inhibited TRPC5 or TRPM3, but not TRPM2. Inhibitory effects of BD1047 were rapid in onset and readily reversed on washout. {"type":"entrez-protein","attrs":{"text":"SKF10047","term_id":"1156210965","term_text":"SKF10047"}}SKF10047 inhibited TRPC5 but not TRPM3 or TRPM2. Depletion of Sig1R did not prevent the inhibitory actions of BD1063 or BD1047 and Sig1R did not co-localize with TRPC5 or TRPM3.

Conclusions and Implications

The data suggest that two types of Sig1R ligand (BD1047/BD1063 and 4-IBP) are inhibitors of receptor- or chemically activated calcium entry channels, acting relatively directly and independently of the Sig1R. Chemical foundations for TRP channel inhibitors are suggested.     

gamma-Aminobutyric acid release from synaptosomes as influenced by Ca2+ and Ca2+ channel blockers

We studied the correlation between the high affinity binding of Ca2+ channel blockers to purified synaptic plasma membranes (SPM) and the effect of these drugs in blocking the 45Ca2+ uptake and the release of [3H]gamma-aminobutyric acid [( 3H]GABA) by preloaded synaptosomes. The Ca2+ channel blocker binding sites were characterized by studying the binding of the dihydropyridine, [3H]nimodipine, and of the phenylalkylamine, (-)-[3H]desmethoxyverapamil, to purified SPM isolated from sheep brain cortex synaptosomes. The purified SPM had high affinity binding sites for both Ca2+ channel blockers. The binding parameters were similar to those previously reported for whole brain homogenates: KD = 0.64 nM and Bmax = 160 fmol/mg of protein for [3H]nimodipine, and KD = 7.9 nM and Bmax = 1,500 fmol/mg of protein for (-)-[3H]desmethoxyverapamil. The Ca2+ channel blockers inhibited the release of [3H]GABA induced by K+ depolarization in the presence or in the absence of Ca2+. The Ca2+-dependent component of [3H]GABA release was inhibited by verapamil, (-)-D 600, d-cis-diltiazem, nifedipine and PY 108-86 with IC50 values of 2.2 X 10(-5) M, 6.3 X 10(-5) M, 3 X 10(-4) M, greater than 10(-4) M and 3 X 10(-5) M, respectively. Furthermore, the Ca2+ channel blockers also inhibited the Ca2+-independent [3H]GABA release which occurred in the presence, but not in the absence, of external Na+. The Ca2+ channel blockers at concentrations which inhibited [3H]GABA release inhibited the entry of Ca2+ through the Ca2+ channels and also the entry of Ca2+ by Na+/Ca2+ exchange. We conclude that the concentrations of Ca2+ blockers necessary to block Ca2+ uptake through the Ca2+ channels and by Na+/Ca2+ exchange coincide with the concentrations at which they inhibit [3H]GABA release, but that their effect on the relationship between Ca2+ uptake and [3H]GABA release is different for the various blockers. The effects of the drugs on Ca2+ movements and [3H]GABA release are not specifically mediated through the high affinity binding of the drugs since relatively high concentrations were necessary (greater than 10(-5) M) for the effects reported here.     

Wortmannin inhibits the myofilament Ca2+ sensitization induced by endothelin-1

Endothelin-1 induces a positive inotropic effect due to a combination of an increase in Ca2+ transients and myofilament Ca2+ sensitivity in rabbit ventricular myocardium. We carried out the experiments to examine the potential contribution of myosin light chain kinase to the Ca2+ sensitization induced by endothelin-1 by use of wortmannin that inhibits myosin light chain kinase at high concentrations (IC50=200 nM). Wortmannin at 3 microM suppressed the basal force of contraction, but did not affect the positive inotropic effect mediated by beta-adrenoceptors. Wortmannin at 1 and 3 microM markedly inhibited the positive inotropic effect of endothelin-1, but did not affect the increase in Ca2+ transients elicited by endothelin-1. The present findings imply that the increase in myofilament Ca2+ sensitivity induced by endothelin-1 may be in part due to activation of myosin light chain kinase in rabbit ventricular myocardium.     

Effects of phosphoinositide 3-kinase on endothelin-1-induced activation of voltage-independent Ca2+ channels and vasoconstriction

We recently demonstrated that endothelin-1 (ET-1) activates two types of Ca(2+)-permeable nonselective cation channel (designated NSCC-1 and NSCC-2) and a store-operated Ca(2+) channel (SOCC) in rabbit basilar artery (BA) vascular smooth muscle cells (VSMCs). In this study, we investigated the effects of phosphoinositide 3-kinase (PI3K) on ET-1-induced activation of these channels and BA contraction by using PI3K inhibitors, wortmannin and LY 249002. To determine which Ca(2+) channels are activated via PI3K, monitoring of intracellular Ca(2+) concentration was performed. Role of PI3K in ET-1-induced vasoconstriction was examined by tension study using rabbit BA rings. Only NSCC-1 was activated by ET-1 in wortmannin- or LY 294002-pretreated VSMCs. In contrast, addition of these drugs after ET-1 stimulation did not suppress Ca(2+) influx. Wortmannin inhibited the ET-1-induced contraction of rabbit BA rings that depends on the Ca(2+) influx through NSCC-2 and SOCC. The IC(50) values of wortmannin for the ET-1-induced Ca(2+) influx and vasoconstriction were similar to those for the ET-1-induced PI3K activation. These results indicate that (1) NSCC-2 and SOCC are stimulated by ET-1 via PI3K-dependent cascade, whereas NSCC-1 is stimulated via PI3K-independent cascade; (2) PI3K is required for the activation of the Ca(2+) entry, but not for its maintenance; and (3) PI3K is involved in the ET-1-induced contraction of rabbit BA rings that depends on the extracellular Ca(2+) influx through SOCC and NSCC-2.     

Analysis of Ca2+ entry channels in endothelin-1-induced contraction of rat aorta using new blockers

1) We have recently shown that endothelin-1 (ET-1) activates two types of Ca(2+)-permeable nonselective cation channels (designated NSCC-1 and NSCC-2) and store-operated Ca2+ channel (SOCC). These channels can be pharmacologically discriminated using Ca2+ channel blockers such as SK&F 96365 and LOE 908. Here we characterized Ca2+ entry channels involved in ET-1-induced contractions of rat thoracic aortic rings and increases in the intracellular free Ca2+ concentration ([Ca2+]i) of single smooth muscle cells using these blockers. 2) LOE 908 or a blocker of voltage-operated Ca2+ channel nifedipine had no effect on the contractions and increases in [Ca2+]i induced by thapsigargin, whereas SK&F 96365 abolished them. 3) The contractions and increases in [Ca2+]i induced by ET-1 depended on extracellular Ca2+ but were resistant to nifedipine. The responses to lower concentrations (< or = 0.1 nM) of ET-1 were abolished by either SK&F 96365 or LOE 908. The responses to higher concentrations (> or = 1 nM) were abolished by SK&F 96365, but were partially resistant to LOE 908. 4) These results show that the contractions and increases in [Ca2+]i of rat aortic smooth muscles at lower concentrations of ET-1 involve only one Ca2+ entry channel which is sensitive to SK&F 96365 and LOE 908 (NSCC-2), whereas those at higher concentrations of ET-1 involve another Ca2+ entry channel which is sensitive to SK&F 96365 but resistant to LOE 908 (SOCC) in addition to the former channel.