Diverse effects of monensin on capacitative Ca2+ entry and release of stored Ca2+ in vascular smooth muscle cells

The effects of monensin, an activator of Na(+)/H(+) exchanger (NHE), on capacitative Ca(2+) entry (CCE) were investigated using A7r5 cells. Capacitative Ca(2+) entry was induced by elevation of extracellular Ca(2+) concentrations of A7r5 cells in which stored Ca(2+) had been depleted by previous administration of thapsigargin. Capacitative Ca(2+) entry was abolished by pretreatment of the cells with SKF-96365 (1-[beta-(3-[4-methoxyphenyl]propoxy)-4-methoxyphenethyl]-1H-imidazole hydrochloride) but was not affected by pretreatment with verapamil. Monensin significantly increased capacitative Ca(2+) entry. On the other hand, 5-hydroxytryptamine-induced inositol monophosphate accumulation and subsequent intracellular Ca(2+) release from its stores were significantly inhibited by monensin, while thapsigargin-induced Ca(2+) release was not affected by monensin. These results suggest that monensin has diverse actions on capacitative Ca(2+) entry and agonist-induced release of stored Ca(2+) in vascular smooth muscle cells.     

Sildenafil inhibits human pulmonary artery smooth muscle cell proliferation by decreasing capacitative Ca2+ entry

Ca(2+) is a pivotal signal in human pulmonary artery smooth muscle cells (PASMCs) proliferation. Capacitative Ca(2+) entry (CCE) via the store-operated channel (SOC), which encoded by the transient receptor potential (TRP) gene, is an important mechanism for regulating intracellular Ca(2+) concentration ([Ca(2+)](i)) in PASMCs. Sildenafil, a potent type 5 nucleotide-dependent phosphodiesterase (PDE) inhibitor, has been proposed as a therapeutic tool to treat or prevent pulmonary arterial hypertension (PAH); however, the mechanism of its antiproliferative effect on PASMCs remains unclear. This study was designed to investigate the possible antiproliferative mechanism of sildenafil on human PASMCs, namely, its effect on the Ca(2+)-signal pathway. Cultured normal PASMCs were treated with endothelin-1 (ET-1) or ET-1 plus sildenafil separately. Cell number and viability were determined with a hemocytometer or MTT assay. [Ca(2+)](i) was measured by loading PASMCs with fura 2-AM. Expression of the TRPC1 gene and protein was detected by RT-PCR and Western blot, respectively. The results show that sildenafil dose-dependently inhibited the proliferation of PASMCs, the enhancement of basal [Ca(2+)](i) level, increase of CCE, and upregulation of TRPC expression induced by ET-1. These results suggest that sildenafil potently inhibits ET-1-induced PASMCs proliferation and downregulation of CCE and TRPC expression may be responsible for its antiproliferative effect.     

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.     

Receptor-activated Ca2+ influx: capacitative Ca2+ entry and TRP proteins]

Receptor-activated Ca2+ channels (RACC) are triggered in response to activation of G protein-coupled receptors or tyrosine kinase-coupled receptors. RACCs, together with voltage-dependent Ca2+ channels, form physiologically the most important Ca2+ influx pathways, being highly diverged in activation mechanisms and Ca2+ permeability. Characterization of mammalian homologues of Drosophila TRP proteins has been an important clue for understanding molecular mechanisms underlying receptor-activated Ca2+ influx in vertebrate cells. Recent issues have been whether any members of the TRP family form capacitative Ca2+ entry (CCE) channels activated by release of Ca2+ from internal stores and their depletion. We have isolated cDNAs that encode seven mouse TRP homologues, TRP1-7. TRP homologues are distributed differently among tissues, although they are all abundant in the brain. Functional characterization of TRP proteins recombinantly expressed in HEK cells indicate that TRP5 is highly permeable to Ca2+, while TRP3 and 7 are non-selective cation channels. The results demonstrate that TRP3,5,7 are capable of generating Ca2+ currents after desensitization of the stimulated G-protein-coupled receptors and replenishment of stores, suggesting that store depletion is not necessary to maintain activity of the TRP homologues. Ca2+ positively regulates TRP channels through Ca(2+)-calmodulin pathways, but via different Ca(2+)-calmodulin-dependent enzymes. Thus, activation of TRP channels is not tightly coupled with store depletion as CCE, suggesting that CCE (or CRAC) channels are molecular entities separate from TRP.     

Ca2+ localization and sensitivity in vascular smooth muscle

An increase in cytosolic Ca2+ level ([Ca2+]i) is a prerequisite for smooth muscle contraction. Simultaneous measurements of [Ca2+]i and muscle tension give direct information on the Ca2+ regulation of smooth muscle. The photoprotein aequorin and the fluorescent Ca2+ indicator fura-2 are widely used for this purpose. Although there are some inconsistencies between the results obtained with these two indicators, comparison between [Ca2+]i and muscle tension in vascular smooth muscle indicates that stimulation of alpha-adrenoceptors increases, whereas stimulation of beta-adrenoceptors decreases, both the Ca2+ sensitivity of contractile elements and [Ca2+]i. Thus, as Hideaki Karaki explains, contractility of vascular smooth muscle may be regulated not only by [Ca2+]i but also by the Ca2+ sensitivity of the contractile elements.     

Monensin augments capacitative Ca2+ entry and subsequent aggregation of platelets via an intracellular alkalosis-mediated mechanism.

Effects of monensin, an ionophore that facilitates the transmembrane exchange of Na+ for H+, on capacitative Ca2+ entry (CCE) of platelets were investigated. CCE of human platelets was induced by addition of Ca2+ to a nominally Ca2+-free medium after release of intracellular stored Ca2+ caused by thapsigargin. CCE was strongly inhibited by SKF-96365 (1-[beta-(3-[4-methoxyphenyl]propoxy)-4-methoxyphenethyl]-1H-imidazole hydrochloride). Monensin significantly increased SKF-96365-sensitive CCE and subsequent platelet aggregation. Monensin also induced a sustained increase in intracellular pH. The augmenting effect of monensin on CCE and subsequent platelet aggregation was not observed in the presence of sodium propionate, which canceled intracellular alkalinization induced by monensin. These results suggest that monensin augments CCE of platelets by a mechanism mediated by intracellular alkalosis.     

Zn2+ increases resting cytosolic Ca2+ levels and abolishes capacitative Ca2+ entry induced by ATP in MDCK cells

The effect of Zn2+ on Ca2+ signaling in Madin Darby canine kidney (MDCK) cells was investigated by measuring the changes in the fluorescence of the Ca2+-sensitive dye fura-2. Zn2+ significantly increased cytoplasmic free Ca2+ levels ([Ca2+]i) at concentrations of 2-100 microM. The maximum response was obtained at concentrations of 25-100 microM. The [Ca2+]i rise induced by 100 microM Zn2+ consisted of a gradual rise and a plateau phase, and was primarily mediated by La3+-sensitive extracellular Ca2+ influx because the [Ca2+]i rise was abolished by pretreatment with 100 microM La3+ or removal of extracellular Ca2+, and that Zn2+ induced Mn2+ quench of fura-2 fluorescence at 360 nm excitation wavelength which was prevented by pretreatment with 100 microM La3+. Pretreatment with 100 microM Zn2+ for 220 s did not reduce the [Ca2+]i rise induced by the endoplasmic reticulum (ER) Ca2+ pump inhibitor, thapsigargin, suggesting that Ca2+ release from the ER played a minor role in the Zn2+-induced [Ca2+]i rise. Zn2+ (100 microM) nearly abolished the capacitative Ca2+ entry induced by ATP (100 microM). We also investigated the effect of Zn2+ pretreatment on the [Ca2+]i rise induced by ATP. Zn2+ (100 microM) affected ATP-induced [Ca2+]i rise by abolishing capacitative Ca2+ entry and increasing [Ca2+]i on its own without altering Ca2+ release from intracellular sources. The effect of Zn2+ on [Ca2+]i was dissociated from changes in membrane potential.     

Ca2+ extrusion in aged smooth muscle cells

We investigated the effects of aging in Ca(2+) extrusion mechanisms in smooth muscle bladder cells from 4 and 20-24-month-old guinea pigs using fluorescence microscopy and fura-2. Cells were challenged with a pulse of KCl immediately before perfusion with a Ca(2+) free solution containing no inhibitors (control, untreated cells) or inhibitors of plasma membrane Ca(2+) pump (PMCA, 1mM La(3+)), Na(+)/Ca(2+) exchanger (NCX, 1 microM SEA0400) or the sarcoendoplasmic Ca(2+) pump (SERCA, 1 microM thapsigargin). Treatment of young adult cells with the inhibitors allowed estimating a relative contribution of 55% for NCX, 27% for PMCA and 31% for SERCA. Combination of two inhibitors at the same time showed the presence of interaction between extrusion mechanisms. In aged cells the [Ca(2+)](i) extrusion was impaired due to decrease of PMCA activity, as revealed by the loss of effect of La(3+), and to inhibitory interactions between NCX and SERCA activities, indicated by acceleration of decay in response to their respective inhibitors. In conclusion, in smooth muscle cells aging decreases the overall Ca(2+) extrusion activity and modifies the interactions between the activities of the main Ca(2+) removing mechanisms.     

Nitric oxide inhibits capacitative Ca2+ entry by suppression of mitochondrial Ca2+ handling

1. Nitric oxide (NO) is a key modulator of cellular Ca(2+) signalling and a determinant of mitochondrial function. Here, we demonstrate that NO governs capacitative Ca(2+) entry (CCE) into HEK293 cells by impairment of mitochondrial Ca(2+) handling. 2. Authentic NO as well as the NO donors 1-[2-(carboxylato)pyrrolidin-1-yl]diazem-1-ium-1,2-diolate (ProliNO) and 2-(N,N-diethylamino)-diazenolate-2-oxide (DEANO) suppressed CCE activated by thapsigargin (TG)-induced store depletion. Threshold concentrations for inhibition of CCE by ProliNO and DEANO were 0.3 and 1 micro M, respectively. 3. NO-induced inhibition of CCE was not mimicked by peroxynitrite (100 micro M), the peroxynitrite donor 3-morpholino-sydnonimine (SIN-1, 100 micro M) or 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP, 1 mM). In addition, the guanylyl cyclase inhibitor 1H-[1,2,4] oxadiazole[4,3-a] quinoxalin-1-one (ODQ, 30 micro M) failed to antagonize the inhibitory action of NO on CCE. 4. DEANO (1-10 micro M) suppressed mitochondrial respiration as evident from inhibition of cellular oxygen consumption. Experiments using fluorescent dyes to monitor mitochondrial membrane potential and mitochondrial Ca(2+) levels, respectively, indicated that DEANO (10 micro M) depolarized mitochondria and suppressed mitochondrial Ca(2+) sequestration. The inhibitory effect of DEANO on Ca(2+) uptake into mitochondria was confirmed by recording mitochondrial Ca(2+) during agonist stimulation in HEK293 cells expressing ratiometric-pericam in mitochondria. 5. DEANO (10 micro M) failed to inhibit Ba(2+) entry into TG-stimulated cells when extracellular Ca(2+) was buffered below 1 micro M, while clear inhibition of Ba(2+) entry into store depleted cells was observed when extracellular Ca(2+) levels were above 10 micro M. Moreover, buffering of intracellular Ca(2+) by use of N,N'-[1,2-ethanediylbis(oxy-2,1-phenylene)] bis [N-[25-[(acetyloxy) methoxy]-2-oxoethyl]]-, bis[(acetyloxy)methyl] ester (BAPTA/AM) eliminated inhibition of CCE by NO, indicating that the observed inhibitory effects are based on an intracellular, Ca(2+) dependent-regulatory process. 6. Our data demonstrate that NO effectively inhibits CCE cells by cGMP-independent suppression of mitochondrial function. We suggest disruption of local Ca(2+) handling by mitochondria as a key mechanism of NO induced suppression of CCE.     

Caffeine induced Ca2+ release and capacitative Ca2+ entry in human embryonic kidney (HEK293) cells

The potential role of endogenous ryanodine receptor (RyR) in modulating Ca2+ handling in HEK293 cells is controversial. Using Fura2/AM, here we provide evidence that caffeine can induce Ca2+ release from inositol 1,4,5-trisphosphate receptor-sensitive stores and Ca2+ entry in early passage numbers of HEK293 cells, but not in late passage ones. Ryanodine blocks caffeine-mediated effect, whereas 4-chloro-m-cresol can mimic these effects. In contrast, an increase in cyclic AMP or activation of voltage-dependent Ca2+ channels does not induce detectable alteration in intracellular Ca2+. Importantly, immunoblotting and staining have revealed that endogenous RyR expression is more abundant in the early than in the late passage cells. Additionally, similar to carbachol, Ca2+ entry in response to caffeine is blocked by capacitative Ca2+ entry inhibitors. These results indicate that the endogenous RyR in HEK293 cells can function as Ca2+ release channels and mediate capacitative Ca2+ entry, but they may be reduced due to cell passage.     

Intracellular alkalinization augments alpha(1)-adrenoceptor-mediated vasoconstriction by promotion of Ca(2+) entry through the non-L-type Ca(2+) channels.

Modulation by intracellular pH of the vasoconstriction induced by alpha-adrenoceptor agonists was investigated in isolated guinea pig aorta. NH(4)Cl (15 mM) increased intracellular pH of aortic smooth muscle cells by about 0.2 pH unit and significantly augmented KCl-induced contraction of aortic strips, whereas simultaneous administration of NH(4)Cl (15 mM) plus Na(+) propionate (30 mM) failed to affect intracellular pH or contractility. NH(4)Cl (15 mM) potentiated contractions induced by alpha-adrenoceptor agonists, norepinephrine, phenylephrine and clonidine. Contraction induced by alpha(1)-selective adrenoceptor agonist, phenylephrine, but not that induced by norepinephrine or clonidine, was insensitive to inhibition by verapamil (1 microM). Phenylephrine-induced contraction was not affected by NH(4)Cl in Ca(2+)-free medium whereas extracellular Ca(2+)-induced contraction of phenylephrine-stimulated aorta was significantly augmented by NH(4)Cl. Consistently, 45Ca(2+)uptake into phenylephrine 1 microM)-stimulated aortic strips was increased by incubation with NH(4)Cl. The potentiating effects of NH(4)Cl on both phenylephrine-induced Ca(2+) entry and contraction were antagonized by Na(+) propionate. These results suggest that intracellular alkalinization facilitates alpha(1)-adrenoceptor-mediated vasoconstriction by facilitation of an agonist-induced Ca(2+) entry pathway that is independent of L-type Ca(2+) channels.     

库容性Ca~(2+)内流介导大鼠远端结肠平滑肌收缩

目的探讨库容性Ca2+内流(capac itative Ca2+entry,CCE)是否参与大鼠远端结肠平滑肌兴奋-收缩偶联过程。方法利用器官离体装置、张力换能器、Powerlab 4/25T数据采集分析系统测定远端结肠平滑肌的张力。结果毒胡萝卜素(thapsigargin,TG,10 nmol.L-1~1μmol.L-1)诱导结肠平滑肌条产生持续的张力性收缩,不同浓度TG所致的同步收缩反应张力不同。在无钙Krebs液(包含1 mmol.L-1EDTA)中使用TG将肌条培养35 m in后,再加入Ca2+2.5mmol.L-1,比未使用TG处理的肌条产生的收缩张力明显提高(99%±28%vs70%±8%)。且TG耗竭胞内钙库后再复钙所致的收缩效应,不受L型钙通道阻断剂verapam il影响,但可被SOC通道阻断剂La3+减弱。结论TG耗竭胞内钙库后再复钙诱导的大鼠远端结肠平滑肌收缩反应由CCE介导,提示CCE是提供大鼠远端结肠平滑肌收缩的激活信号Ca2+的来源之一,参与完成结肠平滑肌兴奋-收缩偶联过程。     

Antisense-inhibition of plasma membrane Ca2+ pump induces apoptosis in vascular smooth muscle cells

The effect of antisense oligodeoxynucleotides (ODNs) of plasma membrane Ca(2+)-pumping ATPase (PMCA) on rat aortic vascular smooth muscle cells (VSMCs) in primary culture was examined. More than 80% of the PMCA expressed in cultured VSMCs was the PMCA-1B subtype. Exposed to antisense ODNs against PMCA-1, not only the expression of the PMCA protein but also mRNA of PMCA-1B was diminished in a concentration-dependent manner. Extracellular Na(+)-independent (45)Ca(2+) efflux catalyzed via PMCA was inhibited with antisense ODNs. Both the resting and ionomycin- or ATP-stimulated levels of intracellular Ca(2+) were increased by antisense ODNs. Furthermore, prolonged treatment with antisense ODNs caused apoptosis in VSMCs. The occurrence of apoptosis was inhibited by FK506, a potent immunosuppressant. These results demonstrate that the PMCA was specifically inhibited by antisense ODNs and suggest that PMCA plays an important role in regulation of intracellular Ca(2+) concentrations, especially at the resting condition to prevent an occurrence of apoptosis that may be induced through the activation of calcineurin.     

Release of Ca2+ from intracellular store in smooth muscle cells of rat portal vein by ATP-induced Ca2+ entry.

1. The action of adenosine 5'-triphosphate (ATP, 10 microM) was studied in single patch-clamped smooth muscle cells of rat portal vein where the free internal Ca2+ concentration in the cell (Cai) was estimated by the emission from the dye indo-1. 2. In the presence of 20 microM gallopamil (D600), a blocker of voltage-dependent Ca2+ channels, ATP applied to cells held at a holding potential of -60 mV evoked a transient inward current and an increase in Cai. 3. The rise in Cai evoked by ATP was completely suppressed in the absence of external Ca2+ although a transient inward current was still observed. 4. ATP-induced responses were not modified by the addition of the inositol 1,4,5-trisphosphate receptor antagonist, heparin (1 mM) in the pipette solution. 5. In the presence of caffeine (5 mM) or ryanodine (100 microM) in the pipette solution, which deplete the intracellular Ca2+ store, the ATP-induced Cai rise was greatly reduced. 6. Our results suggest that in single cells from rat portal vein, ATP releases Ca2+ from intracellular stores without involving InsP3, but via a Ca2+ release mechanism activated by Ca2+ influx through ATP-gated 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.     

Ginsenoside-Rd from panax notoginseng blocks Ca2+ influx through receptor- and store-operated Ca2+ channels in vascular smooth muscle cells

Previously, it was found that total saponins from panax notoginseng inhibited Ca2+ influx coupling to activation of alpha1-adrenoceptor. This study was designed to investigate the effects of ginsenoside-Rd from total saponins of panax notoginseng on receptor-operated (ROCC) and store-operated (SOCC) Ca2+ channels in vascular smooth muscle cells using fura-2 fluorescence, whole cell patch clamp ion channel recording, radio-ligand-receptor binding, 45Ca2+ radio-trace and organ bath techniques. It was found that ginsenoside-Rd reduced phenylephrine-induced contractile responses and Ca2+ influx in normal media without significant effect on these responses in Ca2+ -free media. Ginsenoside-Rd also decreased phenylephrine- and thapsigargin-induced inward Ca2+ currents, and attenuated thapsigargin- and 1-oleoy-2-acetyl-sn-glycerol (OAG)-induced cation entries that are coupled to ROCC and SOCC respectively. Ginsenoside-Rd failed to inhibit KCl-induced contraction of rat aortal rings and Ca2+ influx, and did not alter voltage-dependent inward Ca2+ current (VDCC) which was blocked by nifedipine. Also, ginsenoside-Rd did not change binding site and affinity of [3H]-prazosin for alpha1-adrenoceptor in the vascular plasma membrane. These results suggest that ginsenoside-Rd, as an inhibitor, remarkably inhibits Ca2+ entry through ROCC and SOCC without effects on VDCC and Ca2+ release in vascular smooth muscle cells.     

Role of InsP3 and ryanodine receptors in the activation of capacitative Ca2+ entry by store depletion or hypoxia in canine pulmonary arterial smooth muscle cells

BACKGROUND AND PURPOSE: Experiments were performed to determine if capacitative Ca(2+) entry (CCE) in canine pulmonary arterial smooth muscle cells (PASMCs) is dependent on InsP(3) receptors or ryanodine receptors as induction of CCE is dependent on simultaneous depletion of the functionally separate InsP(3)- and ryanodine-sensitive sarcoplasmic reticulum (SR) Ca(2+) stores in these cells. EXPERIMENTAL APPROACH: Myocytes were isolated from canine pulmonary arteries using enzymatic procedures and were used within 8 h of preparation. Measurements of cytosolic Ca(2+) were made by imaging fura-2 loaded individual myocytes that were perfused with physiological buffered saline solution with or without Ca(2+). KEY RESULTS: Treating myocytes with 10 microM cyclopiazonic acid (CPA), removing extracellular Ca(2+), and briefly applying 10 mM caffeine and 10 microM 5-hydroxytryptamine (5-HT) depleted SR Ca(2+) stores. Extracellular Ca(2+) reintroduction caused cytosolic [Ca(2+)] to elevate above baseline signifying CCE. The InsP(3) receptor inhibitors 2-aminobiphenylborate (50-75 microM; 2-APB) and xestospongin-C (20 microM; XeC) abolished CCE. Yet, CCE was unaffected by 10 microM or 300 microM ryanodine or 10 microM dantrolene, which modify ryanodine receptor activity. Higher dantrolene concentrations (50 microM), however, can inhibit both ryanodine receptors and InsP(3) receptors, did reduce CCE. In contrast, CCE activated by hypoxia was unaffected by XeC (20 microM). CONCLUSIONS AND IMPLICATIONS: The results provide evidence that CCE activated by depletion of both InsP(3) and ryanodine SR Ca(2+) stores in canine PASMCs is dependent on functional InsP(3) receptors, whereas the activation of CCE by hypoxia appears to be independent of functional InsP(3) receptors.     

Protein kinase C potentiates capacitative Ca2+ entry that links to steroidogenesis in bovine adrenocortical cells

I investigated the role of protein kinase C (PKC) in regulation of the capacitative Ca2+ entry and steroidogenesis in bovine adrenocortical (BA) cells. Thapsigargin (TG)-treatment depleted intracellular Ca2+ stores followed by induction of Ca2+ influx from the extracellular pool and also increasing of Mn2+ influx as an indicator of divalent cation influx in BA cells. Calphostin C, a PKC inhibitor, inhibited the TG-induced [Ca2+]i elevation dose-dependently (0.1-1 microM) and attenuated Mn2+ entry. Phorbol 12-myristate 13-acetate (PMA), an activator of PKC, potentiated the elevation of [Ca2+]i and enhanced Mn2+ entry by TG treatment. These results suggest that PKC may modulate capacitative Ca2+ entry in BA cells. In the presence of extracellular Ca2+, TG enhanced cortisol production in BA cells. Calphostin C attenuated the TG-induced steroidogenesis dose-dependently (0.25-1 microM). PMA enhanced the steroidogenesis dose-dependently (1-100 nM). These results suggested that PKC may have a modulatory effect on the capacitative Ca2+ entry that links to steroidogenesis in BA cells.     

Direct block of Ca2+ channels by calmidazolium in cultured vascular smooth muscle cells.

We investigated the action of calmidazolium (CMZ), an inhibitor of calmodulin (CaM), on the L-type Ca2+ currents (ICa(L)) of cultured vascular smooth muscle (VSM) cells (A7r5 cell line), by using the whole-cell voltage-clamp method. All experiments were conducted at room temperature (24-25 degrees C). The peak IBa (Ca2+ channel current with 5 mM Ba2+ as charge carrier) was evoked every 15 s by a test potential to +10 mV from a holding potential of -60 mV. To elevate intracellular free Ca2+ concentration ([Ca]i) to pCa 6.5, the pipette solution contained a Ca2+-EGTA buffer (pCa 6.5) to allow equilibration with the cells. Bath application of 1 microM CMZ reduced the peak amplitude of IBa to 36.7+/-4.9% (n = 8); maximal effect occurred within 7-8 min. Peak IBa continued to decrease even after washing out the CMZ. Recovery of IBa was not observed even after 10 min of washout. Even in presence of an peptide inhibitor of CaM-dependent protein kinase-II (5.2 microM) in the pipette solution, CMZ inhibited IBa to 27.8 +/-5.3% (n = 7). To exclude the possibility that other Ca2+/ CaM-dependent kinases and phosphatases may regulate Ca2+ channel activity, we examined the effect of CMZ on IBa when [Ca]i was reduced by use of Ca2+/EGTA-buffered pipette solutions. At pCa approximately equal to 10 (10 mM EGTA and only contaminant Ca2+), CMZ inhibited IBa to 33.4+/-5.9% (n = 14) with a median inhibitory concentration (IC50) value of 0.29 microM. The activation curve (pCa approximately equal to 10) was shifted in the positive direction by 6.3 mV; the inactivation curve was shifted in the negative direction by 5.0 mV. CMZ decreased IBa progressively during repetitive step depolarizations. CMZ did not slow the rate of recovery from inactivation. In conclusion, CMZ inhibits Ca2+ channel current in a use-dependent manner. This inhibition is independent of CaMK-II and other Ca2+/CaM-dependent pathways. Therefore it is likely due to direct blockade of Ca2+ channels by CMZ. CMZ may reduce the outer surface charge and block the open state of the Ca2+ channels.     

Ca2+ entry activated by emptying of intracellular Ca2+ stores in ileal smooth muscle of the rat.

1. The effects of depletion of intracellular Ca2+ stores on muscle tension and the intracellular Ca2+ concentration ([Ca2+])i were studied in fura-2 loaded longitudinal smooth muscle cells of the rat ileum. 2. After exposure to a Ca(2+)-free solution, application of Ca2+ caused a small contraction and a rise in [Ca2+]i, both of which were potentiated when the muscle was challenged with carbachol or caffeine before the addition of Ca2+. 3. Cyclopiazonic acid (CPA), a specific inhibitor of sarcoplasmic reticulum Ca(2+)-ATPase, dose-dependently decreased tension development and the rises in [Ca2+]i induced by carbachol and caffeine in the Ca(2+)-free solution, but conversely increased the Ca(2+)-induced responses even in the presence of the voltage-dependent Ca2+ channel blockers, methoxyverapamil and nifedipine. 4. The contraction and rise in [Ca2+]i evoked by Ca2+ gradually declined with time after removal of CPA, while the reverse was the case for the responses to carbachol and caffeine. 5. The Ca(2+)-induced contraction and rise in [Ca2+]i in the presence of CPA were inhibited by the replacement of Na+ with K+ or Cs+, and by the addition of Cd2+, Ba2+, Ni2+ or La3+. 6. The influx of Mn2+ was much greater in extent in the presence of CPA than in its absence. 7. These results suggest that the emptying of intracellular Ca2+ stores may activate Ca2+ influx not associated with voltage-dependent Ca2+ channels in the rat ileal smooth muscle.