Activation of two types of Ca2+-permeable nonselective cation channel by endothelin-1 in A7r5 cells

1. In A7r5 cells loaded with the Ca²⁺ indicator fura-2, we examined the effect of a Ca²⁺ channel blocker SK&F 96365 on increases in intracellular free Ca²⁺ concentrations ([Ca²⁺]i) and Mn²⁺ quenching of fura-2 fluorescence by endothelin-1 (ET-1). Whole-cell patch-clamp was also performed.
2. Higher concentrations (⩾10 nM) of ET-1 (higher [ET-1]) evoked a transient peak and a subsequent sustained elevation in [Ca²⁺]i: removal of extracellular Ca²⁺ abolished only the latter. A blocker of L-type voltage-operated Ca²⁺ channel (VOC) nifedipine at 1 μM reduced the sustained phase to about 50%, which was partially sensitive to SK&F 96365 (30 μM).
3. Lower [ET-1] (⩽1 nM) evoked only a sustained elevation in [Ca²⁺]i which depends on extracellular Ca²⁺. The elevation was partly sensitive to nifedipine but not SK&F 96365.
4. In the presence of 1 μM nifedipine, higher [ET-1] increased the rate of Mn²⁺ quenching but lower [ET-1] had little effect.
5. In whole-cell recordings, both lower and higher [ET-1] induced inward currents at a holding potential of −60 mV with linear I-V relationships and reversal potentials close to 0 mV. The current at lower [ET-1] was resistant to SK&F 96365 but was abolished by replacement of Ca²⁺ in the bath solution with Mn²⁺. The current at higher [ET-1] was abolished by the replacement plus SK&F 96365.
6. In a bath solution containing only Ca²⁺ as a movable cation, ET-1 evoked currents: the current at lower [ET-1] was sensitive to Mn²⁺, whereas that at higher [ET-1] was partly sensitive to SK&F 96365.
7. These results indicate that in addition to VOC, ET-1 activates two types of Ca²⁺-permeable nonselective cation channel depending on its concentrations which differ in terms of sensitivity to SK&F 96365 and permeability to Mn²⁺.

     

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.     

Inhibition of a store-operated Ca2+ entry pathway in human endothelial cells by the isoquinoline derivative LOE 908.

1. The novel cation channel blocker, LOE 908, was tested for its effects on Ca2+ entry and membrane currents activated by depletion of intracellular Ca2+ stores in human endothelial cells. 2. LOE 908 inhibited store-operated Ca2+ entry induced by direct depletion of Ca2+ stores with 100 nM thapsigargin or 100 nM ionomycin with an EC50 of 2 microM and 4 microM, respectively. 3. LOE 908 did not affect thapsigargin- or ionomycin-induced Ca2+ release from intracellular stores up to concentrations of 3 microM. 4. LOE 908 reversibly suppressed thapsigargin- as well as ionomycin-induced whole-cell membrane currents. 5. The LOE 908-sensitive membrane conductance corresponded to a cation permeability of 5.5 and 6.9 fold selectivity for Ca2+ over K+ in the presence of thapsigargin and ionomycin, respectively. 6. Our results suggest that the isoquinoline, LOE 908 is a novel, potent inhibitor of the store-operated (capacitive) Ca2+ entry pathway in endothelial cells.     

Pharmacological characterization of Ca2+ entry channels in endothelin-1-induced contraction of rat aorta using LOE 908 and SK&F 96365.

We have recently shown that endothelin-1 (ET-1) activates two types of Ca2+-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. 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 or ionomycin, whereas SK&F 96365 abolished them. 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. SK&F 96365 inhibited the LOE 908-resistant contractions induced by higher concentrations of ET-1 with IC50 values similar to those for contractions induced by thapsigargin or ionomycin. 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.     

The isoquinoline derivative LOE 908 selectively blocks vasopressin-activated nonselective cation currents in A7r5 aortic smooth muscle cells

The effect of (R,S)-(3,4-dihydro6,7-dimethoxyisoquinoline-1-yl)-2-phenyl-N,N-di- [2- (2, 3,4-trimethoxyphenyl)ethyl]-acetamide (LOE 908), a cation channel blocker in HL-60 promyeloblasts, was studied in the A7r5 smooth muscle cell line from rat thoracic aorta, using the whole-cell patch-clamp technique. At a holding potential of –60 mV, application of vasopressin induced a nonselective cation conductance in voltage-clamped A7r5 cells. The current-voltage relation was linear, and currents reversed close to 0 mV regardless of the chloride gradient. The activation of the nonselective cation conductance by vasopressin was not affected by dialysing cells with Ca⁺-free internal solution. LOE 908 blocked this current in a concentration-dependent manner with an IC₅₀ of 560 nM, whereas dihydropyridine-sensitive Ba²⁺ current through voltage-dependent Ca²⁺ channels was blocked with an IC₅₀ of 28 M. Another organic blocker of receptor-mediated Ca^2– entry, 1--[3-(4-methoxyhenyl)-propoxy]-4-methoxyphenethyl-1H-imidazole hydrochloride (SK&F 96365), blocked both, the vasopressin-induced nonselective conductance and the voltage-activated Ba²⁺ current with similar IC₅₀ values of 13 M and 8 M, respectively. The rank order of potency of inorganic blockers on the vasopressin-induced inward current was Gd³⁺>La³⁺>Cd²⁺. Vasopressin-induced nonselective cation current was also observed in pertussis toxin-pretreated A7r5 cells but was completely abolished after infusion of the GDP analogue, guanosine 5-O-[3-thio]diphosphate, from the patch pipette. Furthermore, vasopressin induced a transient outward current, suggesting a Cau²⁺-activated K⁺-current, which overlapped with the nonselective cation conductance. The outward current was blocked by internal Cs⁺ and external Ba²⁺ or TEA.Our data suggest that the activation of nonselective cation current by vasopressin in A7r5 cells involves a pertussis toxin-insensitive G-protein, is independent of the intracellular Ca²⁺ concentration and that LOE 908 selectively blocks this current. Correspondence to: J. Hescheler at the above address     

AH-1058: a novel cardioselective Ca2+ channel blocker

The pharmacologic profile of a cyproheptadine-related compound, 4-(5H-dibenzo[a,d]cyclohepten-5-ylidene)-1-[(E)-3-(3-methoxy-2-nitro)phenyl-2-propenyl]piperidine hydrochloride (AH-1058), was assessed in various in vivo and in vitro models. In guinea pig cardiomyocytes, AH-1058 effectively suppressed L-type Ca2+ channel currents without affecting other ion channel or ion exchange currents. In rat cerebral cortical membranes AH-1058 appears to bind preferentially to L-type Ca2+ channels at phenylalkylamine- and benzothiazepine-binding sites. In canine isolated, blood-perfused heart preparations, AH-1058 exerted negative inotropic, dromotropic, and chronotropic and weak coronary vasodilator effects. In halothane-anesthetized dogs, AH-1058 suppressed ventricular contractility and decreased blood pressure and cardiac output. Total peripheral vascular resistance was hardly affected by the drug, suggesting that in vivo AH-1058 can selectively suppress cardiac, as compared to peripheral vascular, function. In conscious dogs, by intravenous administration AH-1058 reduced systolic blood pressure and maximal upstroke velocity of the left ventricular pressure, while it increased heart rate in a dose-dependent manner. The drug did not affect diastolic blood pressure, which is quite different from cardiovascular properties of well-known Ca2+ channel blockers, verapamil and diltiazem. This unique cardiovascular profile of AH-1058 is expected to be useful in the treatment of certain pathological processes such as the obstructive hypertrophic cardiomyopathy, vasovagal syncope, dissecting aortic aneurysm, and ventricular arrhythmias, in which selective inhibition of the ventricular Ca2+ channels is essential for drug therapy.     

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+ channel blocking activity of lacidipine and amlodipine in A7r5 vascular smooth muscle cells

Inhibition of the K⁺-stimulated increase in cytosolic free Ca²⁺ by a series of 1,4-dihydropyridines was evaluated in A₇r₅ vascular smooth muscle cells loaded with the fluorescent Ca²⁺ indicator fura-2 acetoxymethyl ester. The IC₅₀ of the drugs, added to suspended cells 3 min before 150 mM KCl, gave the following order of potency: lacidipine (2.76 nM) > nitrendipine (3.81 nM) > amlodipine (4.56 nM) > nifedipine (10.08 nM). A₇r₅ cells were also exposed to the 1,4-dihydropyridines, at their IC₅₀, for 25 min, and then repeated washout cycles were performed before adding KCl. The Ca²⁺ channel blocking activity of nifedipine and nitrendipine gradually diminished, disappearing after four washout cycles 25, 55, 115 and 175 min after drug treatment. Amlodipine and lacidipine displayed slow onset and offset of antagonism, their activity becoming stronger with time, in spite of the repeated washes. [³H]Lacidipine was avidly and promptly entrapped in A₇r₅ cells and was not removed by washout. However, its potency as a Ca²⁺ channel blocker was not directly related to the amount of drug locked in the cell since it increased with time, indicating that lacidipine binds to the lipid bilayer of the cell membrane and then gradually diffuses towards a specific binding site. This model can, therefore, predict the Ca²⁺ blocking properties of 1,4-dihydropyridines with slow onset and offset of antagonism and could be employed to evaluate compounds selective for vascular smooth muscle.     

Activation by high potassium of a novel voltage-operated Ca2+ channel in rat spleen

1. High potassium produced a concentration-dependent contraction in rat isolated spleen.
2. The high potassium-induced contraction of rat spleen was abolished in Ca²⁺-free Krebs solution containing 1 mM EGTA, and the subsequent addition of 3 mM Ca²⁺ restored the high potassium-induced contraction to the control level.
3. Nifedipine, verapamil, diltiazem, Cd²⁺, Ni²⁺, Co²⁺, R-(+)-Bay K 8644 and pimozide inhibited and relaxed high potassium-induced contraction of rat spleen with IC₅₀ and EC₅₀ values much higher than those values in rat aorta.
4. In addition, high potassium-stimulated contraction of rat spleen was insensitive to ω-conotoxin GVIA, ω-conotoxin MVIIC and ω-agatoxin IVA.
5. The high potassium-induced contraction of rat spleen was also unaffected by tetrodotoxin (TTX), prazosin, chloroethylclonidine (CEC), yohimbine, propranolol, atropine, diphenhydramine, cimetidine, ketanserin, 3-tropanyl-indole-3-carboxylate, saralasin, indomethacin, nordihydroguaiaretic acid, GR32191B, domperidone, naloxone, chlorpromazine, suramin, (±)-2-amino-5-phosphonopentanoic acid, 6,7-dinitroquinoxaline-2,3-dione (DNQX), L-659,877, L-703,606, lorglumide, PD 135,158 N-methyl-D-glucamine, benextramine, amiloride, dantrolene, TMB-8, econazole, staurosporine and neomycin.
6. Forskolin and sodium nitroprusside relaxed high potassium-induced contraction of rat spleen with EC₅₀ values of 0.55±0.04 and 20.0±2.7 μM, respectively.
7. It is concluded that high potassium may activate a novel, pharmacologically uncharacterized voltage-operated Ca²⁺ channel in rat spleen.

     

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.     

A novel Na+/Ca2+ channel blocker, NS-7, suppresses hypoxic injury in rat cerebrocortical slices

The substance 4-(4-fluorophenyl)-2-methyl-6-(5-piperidinopentyloxy) pyrimidine hydrochloride (NS-7) has been developed recently as a cerebroprotective compound with Na⁺ and Ca²⁺ channel blocking action. In the present study, the effect of NS-7 in an in vitro model of hypoxic injury was examined and the possible involvement of Na⁺ and Ca²⁺ channels in the hypoxic injury subsequently determined. When slices of rat cerebral cortex were exposed to hypoxia/glucose deprivation followed by reoxygenation and restoration of the glucose supply, marked leakage of lactate dehydrogenase (LDH) occurred 3–6 h after reoxygenation. This hypoxia/reoxygenation-induced injury was blocked almost completely by the removal of extracellular Ca²⁺ or by chelating intracellular Ca²⁺ with 1,2-bis(o-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid tetra(acetoxymethyl)ester (BAPTA/AM). In addition, combined treatment with the N-type Ca²⁺ channel blocker ω-conotoxin GVIA and the P/Q-type Ca²⁺ channel blocker ω-agatoxin IVA significantly reduced LDH leakage, although neither of these Ca²⁺ channel blockers alone, nor nimodipine, an L-type Ca²⁺ channel blocker, was effective. On the other hand, several Na⁺ channel blockers, including tetrodotoxin, local anaesthetics and antiepileptics, significantly reduced the hypoxic injury. NS-7 (3–30 μM) concentration-dependently inhibited LDH leakage caused by hypoxia/reoxygenation, but had no influence on the reduction of tissue ATP content and energy charge during hypoxia and glucose deprivation. It is suggested that blockade of Na⁺ and Ca²⁺ channels is implicated in the cerebroprotective action of NS-7. Received: 10 March 1998 / Accepted: 19 April 1998     

Suramin and disulfonated stilbene derivatives stimulate the Ca2+-induced Ca2+ -release mechanism in A7r5 cells

We have described previously a novel Ca2+-induced Ca2+-release (CICR) mechanism in permeabilized A7r5 cells (embryonic rat aorta) and 16HBE14o-cells (human bronchial mucosa) cells (J Biol Chem 278:27548-27555, 2003). This CICR mechanism was activated upon the elevation of the free cytosolic calcium concentration [Ca2+]c and was not inhibited by pharmacological inhibitors of the inositol-1,4,5-trisphosphate (IP3) receptor nor of the ryanodine receptor. This CICR mechanism was inhibited by calmodulin (CaM)1234, a Ca2+-insensitive CaM mutant, and by different members of the superfamily of CaM-like Ca2+-binding proteins. Here, we present evidence that the CICR mechanism that is expressed in A7r5 and 16HBE14o-cells is strongly activated by suramin and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS). We found several indications that both activation mechanisms are indeed two different modes of the same release system. Suramin/DIDS-induced Ca2+ release was only detected in cells that displayed the CICR mechanism, and cell types that do not express this type of CICR mechanism did not exhibit suramin/DIDS-induced Ca2+ release. Furthermore, we show that the suramin-stimulated Ca2+ release is regulated by Ca2+ and CaM in a similar way as the previously described CICR mechanism. The pharmacological characterization of the suramin/DIDS-induced Ca2+ release further confirms its properties as a novel CaM-regulated Ca2+-release mechanism. We also investigated the effects of disulfonated stilbene derivatives on IP3-induced Ca2+ release and found, in contrast to the effect on CICR, a strong inhibition by DIDS and 4'-acetoamido-4'-isothiocyanostilbene-2',2'-disulfonic acid.     

State-dependent inhibition of L-type Ca2+ channels in A7r5 cells by cilnidipine and its derivatives

Using a whole-cell patch-clamp technique, state-dependent inhibition of dihydropyridines (DHP)s was investigated on L-type channels in A7r5 cells. Cilnidipine, its derivatives (D-342 and D-69) and nimodipine inhibited the Ba2+ current. However, cilnidipine and D-342, but not D-69 or nimodipine, accelerated current decay. The apparent rank order for the effects on the DHP-sensitive decaying component was different from that obtained for inhibition of the peak current. The dissociation constants for cilnidipine in the resting and inactivated states were estimated to be 190 and 12 nM, respectively. Cilnidipine, but not other DHP derivatives, created a faster and voltage-independent component (r= 37 ms). The linear relationship between the tau(-1) of the current decay and the cilnidipine concentration provided a value of 471 nM for the dissociation constant in the open state, suggesting that the current decay is mediated by one-to-one lower affinity binding of cilnidipine molecules to their binding site. The present study demonstrates that structurally related DHPs act in distinct ways to inhibit the L-type channel in the resting, open and inactivated states. Cilnidipine and some related DHPs probably exert their blocking action on the open channel by binding to a receptor distinct from the known DHP-binding site.     

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.     

G1 cell cycle arrest by amlodipine, a dihydropyridine Ca2+ channel blocker, in human epidermoid carcinoma A431 cells

We demonstrated previously that amlodipine, a dihydropyridine Ca(2+) channel blocker, exhibits antitumor effects on human epidermoid carcinoma A431 cells both in vitro and in vivo, in part through inhibition of capacitative Ca(2+) entry. In this study, we examined the effects of amlodipine on cell cycle distribution and cell cycle regulatory molecules in A431 cells, since a rise in intracellular Ca(2+) is required at several points during cell cycle progression. Flow cytometric analysis revealed that treatment with amlodipine (20-30muM, for 24h) induced G1 phase cell accumulation. The amlodipine-induced G1 arrest was associated with a decrease in phosphorylation of retinoblastoma protein (pRB), a regulator of G1 to S phase transition, reduction of protein levels of cyclin D1 and cyclin dependent kinase 4 (CDK4), G1 specific cell cycle proteins, and increased expression of p21(Waf1/Cip1), an inhibitory protein of CDK/cyclin complexes. In vitro kinase assay revealed that amlodipine significantly decreased CDK2-, CDK4-, and their partners cyclin E- and cyclin D1-associated kinase activities. The amlodipine-induced reductions in cyclin D1 protein expression and in CDK2 kinase activity were reproduced by a dihydropyridine derivative, nicardipine, having an inhibitory effect on A431 cell growth, but not by nifedipine, lacking the antiproliferative activity. Our results demonstrate that amlodipine caused G1 cell cycle arrest and growth inhibition in A431 cells through induction of p21(Waf1/Cip1) expression, inhibition of CDK/cyclin-associated kinase activities, and reduced phosphorylation of pRB.     

Intracellular angiotensin II elicits Ca2+ increases in A7r5 vascular smooth muscle cells

Recent studies show that angiotensin II can act within the cell, possibly via intracellular receptors pharmacologically different from typical plasma membrane angiotensin II receptors. The signal transduction of intracellular angiotensin II is unclear. Therefore, we investigated the effects of intracellular angiotensin II in cells devoid of physiological responses to extracellular angiotensin II (A7r5 vascular smooth muscle cells). Intracellular delivery of angiotensin II was obtained by using liposomes or cell permeabilisation. Intracellular angiotensin II stimulated Ca2+ influx, as measured by 45Ca2+ uptake and single-cell fluorimetry. This effect was insensitive to extracellular or intracellular addition of losartan (angiotensin AT(1) receptor antagonist) or PD123319 ((s)-1-(4-[dimethylamino]-3-methylphenyl)methyl-5-(diphenylacetyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-6-carboxylate) (angiotensin AT2 receptor antagonist). Intracellular angiotensin II stimulated inositol-1,4,5-trisphosphate (Ins(1,4,5,)P3) production and increased the size of the Ins(1,4,5,)P3 releasable 45Ca2+ pool in permeabilised cells, independent of losartan and PD123319. Small G-proteins did not participate in this process, as assessed by using GDPbetaS. Intracellular delivery of angiotensin I was unable to elicit any of the effects elicited by intracellular angiotensin II. We conclude from our intracellular angiotensin application experiments that angiotensin II modulates Ca2+ homeostasis even in the absence of extracellular actions. Pharmacological properties suggest the involvement of putative angiotensin non-AT1-/non-AT2 receptors.     

Molecular mechanisms for the activation of voltage-independent Ca2+ channels by endothelin-1 in chinese hamster ovary cells stably expressing human endothelin(A) receptors

We demonstrated recently that in Chinese hamster ovary cells stably expressing human recombinant endothelin(A) receptors (CHO-ET(A)R), endothelin-1 (ET-1) activates two types of Ca2+-permeable nonselective cation channels (designated NSCC-1 and NSCC-2) and a store-operated Ca2+ channel (SOCC), which can be distinguished by Ca(2+) channel blockers such as 1-[beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenylethyl]-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). We also reported that CHO-ET(A)R couples with G12 in addition to G(q) and G(s). The purpose of the present study was to identify the G proteins involved in the activation of these Ca2+ channels by ET-1, using mutated ET(A)Rs with coupling to either G(q) or G(s)/G12 (designated ET(A)RDelta385 and SerET(A)R, respectively) and a dominant-negative mutant of G12 (G12G228A). ET(A)RDelta385 is truncated immediately downstream of Cys385 in the C terminus as palmitoylation sites, whereas SerET(A)R is unpalmitoylated because of substitution of all the cysteine residues to serine (Cys383Cys385-388 --> Ser383Ser385-388). In CHO-ET(A)RDelta385, stimulation with ET-1 activated only SOCC. In CHO-SerET(A)R or CHO-ET(A)R pretreated with U73122, an inhibitor of phospholipase C (PLC), ET-1 activated only NSCC-1. Dibutyryl cAMP alone did not activate any Ca2+ channels in the resting and ET-1-stimulated CHO-SerET(A)R. Microinjection of G12G228A abolished the activation of NSCC-1 and NSCC-2 in CHO-ET(A)R and that of NSCC-1 in CHO-SerET(A)R. These results indicate that ET(A)R activates three types of Ca2+ channels via different G protein-related pathways. NSCC-1 is activated via a G12-dependent pathway, NSCC-2 via G(q)/PLC- and G12-dependent pathways, and SOCC via a G(q)/PLC-dependent pathway.     

Ca2+ influx mediates apoptosis induced by 4-aminopyridine, a K+ channel blocker, in HepG2 human hepatoblastoma cells

Apoptosis appears to be implicated in the pathogenesis and therapeutic applications of cancer. In this study we investigated the induction of apoptosis by 4-aminopyridine (4-AP), a K(+) channel blocker, and its mechanism in HepG2 human hepatoblastoma cells. 4-AP reduced cell viability and induced DNA fragmentation, a hallmark of apoptosis, in a dose-dependent manner. In addition, 4-AP induced a sustained increase in intracellular Ca(2+) concentration, which was completely inhibited by the extracellular Ca(2+) chelation with EGTA. 4-AP also induced Mn(2+) influx, indicating that the 4-AP-induced increased intracellular Ca(2+) levels were due to activation of Ca(2+) influx pathway. 4-AP also depolarized membrane potential that was measured by using di-O-C(5)(3), a voltage-sensitive fluorescent dye. 4-AP-induced Ca(2+) influx was significantly inhibited not by voltage-operative Ca(2+) channel blockers (nifedipine or verapamil), but by flufenamic acid (FA), a known nonselective cation channel blocker. Quantitative analysis of apoptosis by the flow cytometry revealed that treatment with either FA or BAPTA, an intracellular Ca(2+) chelator, significantly inhibited the 4-AP-induced apoptosis. Taken together, these results suggest that the observed 4-AP-induced apoptosis in the HepG2 cells may result from Ca(2+) influx through the activation of voltage-sensitive Ca(2+)-permeable non-selective cation channels. These results further suggest that membrane potential change by modulation of K(+) channel activity may be involved in the mechanism of apoptosis in human hepatoma cells.     

Inhibition of the fast Na+ inward current by the Ca2+ channel blocker tiapamil

The inhibitory effect of the phenylalkylamine-type Ca2+-entry blocker, tiapamil, on the fast Na+ inward current was investigated in guinea-pig papillary muscles by measuring the maximum upstroke velocity (dV/dt)max of transmembrane action potentials. Tiapamil inhibited (dV/dt)max at concentrations above 10(-6) M, with an IC50 value of 7 X 10(-5) M (1 Hz stimulation frequency, 5.9 mM extracellular K+). Verapamil was less potent in depressing upstroke velocity. Inhibition of the dV/dtmax strongly depended on the frequency at which the muscles were stimulated ("use-dependent" effect). There was no evidence that tiapamil acts in a potential-dependent manner like local anesthetics. The results indicate that the Ca2+-entry blocker tiapamil has additional pharmacological properties, which may contribute to its usefulness in the treatment of ventricular arrhythmias.