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We previously reported synergistic induction of apoptosis by IFN-gamma plus either cyclosporin A (CsA) or tacrolimus (FK506) in gastric carcinoma cells. In this study, we aimed to elucidate the mechanism for this synergistic induction of apoptosis. IFN-gamma plus CsA synergistically induced caspase-3 mediated apoptosis in gastric carcinoma cells. Although IFN-gamma induced activation of signal transducer and activator of transcription1 (STAT1) and expression of interferon regulatory factor-1 (IRF-1) mRNA, IFN-gamma alone was not able to induce caspase-3 activation and apoptosis. When gastric carcinoma cells were treated with cyclohexamide, a protein synthesis inhibitor, following IFN-gamma pretreatment, caspase-3 was activated, and apoptosis was markedly induced. These findings suggest the existence of IFN-gamma-induced anti-apoptotic pathway and we evaluated the effect of IFN-gamma and CsA on calcium-sensitive nuclear factor-kappa B (NF-kappa B) activation. IFN-gamma increased intracellular calcium ion concentration ([Ca(2+)](i)) consisting of a spike and a sustained phase, and the latter was completely abrogated by CsA. Activation of NF-kappa B occurred in response to IFN-gamma, and which was markedly inhibited by either CsA or FK506. NF-kappa B decoy also enhanced the cytotoxic effect of IFN-gamma. These results suggest that IFN-gamma may simultaneously induce the STAT1-mediated apoptotic pathway and the anti-apoptotic pathway through calcium-activated NF-kappa B and that inhibition of the latter by CsA may result in dominance of the apoptosis-inducing pathway.

In endothelial cells in situ and in primary culture, immunoblot analysis revealed an expression of the 130-kDa subunit of myosin phosphatase, similar to the myosin phosphatase targeting subunit (MYPT) of smooth muscle. Screening of an endothelial cell cDNA library yielded a clone encoding an NH2-terminal fragment of 89.6 kDa, closely related to smooth muscle MYPT1. Two isoforms differing by a central insert of 56 residues were detected. In growing cells, MYPT1 was localized on stress fiber, but at confluence the localization pattern changed and MYPT1 was distributed close to the cell membrane and at cell-cell contacts. The membrane localization of MYPT1 suggested a target other than myosin and raised the possibility that MYPT1 may be involved in dephosphorylation of alternative substrate(s). These distinct mechanisms would also be dependent on the growth state of the endothelial cells, i.e., regulation of actin-myosin interactions in growing cells and an unknown function in cells at confluence.

It has recently been suggested that rho A and rho kinase (ROK) play a role in the increase in the Ca2+ sensitivity of the smooth muscle myofilaments. In the present study, we investigated the mRNA expressions of rho A and two types of ROK (alpha and beta) in the myometrium obtained from both non-pregnant and pregnant rats. Reverse transcription polymerase chain reaction (RT-PCR) experiments using total RNA from these tissue specimens and the specific primers revealed rho A and both types of ROK mRNAs to be expressed in the rat myometrium. The mRNA expressions of rho A, ROK alpha and ROK beta in the pregnant myometrium were found to increase in comparison to those in the non-pregnant myometrium. These results thus support the idea that the up-regulation of these proteins might be involved in the mechanism underlying the increased contractility of the pregnant myometrium.

It has recently been indicated that rho A and rho kinase may be involved in the mechanism for the increase in Ca2+sensitivity in smooth muscle myofilaments. In the present study, we investigated the mRNA expression of rho A and rho kinase in porcine vascular smooth muscle cells (aorta, coronary artery, pulmonary artery, and pulmonary vein). In reverse transcription–polymerase chain reaction (RT–PCR) experiments, using total RNA from these tissue specimens and the primers designed in the conserved regions of each mRNA in human and bovine, rho A and rho kinase mRNAs were detected in all of these smooth muscle cells. An analysis of these PCR products by direct sequencing indicated that they were derived from each mRNA. The finding that both rho A and rho kinase mRNAs were expressed in the various porcine vascular smooth muscle cells strongly supports the idea that these proteins are involved in the mechanism dealing with the increase in Ca2+sensitivity in the myofilaments of vascular smooth muscle cells.

We tested the hypothesis that adrenomedullin (ADM), a recently isolated hypotensive peptide, is an autocrine transmitter in renal arterial smooth muscle cells (RASMCs). Fura-2 fluorometry revealed that ADM decreases both the cytosolic Ca 2+ concentration ([Ca 2+ ]i) and tension induced by 0.3 μM phenylephrine (PE) in porcine RASMCs. The expression of ADM mRNA in RASMCs was assessed by the reverse transcription polymerase chain reaction (RT-PCR), using the total RNA from the RASMCs and the specific primers for the porcine ADM mRNA. A band with an expected size could be detected only when the reverse transcriptase was added, thus indicating that the porcine RASMCs express ADM mRNA. These results therefore suggested that ADM plays an important role in the regulation of the renal vascular tone, not only as a circulating hormone, but also as an autocrine transmitter.

The effects of high (28mM) D-glucose (HG) on the cell cycle progression were investigated in rat aorta vascular smooth muscle cells (VSMCs) in primary culture, using an immunocytochemical analysis of cell-cycle-specific nuclear antigens. HG had no effect on the cell cycle of the serum-deprived G0 cells, whereas platelet-derived growth factor (PDGF) stimulated the entry of the G0 cells to the G1 phase without a further progression to the S and M phases. HG, but neither mannitol nor L-glucose, stimulated the progression of the PDGF-pretreated G1 cells to the S and M phases, which was blocked by calphostin-C, a protein kinase C (PKC) blocker. HG did not affect the cytosolic Ca2+ concentration ([Ca2+]i). These data suggest that HG has no competent effect on the G0 cells and acts as a progression growth factor (to stimulate the cell cycle from the G1 to the S/M) in VSMCs through the mechanism, which may be insensitive to [Ca2+]i and mediated by PKC.

Using front-surface fluorometry with fura-2 and porcine aortic valvular strips, we investigated the effect of motilin, a gastrointestinal peptide, on the cytosolic Ca 2+ concentration ([Ca 2+ ] i ) of endothelial cells in situ . Motilin induced a biphasic elevation of [Ca 2+ ] i of the endothelial cells on the porcine aortic valvular strips. To investigate the physiological role of these Ca 2+ transients in the endothelial cells, we determined the effect of motilin on [Ca 2+ ] i as well as the tension of the smooth muscles in the porcine coronary strips with an intact endothelium. Motilin decreased [Ca 2+ ] i and the tension of the coronary smooth muscle precontracted by U46619, a thromboxane A2 analogue, in an endothelium-dependent manner. In the presence of indomethacin (a cycloxygenase inhibitor), motilin induced an endothelium-dependent relaxation of the coronary strips which was partially inhibited by N ω -nitro-L-arginine (L-NNA; a NO synthase inhibitor). These results thus indicate that motilin induces Ca 2+ transients of the endothelial cells while it also induces vasorelaxation, which may be mediated by both L-NNA sensitive and resistant factors that are derived from the endothelium.

Platelet derived growth factor (PDGF) has been shown to induce c-fos and c-myc protooncogenes. In the present study, we investigated the effects of genistein, a tyrosine kinase inhibitor, and NiCl2, a Ca2+ influx blocker, on PDGF-induced Ca2+ transient and on expression of c-fos and c-myc mRNA. In the presence of extracellular Ca2+, PDGF induced elevation of intracellular Ca2+ concentration ([Ca2+]i) and increases in c-fos and c-myc mRNA, as detected by reverse transcription polymerase chain reaction (RT PCR) and Northern hybridization. PDGF-induced [Ca2+]i elevation was composed of an initial transient increase(first component) followed by steady state elevation (second component). Genistein (10,μ M) blocked the 1st, but not the 2nd, component of [Ca2+]i elevation induced by PDGF. NiCl2 (1mM) and removal of extracellular Ca2+ inhibited the 2nd, but not the 1st, component. In the presence of 10 μM genistein and 1 mM NiCl2, PDGF induced c-fos and c-myc mRNA, although the [Ca2+]i elevation could be completely blocked by these two agents. These results indicate that elevation of [Ca2+]i is not a prerequisite condition for PDGF to induce c-fos and/or c-myc mRNA in rat aortic smooth muscle cells in primary culture.

The effects of cAMP on the expression of ETA (ET-1 selective type) receptor mRNA and on the response to endothelin-1 (ET-1) were investigated in rat aortic smooth muscle cells in primary culture, using reverse transcription polymerase chain reaction (RT-PCR) and fura-2 microfluorometry of cytosolic Ca2+ concentrations ([Ca2+]i). Incubation of the smooth muscle cells with 10 μM forskolin and 10 μM 3-isobutyl-1-methyl-xanthine (IBMX) induced an increase in ETA receptor mRNA by 420 % of control after 24 hours. ET-1 induced a biphasic increase in [Ca2+]i, first transient and second sustained phases. Incubation of the smooth muscle cells with forskolin and IBMX for 24 hours increased the [Ca2+]i response by 849 % in the first phase and 286 % in the second phase, compared with time-matched controls. From these results, we conclude that cAMP induces an up-regulation of the ETA receptor mRNA and increases responsiveness to ET-1 of rat aortic smooth muscle cells in primary culture.

Using front-surface fluorometry of fura-2 and valvular strips of the pig aorta, we recorded changes in the cytosolic Ca2+ concentration, [Ca2+]i, of endothelial cells in situ, quantitatively, and investigated the effects of endothelin-1 and -3 on these endothelial cells. Both endothelin-1 and -3 elevated [Ca2+]i of a peak (the first phase) and sustained type. This first phase is considered to be due to a release of Ca2+ from intracellular storage sites. The sustained phase depended on extracellular Ca2+ and is considered to be due to an influx of Ca2+ through the plasma membrane. At equimolar concentrations, the peak elevations of [Ca2+]i induced by endothelin-1 were much higher than those induced by endothelin-3. We suggest that, in endothelial cells in situ, endothelin-1 mobilizes stored Ca2+ and may activate Ca(2+)-sensitive pathways, including the release of prostacyclin and endothelium-derived relaxing factors, more potently than does endothelin-3.

We investigated the involvement of G i/o protein in NO production following the activation of proteinase-activated receptor-4 (PAR-4) in cultured bovine aortic endothelial cells. AYPGKF-NH 2 (PAR-4 activating peptide), thrombin, and ionomycin induced a concentration-dependent NO production, with the maximal production seen at 30 μM, 0.1 U/ml, and 1 μM, respectively. Ionomycin elevated [Ca 2+ ] i in a concentration-dependent manner. However, AYPGKF-NH 2 and thrombin induced no [Ca 2+ ] i elevation. The loading of cells with BAPTA almost completely inhibited both the NO production and [Ca 2+ ] i elevation induced by 1 μM ionomycin, while it had no significant effect on the AYPGKF-NH 2 -induced NO production. Treatment with pertussis toxin inhibited the AYPGKF-NH 2 -induced NO production, while it had no effect on the ionomycin-induced NO production. Our findings thus demonstrate, for the first time, that PAR-4 activation induced NO production in a manner mostly independent of the Ca 2+ signal and also that G i/o is involved in such NO production in vascular endothelial cells.

Estrogen has been reported to inhibit apoptosis in vascular endothelial cells. However, its precise mechanism still remains to be elucidated. Here we determined the role of Akt in the anti-apoptotic effect of estrogen. 17β-Estradiol prevented the apoptosis induced by TNF-α in bovine aortic endothelial cells, as evaluated by double staining with fluorescein isothiocyanate-conjugated annexin V and propidium iodide. Introducing a dominant negative mutant of Akt by using a cell-penetrating peptide of Tat protein inhibited the anti-apoptotic effect of estrogen in a concentration-dependent manner, and resulted in the complete inhibition of the anti-apoptotic effect of 17β-estradiol at 1 nM and higher concentrations. The dominant negative mutant without the cell-penetrating peptide and Tat peptide-conjugated protein A had no effect. The intracellular protein transduction was confirmed by immunoblot analysis. Our observations thus provide first direct evidence that Akt plays a central role in the anti-apoptotic effect of estrogen in vascular endothelial cells.

p27(Kip1) is a cyclin-dependent kinase inhibitor, and its nuclear localization is a prerequisite for it to function as a cell cycle regulator. In the present study, the minimal requirement for the nuclear localization signal (NLS) of p27(Kip1) was determined by analyzing the localization of various mutants of p27(Kip1) tagged with green fluorescent protein (GFP) in HeLa cells and porcine aortic endothelial cells. Wild-type p27(Kip1) exclusively localized into nucleus, while GFP alone localized in both cytosol and nucleus. A comparison of various truncation mutants revealed residues 153-166 to be the minimal region necessary for nuclear localization. However, a fusion of this region to GFP showed cytoplasmic retention in addition to nuclear localization, thus suggesting that some extension flanking this region is required to achieve a full function of NLS. The site-directed mutation of the full-length p27(Kip1) therefore showed that four basic residues (K153, R154, K165, R166), especially R166, play a critical role in the nuclear localization of p27(Kip1).

The effects of the small noncatalytic subunit of myosin light chain phosphatase (MLCPsr) on the Ca2+-induced contraction of smooth muscle were investigated in the Triton X-100-permeabilized porcine renal artery. The full-length recombinant chicken MLCPsr obtained by the bacterial expression system induced an additional contraction at a constant [Ca2+]i and shifted the [Ca2+]i-force relation curve to the left. A deletion mutant containing the N-terminal 78 amino acids of MLCPsr retained the full action, compared with the full-length MLCPsr, while the deletion of this region completely abolished its effect. The process of relaxation was also delayed by the fragment containing the N-terminal 78 amino acids. These results indicated that MLCPsr increases the Ca2+ sensitivity of the contractile apparatus while the N-terminal 78 amino acids are responsible for this effect in vascular smooth muscle.

Using reverse transcription polymerase chain reaction (RT-PCR), we determined the expression of the mRNAs for basic calponin isoforms, namely h 1 and h 2 forms, in the porcine aortic endothelial cells (PAECs) and aortic smooth muscle cells (PASMCs). The mRNAs for both h 1 and h 2 calponin isoforms were expressed in PAECs as well as in PASMCs. However, the expression of h 2 isoform mRNA was more abundant than h 1 isoform in PAECs, while h 1 isoform was more abundant than h 2 in PASMCs. These findings indicated the existence of calponin mRNA and the predominance of h 2 over h 1 calponin isoform in PAECs. It is suggested that h 2 as well as h 1 , calponin isoform might have a physiological role in vascular endothelial cell. This is the first report which describes the existence of calponin h 2 -dominant cells.

The regulation of the cell cycle by extracellular UTP was investigated in rat aortic smooth muscle cells in primary culture (VSMCs) by means of an immunocytochemical analysis of cell cycle-specific nuclear antigens. UTP induced a rise in the cytosolic Ca 2+ concentration ([Ca 2+ ]i) of VSMCs, which was desensitized by pretreatment with ATP, but not with 2-methylthioATP nor α,β-methyleneATP. The incubation of serum-deprived G 0 cells with platelet derived growth factor (PDGF) induced cell cycle progression into the G 1 phase without any further progression into the S and M phases, while none of the nucleotides had any effect on the cell cycle of the G 0 cells. The incubation of the PDGF-pretreated cells at the G 1 phase with 2-methylthioATP or α,β-methyleneATP had no effect on the cell cycle of G 1 cells, while the incubation of the G 1 cells with UTP, ATP, and ATPγS stimulated cell cycle progression into the S and M phases. These results thus indicate that P 2U purinergic activation mediates a [Ca 2+ ]i transient and a progression growth factor effect of nucleotides in VSMCs.

Using front-surface fluorometry and fura-2-loaded porcine coronary arterial strips with an intact endothelium, changes in cytosolic Ca2+ concentrations ([Ca2+]i) and tension of smooth muscle were simultaneously monitored in an attempt to determine the vasoactive properties of endothelin-1 (ET-1). ET-1 in low concentrations (0.1–1nM) caused a significant transient decrease in [Ca2+]i and tension of the strips precontracted with 10−7M U-46619. The maximal decreases in [Ca2+]i and tension were obtained with 0.6nM ET-1. In higher concentrations (1nM–100nM), there was no reduction in [Ca2+]i or tension; the contraction induced by U-46619 was potentiated. The decreases in [Ca2+]i and tension induced by ET-1 were inhibited by the mechanical removal of the endothelium or by pretreatment with NG-nitro-L-arginine and were slightly attenuated by indomethacin. Thus, ET-1 in low concentrations can induce endothelium-dependent transient relaxations accompanied by transient reductions of [Ca2+]i in isolated porcine coronary arteries. This effect is mainly mediated by the release of endothelium-derived relaxing factor.

In cultured rat arterial smooth muscle cells treated with quin 2, cytosolic Ca 2+ transients induced by norepinephrine were recorded microfluorometrically. In the presence or absence of extracellular Ca 2+ , norepinephrine induced transient and dose-dependent elevations in cytosolic Ca 2+ , with a similar time course, the peak levels being observed at 2 min. These transient elevations in cytosolic Ca 2+ were dose-dependently inhibited by alpha-adrenergic antagonists, the order of potency being prazosin > phentolamine > yohimbine, irrespective of the presence of extracellular Ca 2+ . We propose that with or without extracellular Ca 2+ , norepinephrine activates mainly alpha-1 adrenoceptors leading to a release of Ca 2+ from intracellular stores. This would explain the transient elevation in cytosolic Ca 2+ in rat aortic vascular smooth muscle cells in primary culture.

Summary Front surface fluorometry and porcine coronary arterial strips loaded with fura-2 were used to investigate the effect of endothelin on cytosolic Ca concentrations, [Ca]i, and on contractile force, the objective being to elucidate the mechanism of action. Both in the presence and absence of extracellular Ca, endothelin induced rapid and dose-dependent increases in [Ca]i and in contraction. When caffeine-sensitive and histamine-sensitive intracellular Ca stores were depleted, in Ca-free medium, a transient contraction but no increase in [Ca]i followed the subsequent application of endothelin. This Ca-independent component was largely inhibited by the relative protein kinase C inhibitor, H-7, but not inhibited by W-7, calmodulin antagonist. This component is probably linked to activation of protein kinase C.

Effects of pertussis toxin on Ca2+ transients in rat arterial smooth muscle cells in primary culture were monitored, using quin 2-microfluorometry. In the presence or the absence of extracellular Ca2+, norepinephrine, histamine, caffeine and high extracellular K+ induced elevations in cytosolic Ca2+ concentration. Cytosolic Ca2+ elevations induced by norepinephrine and histamine were inhibited by pretreatment of the cells with pertussis toxin, time- and dose-dependently. However, elevations induced by caffeine and K+-depolarization were unaffected by the pretreatment with this toxin. Thus, it is suggested that GTP binding protein, a pertussis toxin substrate and involved in the receptor-mediated cytosolic Ca2+ transients, is not involved in transient elevations in cytosolic Ca2+ induced by caffeine and K+-depolarization in cultured vascular smooth muscle cells.

We studied the subcellular distribution and the properties of [3H] mepyramine binding in the porcine vascular smooth muscle. A close correlation was observed between the specific binding activity of [3H] mepyramine and the extent of the enrichment of sarcolemmal marker enzyme in 4 subfractions obtained by sucrose density gradient centrifugation of the aortic microsome. In the binding isotherm in the sarcolemmal fractions from the aorta and coronary artery, there was no difference in the Kd value, but the Bmax of the coronary artery was significantly lower than that of the aorta. Thus, there is a single type of high affinity mepyramine binding site in the porcine vascular smooth muscle sarcolemma and the number of H1-receptors of the coronary artery may be smaller than that of the aorta.

Summary In the presence of endothelin, there was a rapid increase in the 45 Ca ++ efflux from primary cultured rat vascular smooth muscle cells, both in physiological salt solution and in calcium free medium containing 2 mM EGTA. The 45 Ca ++ influx was not affected. The endothelin-induced, transient increase in cytosolic calcium concentration is probably mainly due to release of calcium from the intracellular store in vascular smooth muscle cells.

Using an intracellularly trapped dye, quin 2, effects of K+-depolarization on cytosolic free calcium concentrations were recorded microfluorometrically in rat aorta vascular smooth muscle cells in primary culture. When the cells were exposed to high extracellular K+ in Ca+-free media containing 2mM EGTA, there was a transient and dose-dependent elevation of cytosolic Ca2+ concentrations. However, the concentration of the cytosolic Ca2+ was not elevated when the intracellularly stored Ca2+ was depleted by the repetitive treatment with caffeine prior to the application of high K+. Thus, depolarization of plasma membrane, per se, directly induces a release of Ca2+ from intracellular storage sites in vascular smooth muscle cells, and the main fraction of this released Ca2+ is derived from the caffeine sensitive storage sites; perhaps from the sarcoplasmic reticulum.

Summary We made use of quin2 microfluorometry to determine the effects of endothelin (ET) on cytosolic free Ca 2+ concentrations ([Ca 2+ ] i ) in rat aortic smooth muscle cells in primary culture. In Ca 2+ -containing medium, ET induced a rapid and sustained elevation of [Ca 2+ ] i . In the latter component, in particular, the elevation of [Ca 2+ ] i was inhibited by diltiazem. In Ca 2+ -free medium, ET induced a rapid and transient [Ca 2+ ] i elevation, which was not inhibited by diltiazem. When the caffeine-sensitive intracellular Ca 2+ store was practically depleted by repeated treatment with caffeine in Ca 2+ -free media, ET did not elevate [Ca 2+ ] i . Thus, it was suggested that ET induces [Ca 2+ ] i elevation not only by extracellular Ca 2+ -dependent mechanisms but also by releasing Ca 2+ from the intracellular store, and that the ET-sensitive Ca 2+ store may overlap with the caffeine-sensitive one, in cultured vascular smooth muscle cells.

Using an intracellularly trapped dye, quin 2, the effects of histamine on cytosolic free calcium concentrations in rat aortic vascular smooth muscle cells in primary culture were recorded, microfluorometrically. When the cells were exposed to histamine, both in the presence and the absence of extracellular Ca2+, there was a rapid, transient and dose-dependent elevation of cytosolic Ca2+ concentrations, with a similar time course. This elevation of cytosolic Ca2+ was dose-dependently inhibited by mepyramine, but not by cimetidine. Thus, histamine activates H1- but not H2- receptors to mediate a release of Ca2+ from the store sites, and there is a rapid and transient elevation of cytosolic Ca2+.

Serotonin induced a transient elevation in the levels of cytosolic calcium in cultured rat vascular smooth muscle cells. Ketanserin, a selective antagonist of serotonin 2 receptors, dose-dependently inhibited the elevation of cytosolic calcium induced by serotonin, and ultimately unmasked a serotonin-induced decrease in the levels of cytosolic calcium. These observations show that serotonin has direct and dual effects, that is, it increases and decreases cytosolic free calcium concentrations in vascular smooth muscle cells, in culture. Knowledge of such events is important because serotonergic inhibitors may prove to be useful drugs for treating clinical hypertension and vasospastic disorders.