Cell Line A7r5 Research Articles

Actin filament disruption inhibits L-type Ca(2+) channel current in cultured vascular smooth muscle cells.

To clarify interactions between the cytoskeleton and activity of L-type Ca(2+) (Ca(L)) channels in vascular smooth muscle (VSM) cells, we investigated the effect of disruption of actin filaments and microtubules on the L-type Ca(2+) current [I(Ba(L))] of cultured VSM cells (A7r5 cell line) using whole cell voltage clamp. The cells were exposed to each disrupter for 1 h and then examined electrophysiologically and morphologically. Results of immunostaining using anti-alpha-actin and anti-alpha-tubulin antibodies showed that colchicine disrupted both actin filaments and microtubules, cytochalasin D disrupted only actin filaments, and nocodazole disrupted only microtubules. I(Ba(L)) was greatly reduced in cells that were exposed to colchicine or cytochalasin D but not to nocodazole. Colchicine even inhibited I(Ba(L)) by about 40% when the actin filaments were stabilized by phalloidin or when the cells were treated with phalloidin plus taxol to stabilize both cytoskeletal components. These results suggest that colchicine must also cause some inhibition of I(Ba(L)) due to another unknown mechanism, e.g., a direct block of Ca(L) channels. In summary, actin filament disruption of VSM cells inhibits Ca(L) channel activity, whereas disrupting the microtubules does not.

Regulation of corticotropin-releasing factor receptor type 2 beta messenger ribonucleic acid in the rat cardiovascular system by urocortin, glucocorticoids, and cytokines.

CRF receptor type 2 (CRF R2) messenger RNA (mRNA) expression in the rodent heart is modulated by exposure to both the bacterial endotoxin lipopolysaccharide (LPS) and glucocorticoids. In this study we examined the roles of glucocorticoids, cytokines, and CRF R2beta ligands in the regulation of CRF R2beta expression in the cardiovascular system both in vivo and in vitro. Using ribonuclease protection assays, we found that, in addition to the injection of LPS or corticosterone, physical restraint caused a decrease in CRF R2beta mRNA levels in the rat heart and aorta. Adrenalectomy with corticosterone replacement at constant levels partially blocked LPS-induced decreases in CRF R2beta mRNA expression in the heart. Thus, elevations of endogenous circulating corticosterone could contribute to the down-regulation of CRF R2beta mRNA expression in heart. To identify other putative modulating factors, we examined CRF R2beta expression in the aorta-derived A7R5 cell line. Incubation with CRF R2 ligands or dexamethasone reduced CRF R2beta mRNA levels. In addition, incubation with a variety of cytokines, proteins released during immune challenge, also reduced CRF R2beta mRNA expression. The multifactorial regulation of CRF R2beta mRNA expression in the cardiovascular system may serve to limit the inotropic and chronotropic effects of CRF R2 agonists such as urocortin during prolonged physical or immune challenge.

Labedipinedilol-A: A vanilloid-based ?/?-adrenoceptor blocker with calcium entry blocking and long-acting antihypertensive properties

The combination of β-adrenoceptor blockade and vasodilator action have proved highly useful in antihypertensive therapy. Studies of the mechanisms of action of labedipinedilol-A that combine these effects within a single molecule are described in this report. Intravenous labedipinedilol-A (0.1–1.0 mg/kg) produced dose-dependent hypotensive and bradycardia responses for above 1.0 h, significantly different from nifedipine (0.5 mg/kg, i.v.)-induced hypotensive and reflex tachycardia activities in pentobarbital-anesthetized Wistar rats. Pretreatment with labedipinedilol-A also inhibited phenylephrine (20 μg/kg, i.v.)-induced hypertensive and (-)isoprenaline (0.5 μg/kg, i.v.)-induced tachycardia effects. Oral administration of labedipinedilol-A (5–50 mg/kg) in spontaneously hypertensive rats (SHR) reduced the blood pressure and heart rate for 24 h but did not increase heart rate. Labedipinedilol-A (10–7–10–5 M) competitively antagonized (-)isoprenaline (10–10–10–4M)-induced positive chronotropic and inotropic effects of the isolated rat atria and tracheal relaxation responses of the isolated guinea pig tissues. Labedipinedilol-A also prevented the rate-increasing effects of increased extracellular Ca2+ (3.0–9.0 mM) in a concentration-dependent manner. In the isolated rat aorta, labedipinedilol-A competitively antagonized CaCl2 and norepinephrine-induced contractions with pKCa–1 and pA2 values of 8.46 ± 0.05 and 8.28 ± 0.03 and had a potent effect of inhibiting high K+-induced vasocontraction. Furthermore, labedipinedilol-A, in an equal antagonist activity, inhibited norepinephrine-induced phasic and tonic contraction. In the cultured blood vessel smooth muscle cell (A7r5 cell line), KCl, norepinephrine, and Bay K 8644-induced intracellular calcium changes were decreased after application of labedipinedilol-A (10–9–10–6 M). The binding characteristics of labedipinedilol-A were evaluated in [3H]CGP-12177 binding to ventricle and lung and [3H]nitrendipine and [3H]prazosin binding to brain membranes in rats. The -logIC50 values of labedipinedilol-A for β1-, β2-, and α1-adrenoceptor and calcium channel, were 8.17 × 10–7 M, 8.20 × 10–7 M, 2.20 × 10–8 M, and 2.46 × 10–8 M, respectively. Labedipinedilol-A-induced sustained depressor effect was mainly attributed to its calcium entry and α-adrenoceptor blocking activities in the blood vessel. Sustained bradycardia effect resulted from β-adrenoceptor and calcium entry blocking, which deleted the sympathetic activation-associated reflex tachycardia in the heart. Drug Dev. Res. 49:94–108, 2000. © 2000 Wiley-Liss, Inc.

Vanidipinedilol: a vanilloid-based beta-adrenoceptor blocker displaying calcium entry blocking and vasorelaxant activities.

Calcium channel and beta-adrenoceptor blockade have proved highly useful in antihypertensive therapy. Studies of the mechanisms of action of vanidipinedilol that combine these effects within a single molecule are described here. Intravenous injection of vanidipinedilol (0.1, 0.25, 0.5, 1.0, and 2.0 mg/kg) produced dose-dependent hypotensive and bradycardic responses, significantly different from nifedipine-induced (0.5 mg/kg, i.v.) hypotensive and reflex tachycardic effects in pentobarbital-anesthetized Wistar rats. A single oral administration of vanidipinedilol at doses of 10, 25, and 50 mg/kg dose-dependently reduced blood pressure with a decrease in heart rate in conscious spontaneously hypertensive rats (SHRs). In the isolated Wistar rat atrium, vanidipinedilol (10(-7), 10(-6), and 10(-5) M) competitively antagonized the (-)isoproterenol-induced positive chronotropic and inotropic effects and inhibited the increase in heart rate induced by Ca2+ (3.0-9.0 mM) in a concentration-dependent manner. The parallel shift to the right of the concentration-response curve of (-)isoproterenol and CaCl2 suggested that vanidipinedilol possessed beta-adrenoceptor-blocking and calcium entry-blocking activities. On tracheal strips of reserpinized guinea pig, cumulative doses of vanidipinedilol (10(-10) to 3x10(-6) M) produced dose-dependent relaxant responses. Preincubating the preparation with ICI 118,551 (10(-10), 10(-9), 10(-8) M), a beta2-adrenoceptor antagonist, shifted the vanidipinedilol concentration-relaxation curve significantly to a region of higher concentrations. These results implied that vanidipinedilol had a partial beta2-agonist activity. In the isolated thoracic aorta of rat, vanidipinedilol had a potent effect inhibiting high-K+-induced contractions. KCI-induced intracellular calcium changes of blood vessel smooth muscle cell (A7r5 cell lines) determined by laser cytometry also was decreased after administration of vanidipinedilol (10(-8), 10(-7), 10(-6) M). Furthermore, the binding characteristics of vanidipinedilol and various antagonists were evaluated in [3H]CGP-12177 binding to ventricle and lung and [3H]nitrendipine binding to cerebral cortex membranes in rats. The order of potency of beta1- and beta2-adrenoceptor antagonist activity against [3H]CGP-12177 binding was (-)propranolol (pKi, 8.59 for beta1 and 8.09 for beta2) > vanidipinedilol (pKi, 7.09 for beta1 and 6.64 for beta2) > atenolol (pKi, 6.58 for beta1 and 5.12 for beta2). The order of potency of calcium channel antagonist activity against [3H]nitrendipine binding was nifedipine (pKi, 9.36) > vanidipinedilol (pKi, 8.07). The ratio of beta1-adrenergic-blocking/calcium entry-blocking selectivity is 0.1 and indicated that vanidipinedilol revealed more in calcium entry-blocking than in beta-adrenergic-blocking activities. It has been suggested that vanidipinedilol-induced smooth muscle relaxation may involve decreased entry of Ca2+ and partial beta2-agonist activities. In conclusion, vanidipinedilol is a nonselective beta-adrenoceptor antagonist with calcium channel blocking and partial beta2-agonist associated vasorelaxant and tracheal relaxant activities. Particularly, the vasodilator effects of vanidipinedilol are attributed to a synergism of its calcium entry blocking and partial beta2-agonist activities in the blood vessel. A sustained bradycardic effect results from beta-adrenoceptor blocking and calcium entry blocking, which blunts the sympathetic activation-associated reflex tachycardia in the heart.

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.

Receptors linked to polyphosphoinositide hydrolysis stimulate Ca2+ extrusion by a phospholipase C-independent mechanism

In A7r5 cells with empty intracellular Ca(2+) stores in which the cytosolic free Ca(2+) concentration ([Ca(2+)](i)) had been increased by capacitative Ca(2+) entry, stimulation of receptors linked to phospholipase C (PLC), including those for Arg(8)-vasopressin (AVP) and platelet-derived growth factor (PDGF), caused a decrease in [Ca(2+)](i.) This effect was further examined in a stable variant of the A7r5 cell line in which the usual ability of hormones to stimulate non-capacitative Ca(2+) entry is not expresssed. In thapsigargin-treated cells, neither AVP nor PDGF affected capacitative Mn(2+) or Ba(2+) entry, but both stimulated the rate of Ca(2+) extrusion, and their abilities to decrease [Ca(2+)](i) were only partially inhibited by removal of extracellular Na(+). These results suggest that receptors linked to PLC also stimulate plasma membrane Ca(2+) pumps. Activation of protein kinase C by phorbol 12, 13-dibutyrate (PDBu, 1 microM) also caused a decrease in [Ca(2+)](i) by accelerating Ca(2+) removal from the cytosol; the effect was again only partially inhibited by removal of extracellular Na(+). An inhibitor of PKC, Ro31-8220 (10 microM), abolished the ability of PDBu to decrease [Ca(2+)](i), without affecting the response to maximal or submaximal concentrations of AVP. Similar experiments with PDGF were impracticable because Ro31-8220, presumably by inhibiting the tyrosine kinase activity of the PDGF receptor, abolished all responses to PDGF. U73122 (10 microM), an inhibitor of PLC, completely inhibited PDGF- or AVP-evoked Ca(2+) mobilization, without preventing either stimulus from causing a decrease in [Ca(2+)](i). We conclude that receptors coupled to PLC, whether via G-proteins or protein tyrosine kinase activity, also share an ability to stimulate the plasma membrane Ca(2+) pump via a mechanism that does not require PLC activity.

Bradykinin modulates arginine vasopressin-induced calcium influx in vascular myocytes

We investigated direct, endothelium-independent effects of bradykinin on arginine vasopressin-induced calcium influx in vascular smooth muscle cells. We studied cultured rat vascular smooth muscle cells by using the whole-cell voltage-clamp and calcium fluorescence imaging methods. Exposing cultured vascular smooth muscle cells (A7r5 cell line) to arginine vasopressin (100 nM) produced a transient increase in [Ca 2+] i, followed by a sustained increase in [Ca 2+] i. This was readily reversible ( n=28). At a holding potential of −40 to −60 mV, arginine vasopressin induced a sustained inward current correlated with a sustained increase in [Ca 2+] i. Bradykinin (30 nM to 30 μM) had no effect on arginine vasopressin-induced [Ca 2+] i transients. However, during the sustained phase of increased [Ca 2+] i, bradykinin reversibly attenuated relative fluorescence and inward current in the presence of arginine vasopressin ( n=14). This was concentration dependent and inhibited by [ d-Phe 7]-bradykinin (30 μM), a kinin receptor antagonist. Also, sustained arginine vasopressin-mediated increases in [Ca 2+] i and inward current were attenuated by Ca 2+-free or La 3+-supplemented perfusate but not by nifedipine ( n=5). Conclusions: (1) Bradykinin can attenuate arginine vasopressin-induced and sustained Ca 2+ influx and sustained inward current through a novel endothelium-independent process. (2) The direct effect of bradykinin on arginine vasopressin-induced increases in [Ca 2+] i sustained Ca 2+ influx in vascular smooth muscle cells is concentration dependent and kinin-receptor mediated. (3) Arginine vasopressin-induced sustained [Ca 2+] i elevation correlates with the activation of a dihydropyridine-insensitive, Ca 2+-conducting inward current.

Inhibition of serum diamine oxidase discloses a constitutive putrescine release from cultured vascular smooth muscle cells.

To determine if putrescine and the higher polyamines spermidine and spermine are released from cultured vascular smooth muscle cells. Vascular smooth muscle cell line A7r5. Cells were treated with aminoguanidine (10 or 100 microM) for 1 to 24 h with or without fetal calf serum (10%) present in the culture medium. Cellular and medium concentrations of polyamines were determined by liquid chromatography. Total cellular protein was determined by the Bradford procedure. Student's two-tailed t-test was used for statistical calculations. A constitutive release of putrescine was disclosed within 5 h if serum diamine oxidase was inhibited by 10 microM aminoguanidine. The release was linear with time for 24 h and specific for putrescine in the sense that the higher polyamines spermidine and spermine, despite similar cellular concentrations, were not released. Similar amounts of putrescine were released from the smooth muscle cells whether or not culture medium contained serum. Cells, that had been cultured in medium from which fetal calf serum had been omitted for last 48 h, contained less putrescine, spermidine and protein than those that persisted in medium that contained serum. A constitutive putrescine release from vascular smooth muscle cells is disclosed in the presence of aminoguanidine.

Intracellular Application of Calmidazolium Increases Ca2+ Current through Activation of Protein Kinase A in Cultured Vascular Smooth Muscle Cells

In the present study, we investigated the actions of calmodulin (CaM) and CaM-dependent protein kinase II (CaMK-II) on the L-type Ca²⁺ currents (I_Ca(L)) of cultured vascular smooth muscle (VSM) cells (A7r5 cell line), using the whole-cell voltage clamp method. The peak I_Ba (Ca²⁺ channel using 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 test the effect of CaM on I_Ba, 1 µM calmidazolium (CMZ), an inhibitor of CaM, was added to the pipette solution (pCa of 6.5 or 300 nM [Ca]_i). The amplitude of maximally activated I_Ba was –4.3 ± 0.5 pA/pF (n = 13) for control and –8.1 ± 0.9 pA/pF (n = 14) in the presence of CMZ. This difference was statistically significant (p = 0.016). The CMZ stimulation of I_Ba was not abolished when 5 µM KN-62, a specific inhibitor of CaMK-II, was included in the pipette (–9.5 ± 1.1 pA/pF; n = 10). Introduction of CaMK-II itself intracellularly had no effect on the basal I_Ba. On the other hand, the CMZ stimulation of I_Ba was prevented by both H-7, a nonspecific protein kinase inhibitor, and H-89, a specific inhibitor of protein kinase A (PK-A). Since CMZ is a strong inhibitor of Ca²⁺/CaM-dependent phosphodiesterase (type I PDE), we studied the effect of 8-methoxymethyl-3-isobutyl-1-methylxanthine (MIBMX), another specific inhibitor of the PDE. MIBMX, like CMZ, stimulated I_Ba: control, –4.6 ± 0.4 pA/pF (n = 10); MIBMX, –9.6 ± 1.2 (n = 8), and CMZ, –7.9 ± 0.9 (n = 15). 0.1 mM 8Br-cAMP, a membrane permeable cAMP analogue, stimulated I_Ba by +42%: before, –3.7 ± 0.7 pA/pF; after, –5.2 ± 1.0 (n = 6). In conclusion, Ca²⁺ channels of VSM cells might not be directly regulated by the CaM/CaMK-II pathway. Therefore, the CMZ stimulation of I_Ba might occur due to the increase in intracellular concentration of cAMP produced by inhibition of CaM-dependent PDE.

Role of intracellular calcium ([Ca2+]i) and tyrosine phosphorylation in adhesion of cultured vascular smooth muscle cells to fibrinogen.

Fibrinogen is an independent risk factor for cardiovascular disease. This study has investigated the role of intracellular Ca2+ ([Ca2+]i) and tyrosine phosphorylation in the attachment of human and rat-derived cultured vascular smooth muscle cells to fibrinogen. Cells were cultured from human saphenous vein segments (HVSMC) and from an established rat aortic cell line (A7r5). [Ca2+]i was measured using fura-2 and adhesion was studied using pre-coated 96 well polystyrene plates. Fibrinogen increased [Ca2+]i in both cell types. In A7r5 cells fibrinogen-induced increases in [Ca2+]i were partially inhibited by a peptide containing the amino acid sequence Arg-Gly-Asp (RGD) which interferes with binding to integrins. In contrast RGD increased [Ca2+]i in HVSMC, but did not inhibit responses to fibrinogen. Ni2+, an inorganic calcium channel blocker largely abolished the rise in [Ca2+]i, but blockers of voltage-operated calcium channels failed to affect [Ca2+]i responses to fibrinogen in either cell type. Genistein, an inhibitor of tyrosine kinase inhibited fibrinogen-induced rises in [Ca2+]i, while daidzein, an inactive analogue, was without effect. Adhesion of cells to fibrinogen was concentration- and time-dependent. Cell adhesion to fibrinogen was partially inhibited by RGD peptide in both cell types. Adhesion of cell to fibrinogen was inhibited by chelation of [Ca2+]i with BAPTA-AM, inhibition of Ca2+ entry by Ni2+, and inhibition of tyrosine kinases by genistein, but heparin had no effect on adhesion. Vascular smooth muscle cells attach to fibrinogen in part through RGD-type interactions. Activation of tyrosine kinase(s) and a subsequent rise in [Ca2+]i appear to be important signals mediating the response to fibrinogen.

Effect of curcumin on cell cycle progression and apoptosis in vascular smooth muscle cells.

1. The possible mechanisms of the antiproliferative and apoptotic effects of curcumin (diferuloylmethane), a polyphenol in the spice turmeric, on vascular smooth muscle cells were studied in rat aortic smooth muscle cell line (A7r5). 2. The proliferative response was determined from the uptake of [3H]-thymidine. Curcumin (10(-6)-10(-4) M) inhibited serum-stimulated [3H]-thymidine incorporation of both A7r5 cells and rabbit cultured vascular smooth muscle cells in a concentration-dependent manner. Cell viability, as determined by the trypan blue dye exclusion method, was unaffected by curcumin at the concentration range 10(-6) to 10(-5) M in A7r5 cells. However, the number of viable cells after 10(-4) M curcumin treatment was less than the basal value (2 x 10(5) cells). 3. To analyse the various stages of the cell cycle, [3H]-thymidine incorporation into DNA was determined every 3 h. After stimulation with foetal calf serum, quiescent A7r5 cells started DNA synthesis in 9 to 12 h (G1/S phase), then reached a maximum at 15 to 18 h (S phase). Curcumin (10(-6)-10(-4) M) added during either the G1/S phase or S phase significantly inhibited [3H]-thymidine incorporation. 4. Following curcumin (10(-6)-10(-4) M) treatment, cell cycle analysis utilizing flow cytometry of propidium iodide stained cells revealed a G0/G1 arrest and a reduction in the percentage of cells in S phase. Curcumin at 10(-4) M also induced cell apoptosis. It is suggested that curcumin arrested cell proliferation and induced cell apoptosis, and hence reduced the [3H]-thymidine incorporation. 5. The apoptotic effect of 10(-4) M curcumin was also demonstrated by haematoxylin-eosin staining, TdT-mediated dUTP nick end labelling (TUNEL), and DNA laddering. Curcumin (10(-4) M) induced cell shrinkage, chromatin condensation, and DNA fragmentation. 6. The membranous protein tyrosine kinase activity stimulated by serum in A7r5 cells was significantly reduced by curcumin at the concentration range 10(-5) to 10(-4) M. On the other hand, the cytosolic protein kinase C activity stimulated by phorbol ester was reduced by 10(-4) M curcumin, but unaffected by lower concentrations (10(-6)-10(-5) M). 7. The levels of c-myc, p53 and bcl-2 mRNA were analysed using a reverse transcription-polymerase chain reaction (RT-PCR) technique. The level of c-myc mRNA was significantly reduced by curcumin (10(-5)-10(-4) M) treatment. And, the level of bcl-2 mRNA was significantly reduced by 10(-4) M curcumin. However, the alteration of the p53 mRNA level by curcumin (10(-5)-10(-4) M) treatment did not achieve significance. The effects of curcumin on the levels of c-myc and bcl-2 mRNA were then confirmed by Northern blotting. 8. Our results demonstrate that curcumin inhibited cell proliferation, arrested the cell cycle progression and induced cell apoptosis in vascular smooth muscle cells. Curcumin may be useful as a template for the development of drugs to prevent the pathological changes of atherosclerosis and post-angioplasty restenosis. Our results suggest that the antiproliferative effect of curcumin may partly be mediated through inhibition of protein tyrosine kinase activity and c-myc mRNA expression. And, the apoptotic effect may partly be mediated through inhibition of protein tyrosine kinase activity, protein kinase C activity, c-myc mRNA expression and bcl-2 mRNA expression.

Genomic organization of the rat connexin40 gene: identical transcription start sites in heart and lung.

The gap junction protein connexin(Cx)40 is developmentally and tissue-specifically expressed. How Cx40 expression is regulated is unknown. We therefore set out to characterize the 5'-untranslated end of both the Cx40 gene and mRNA from different tissues and ages and to identify the Cx40 promoter region. The PCR method 5'-RACE was used to amplify the 5'-end of rat Cx40 mRNAs. Genomic rat Cx40 clones were isolated from a lambda EMBL3 library. The promoter sequence was isolated by long distance PCR. The transcription start site was identified by primer extension and RNase protection assays. Comparison of Cx40 genomic DNA and mRNA sequences revealed that the Cx40 gene contains a small untranslated exon, exon I, which is separated from the coding sequences by an intron of at least 5.5 Kb. The untranslated 5'-end of Cx40 mRNA sequences from adult rat lung, neonatal and adult rat heart and the rat aortic smooth muscle cell line A7r5 were identical. While the same transcription start site was found for the Cx40 mRNAs from different tissues and ages, and amount of Cx40 mRNA differed between tissues as follows: A7r5 cells > neonatal lung > adult lung > or = neonatal atrium > neonatal ventricle; Cx40 mRNA from adult atrium and ventricle was not readily detected by primer extension and RNase protection analyses. The genomic sequence upstream of the transcription start site contains multiple consensus binding sites for transcription factors putatively responsible for spatio-temporal control of Cx40 gene expression. Similar to other connexin genes, the Cx40 gene contains two exons. The same exon I sequence is present in all tissues and developmental stages examined and the relative amounts of Cx40 mRNA in these compare well with published data. Together our data suggest that tissue-specific and developmentally regulated expression of the Cx40 gene is controlled within the same promoter region by mechanisms that have yet to be detailed.

Inhibition of vascular smooth muscle cell growth by inhibition of fibronectin matrix assembly.

The regulation of vascular smooth muscle cell (VSMC) proliferation by the fibronectin matrix was tested by treating human umbilical artery smooth muscle cells (HUASMCs) with a recombinant fragment of fibronectin (protein III1-C) that has previously been shown to modulate fibronectin matrix assembly. III1-C inhibited HUASMC proliferation by 75% to 90%. The inhibition of growth was time dependent; III1-C had no effect on DNA synthesis after 0 to 5 hours of treatment but did have an effect at 24 hours and beyond. III1-C did not stimulate apoptosis in these cells, indicating that the inhibition of proliferation was not due to an induction of programmed cell death. The effects of III1-C on cell growth were only specific for normal diploid smooth muscle cells. III1-C had no effect on the proliferation of IMR-90 fibroblasts, endothelial cells, NIH 3T3 cells, or the rat aortic smooth muscle cell line A7r5. However, III1-C did inhibit proliferation by primary rat aortic smooth muscle cells. An analysis of HUASMC fibronectin receptor (integrin alpha5beta1) distribution revealed that III1-C did not inhibit alpha5beta1 localization to focal contacts. Moreover, III1-C had no effect on the relative expression levels of seven different integrin subunits on HUASMCs. However, III1-C did inhibit fibronectin matrix assembly by rat aortic smooth muscle cells, HUASMCs, A7r5 cells, IMR-90 cells, and endothelial cells. An analysis of fibronectin synthesis indicated that the inhibition of fibronectin matrix assembly by III1-C was not due solely to a decrease in fibronectin synthesis. Finally, treatment of HUASMCs with anti-fibronectin monoclonal antibody L8 (which is known to inhibit fibronectin matrix assembly) also decreased the rate of HUASMC DNA synthesis. These results demonstrate that III1-C inhibits VSMC proliferation and suggest that this effect may be mediated by the inhibition of fibronectin matrix assembly.

Effects of Protein Kinase Cα Overexpression on A7r5 Smooth Muscle Cell Proliferation and Differentiation

Smooth muscle cell differentiation and proliferation are increasingly seen to be intimately tied to the etiology of atherosclerosis and hypertension. To determine the role of PKCα in the regulation of smooth muscle cell differentiation and proliferation, the rat embryonic smooth muscle cell line A7r5 was transfected with an expression vector containing the full-length PKCα cDNA. Neomycin-resistant clones which exhibited increased PKCα levels compared to wild-type cells were selected. The A7r5 cells overexpressing PKCα had altered morphology and decreased growth rates compared to wild-type cells and cells transfected only with the neomycin resistance gene. Electrophoretic mobility shift assays showed that nuclear extracts from overexpressing clones gave a different pattern of protein–DNA binding to an AP-1 consensus oligonucleotide compared to wild-type cells. In contrast to the growth characteristics of these clones, their levels of cell differentiation marker proteins such as vinculin and desmin were not affected by PKCα overexpression. Moreover, the smooth muscle-specific differentiation marker α-actin was markedly reduced, while β-actin levels were found to remain unchanged. Northern blot analysis confirmed that α-actin downregulation occurred at the RNA level. Western blot analysis revealed that A7r5 cells have five different PKC isoforms and that these isoform protein levels were not changed by PKCα overexpression. These findings suggest that PKCα regulates growth and differentiation of A7r5 smooth muscle cells and that these changes might result from altered expression/function of AP-1 transcription factors.

Domains of high Ca2+ beneath the plasma membrane of living A7r5 cells.

Theoretical models and indirect experimental observations predict that Ca2+ concentrations at the inner surface of the plasma membrane may reach, upon stimulation, values much higher than those of the bulk cytosol. In the past few years, we have shown that the Ca2+-sensitive photoprotein aequorin can be intracellularly targeted and utilized for specifically monitoring the [Ca2+] of various organelles. In this work, we extend this approach to the study of the cytoplasmic rim beneath the plasma membrane. We have constructed a new aequorin chimera by fusing the photoprotein with SNAP-25, a neuronal protein which is recruited to the plasma membrane after the post-translational addition of a lipid anchor. The SNAP-25-aequorin chimera, expressed in the rat aortic smooth muscle cell line A7r5, appears correctly sorted as revealed by immunocytochemistry. Using this probe, we demonstrate that the mean [Ca2+] of this cytoplasmic region ([Ca2+]pm) can reach values >10-fold higher than those of the bulk cytosol ([Ca2+]c) upon activation of Ca2+ influx through plasma membrane channels. In unstimulated cells, the mean [Ca2+]pm appears also to be higher than the bulk cytosol, presumably reflecting the existence of microdomains of high [Ca2+].

Acetaldehyde Inhibits Current through Voltage-Dependent Calcium Channels

Ethanol consumption is often accompanied by an increase in both cardiac and vascular dysfunction. Underlying mechanisms may include direct actions of acetaldehyde (ACA), the principal by-product of ethanol metabolism, which has previously been shown to decrease both KCl- and nonrepinephrine-elicited contractions of isolated aortic rings. To determine whether ACA reduces vascular contractility through a direct action on sarcolemmal Ca2+currents of vascular smooth muscle cells, Ca2+channel currents in an aortic smooth muscle cell line (A7r5) were studied using the whole-cell patch clamping technique. With Ba2+as the major charge carrier, Ca+in the electrode, and TEA to block K+currents, ramp depolarization activated an inward current consisting mostly of current through L-type Ca2+channels. ACA caused a progressive decline in inward current, causing a significant reduction in 30 mmACA of 21.2 ± 4.3% (n= 6 cells;p< 0.01) within 4 min and 39.4 ± 6.8% (n= 5 cells,p< 0.001) reduction within 8 min. Although the decline in inward current in 10 mmACA was not significant at 4 min, significant (p< 0.05) reductions in 10 mmACA were present at 8 min (15.5 ± 3.5%,n= 9 cells) and 12 min (25.2 ± 6.7%,n= 3 cells). There was no apparent shift in the voltage dependence of the current in response to ACA. The results of this study support the hypothesis that one of the underlying causes of ACA inhibition of potassium-elicited contraction is inhibition of voltage-dependent Ca2+currents in smooth muscle cells.

Extracellular Mg 2+ inhibits receptor-mediated Ca 2+-permeable non-selective cation currents in aortic smooth muscle cells

The effects of extracellular Mg 2+ on receptor-mediated Ca 2+-permeable non-selective cation currents were investigated in a cultured aortic smooth muscle cell line (A7r5) from rat thoracic aorta, using the whole-cell voltage-clamp technique. Under the Cs +-containing internal solution, both vasopressin and endothelin-1 (100 nM) activated a long-lasting inward current with a high noise level. The reversal potential of these agonists-induced current was approximately +0 mV, and was not significantly altered by the replacement of [Cl −] i or [Cl −] o, suggesting that the inward current was a cation-selective channel. La 3+ and Cd 2+ (1 mM) almost completely abolished the vasopressin or endothelin-induced non-selective cation current; however, nifedipine (10 μM) failed to inhibit it significantly. Extracellular Mg 2+ (3–20 mM) also markedly inhibited the vasopressin- or endothelin-induced non-selective cation current in a concentration-dependent manner. When a non-hydrolysable GTP-analogue, GTPγS (1 mM), was applied from the patch pipette, the non-selective cation current was gradually activated even in the absence of agonist (vasopressin or endothelin-1), probably due to the direct activation of GTP-binding proteins coupled to the receptors. Extracellular Mg 2+ (3–20 mM) also suppressed the activation of non-selective cation current induced by GTPγS, suggesting that the inhibitory sites of Mg 2+ are not located on the receptors. These results suggest that extracellular Mg 2+ inhibits receptor-mediated non-selective cation current, which may contribute to the relaxation effects of Mg 2+ in vascular smooth muscle cells.

Differential effects of steroids on vascular tone and smooth muscle cell Ca2+-homeostasis

The protective effects of 17p oestradiol against the onset of atherosclerosis in women can only partly be explained by altered lipid metabolism. The vessel wall itself is also a target organ for sex steroids. 17p oestradiol-induced increases in the expression of endothelial cell nitric oxide synthase [l] and proliferation of endothelial cells [2], as well as the decrease in the proliferation of smooth muscle cells [3] require de now protein synthesis. These effects of 17p oestradiol appear to be mediated by the classical ICI 182,780-sensitive nuclear oestrogen receptors. In contrast, 17p oestradiol induces a rapid and reversible endotheliumindependent relaxation of preconstricted vascular rings [4]. This latter response to 17p oestradiol is independent of changes in protein synthesis and has been attributed to an alteration of smooth muscle cell Ca2'-homeostasis, presumably involving a reduction in Ca2' influx through voltage operated L-type channels [5]. Recent studies have shown that progesterone also acts as a vasodilator [6], suggesting that the novel steroid receptor in the plasmalemma of cells is less selective than classical, nuclear ones. In the present study we have investigated the effects of 17poestradiol and progesterone on coronary vascular tone in isolated perfised Langendofl rat hearts [7] and on intracellular Ca2'-homeostasis in a rat fetal aortic smooth muscle cell line A7r5. Isolated perfused hearts: Male Sprague-Dawley rats (>350g) were killed by cervical dislocation, the heart excised and the aorta cannulated. The coronary vasculature was perfised retrogradely through the aorta at a constant flow (10 ml.min-') with a heated (37°C) Krebs-Henseleit bicarbonate buffer [7]. Coronary perfision pressure (CPP) was recorded as an index of vascular resistance. As CPP is generally low in isolated hearts perfised with a blood-free solution, CPP was raised with the thromboxane A2 mimetic U46619 (5 nM). Bolus injections (10 pl) of bradykinin and sodium nitroprusside (a donor of nitric oxide in aqueous solution) were then administered into the coronary perfusate. Close-arterial injections of bradykinin (BK, 0.1 nmol) and sodium nitroprusside (SNP, 10 nmol) caused a rapid and transient fall in CPP: 4 1 s mmHg (meankSE, n=15) for BK and 41k3 mmHg (n=15) for S N P . These vasodilator responses to BK and S N P confirmed the integrity of endothelial and smooth muscle cell layers [7]. 17p oestradiol and progesterone caused a rapid, dose-dependent (Fig. 1) and transient reduction in CPP. At the maximal dose used (1 00 nmol), 17p oestradiol and progesterone reduced CPP by 34+3 mmHg (n=15) and 19M mmHg (n=8), respectively. Disruption of the endothelium with 1.5 ml air reversed the normal vasodilator response to BK (0.1 nmol) to a vasoconstriction (754 mmHg, n=4), but did not alter the vasodilator responses to SNP (10 nmol, 50+7 mmHg), 17p oestradiol (100 nmol, 3 7 H mmHg, n=4) or progesterone (100 nmol, 20*6 mmHg, n=4). These findings suggest that 17p oestradiol and progesterone are acting directly on coronary vascular smooth muscle cells 40 1

High permeation of L-type Ca2+ channels at physiological [Ca2+]: homogeneity and dependence on the alpha 1-subunit.

Molecular cloning has identified multiple isoforms of dihydropyridine-sensitive C-class L-type Ca2+ channels. We tested the hypotheses that L-type (C-class) channels exhibit homogeneous high permeation properties at physiological Ca2+ concentrations and membrane potentials. We measured unitary currents through single dihydropyridine-sensitive omega-conotoxin-insensitive endocrine and smooth muscle L-type Ca2+ channels in rat pituitary GH3 and rat aortic A7r5 cell lines. We also measured unitary currents through smooth muscle (Cb) Ca2+ channel alpha 1-subunits in Chinese hamster ovary (CHO) cells. Our results show that single channel conductances of all three L-type (C-class) channels are uniform with high Ba2+ concentrations, e.g., approximately 23 pS with 110 mM Ba2+. The single channel conductances were reduced to similar values when the Ba2+ concentration was lowered to near-physiological values: 11.1, 9.3, and 8.4 pS in GH3, A7r5, and CHO cells at 2 mM Ba2+, respectively. The single channel conductances were not significantly different with near-physiological Ca2+ concentrations: 5.5, 5.9, and 4.9 pS in GH3, A7r5, and CHO cells at 2 mM Ca2+, respectively. The data suggest that L-type (C-class) channels are homogeneous in terms of Ca2+ permeation at physiological charge carrier concentrations and membrane potentials. Furthermore, the data indicate that the relatively high Ca2+ permeation under physiological conditions is determined by the intrinsic properties of the pore-forming Ca2+ channel alpha 1-subunit.

Regulation of the L-type calcium channel alpha-1 subunit by chronic depolarization in the neuron-like PC12 and aortic smooth muscle A7r5 cell lines.

The regulation of L-type voltage-dependent Ca2+ channels by chronic depolarization was studied in the aortic smooth muscle A7r5 and neuron-type PC12 cell lines, by probing the expression and the functional state of their constitutive alpha-1 subunits. PC12 cells showed, after prolonged exposure to a high-K+ depolarizing solution, a 25% reduction of the functional Ca2+ channel density which was accompanied by a decrease of the alpha-1 subunit mRNA expression. In A7r5 cells submitted to a similar protocol of depolarization, 45Ca2+ uptake measurements revealed a fall in the functional activity of L-type Ca2+ channels which was not related to a modulation of their mRNA expression, but arose from a long-term voltage-dependent channel inactivation. Accordingly, the lag time and the mechanisms of recovery were different in the two cell types. In PC12 cells, when restoring physiological culture conditions, de novo synthesis of alpha-1 subunits allowed the recovery of the original density of L-type Ca2+ channels at the membrane surface. As for the A7r5 cells, we showed that after chronic depolarization, the complete restoration of the resting membrane potential and the related Ca2+ channel activity required a 2-day incubation in physiological medium and could probably be related to a normalization of the increased intracellular Ca2+ concentration. In contrast, it is noteworthy that, in PC12 cells, the only transient increase of intracellular Ca2+ content in the first hours of depolarization could account for the long-term down-regulation of L-type Ca2+ channels.