Cytoplasmic Ca2+ oscillations evoked by receptor stimulation, G-protein activation, internal application of inositol trisphosphate or Ca2+: simultaneous microfluorimetry and Ca2+ dependent Cl- current recording in single pancreatic acinar cells.

The effects of acetylcholine (ACh), cholecystokinin (CCK), internally applied GTP-gamma-S, inositol trisphosphate [Ins (1,4,5) P3] or Ca2+ on the cytoplasmic free Ca2+ concentration [( Ca2+]i) were assessed by simultaneous microfluorimetry (fura-2) and measurement of the Ca2(+)-dependent Cl- current (patch-clamp whole-cell recording) in single internally perfused mouse pancreatic acinar cells. ACh (0.1-0.2 microM) evoked an oscillating increase in [Ca2+]i measured in the cell as a whole (microfluorimetry) which was synchronous with oscillations in the Ca2(+)-dependent Cl- current reporting [Ca2+]i close to the cell membrane. In the same cells a lower ACh concentration (0.05 microM) evoked shorter repetitive Cl- current pulses that were not accompanied by similar spikes in the microfluorimetric recording. When cells did not respond to 0.1 microM ACh, caffeine (1 mM) added on top of the sustained ACh stimulus resulted in [Ca2+]i oscillations seen synchronously in both types of recording. CCK (10 nM) also evoked [Ca2+]i oscillations, but with much longer intervals between slightly broader Ca2+ pulses. Internal perfusion with 100 microM GTP-gamma-S evoked [Ca2+]i oscillations with a similar pattern. Ins (1,4,5) P3 (10 microM) evoked repetitive shortlasting spikes in [Ca2+]i that were only seen in the Cl- current traces, except in one small cell where these spikes were also observed synchronously in the microfluorimetric recording. Caffeine (1 mM) broadened these Ca2+ pulses. [Ca2+]i was also directly changed, bypassing the normal signalling process, by infusion of a low or high Ca2+ solution into the pipette.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of endothelium-dependent vascular relaxation by tetrandrine.

The effects of tetrandrine, a Ca2+ antagonist of bis-benzylisoquinoline alkaloid origin, on endothelium-dependent and -independent vascular responsiveness were investigated in perfused rat mesenteric artery. In endothelium-intact preparations pre-contracted with 3 microM phenylephrine and fully relaxed by 0.3 microM acetylcholine tetrandrine caused a rapid transient contraction. In endothelium-denuded preparations, tetrandrine caused only vasorelaxation of phenylephrine-contraction. The biphasic effect of tetrandrine in acetylcholine-relaxed preparations could also be mimicked by sequential applications of atropine/tetrandrine or N(G)-nitro-L-arginine-methylester (L-NAME)/tetrandrine, but atropine or L-NAME alone caused only vasoconstriction. This tetrandrine-induced transient vasoconstriction was also observed in preparations relaxed with ATP, histamine or thapsigargin (TSG), but not those relaxed with A23187, sodium nitroprusside or nifedipine. The present results suggest that tetrandrine, in addition to its known inhibitory effects on vascular smooth muscle by virtue of its Ca2+ antagonistic actions, also inhibits NO production by the endothelial cells possibly by blockade of Ca2+ release-activated Ca2+ channels.     

Activation of the human intermediate-conductance Ca(2+)-activated K(+) channel by 1-ethyl-2-benzimidazolinone is strongly Ca(2+)-dependent.

Modulation of the cloned human intermediate-conductance Ca(2+)-activated K(+) channel (hIK) by the compound 1-ethyl-2-benzimidazolinone (EBIO) was studied by patch-clamp technique using human embryonic kidney cells (HEK 293) stably expressing the hIK channels. In whole-cell studies, intracellular concentrations of free Ca(2+) were systematically varied, by buffering the pipette solutions. In voltage-clamp, the hIK specific currents increased gradually from 0 to approximately 300 pA/pF without reaching saturation even at the highest Ca(2+) concentration tested (300 nM). In the presence of EBIO (100 microM), the Ca(2+)-activation curve was shifted leftwards, and maximal currents were attained at 100 nM Ca(2+). In current-clamp, steeply Ca(2+)-dependent membrane potentials were recorded and the cells gradually hyperpolarised from -20 to -85 mV when Ca(2+) was augmented from 0 to 300 nM. EBIO strongly hyperpolarised cells buffered at intermediate Ca(2+) concentrations. In contrast, no effects were detected either below 10 nM (no basic channel activation) or at 300 nM Ca(2+) (V(m) close to E(K)). Without Ca(2+), EBIO-induced hyperpolarisations were not obtainable, indicating an obligatory Ca(2+)-dependent mechanism of action. When applied to inside-out patches, EBIO exerted a Ca(2+)-dependent increase in the single-channel open-state probability, showing that the compound modulates hIK channels by a direct action on the alpha-subunit or on a closely associated protein. In conclusion, EBIO activates hIK channels in whole-cell and inside-out patches by a direct mechanism, which requires the presence of internal Ca(2+).     

Activation of a small-conductance Ca(2+)-dependent K+ channel contributes to bradykinin-induced stimulation of nitric oxide synthesis in pig aortic endothelial cells.

Bradykinin-induced K+ currents, membrane hyperpolarization, as well as rises in cytoplasmic Ca2+ and cGMP levels were studied in endothelial cells cultured from pig aorta. Exposure of endothelial cells to 1 microM bradykinin induced a whole-cell K+ current and activated a small-conductance (approximately 9 pS) K+ channel in on-cell patches. This K+ channel lacked voltage sensitivity, was activated by increasing the Ca2+ concentration at the cytoplasmic face of inside-out patches and blocked by extracellular tetrabutylammonium (TBA). Bradykinin concomitantly increased membrane potential and cytoplasmic Ca2+ of endothelial cells. In high (140 mM) extracellular K+ solution, as well as in the presence of the K(+)-channel blocker TBA (10 mM), bradykinin-induced membrane hyperpolarization was abolished and increases in cytoplasmic Ca2+ were reduced to a slight transient response. Bradykinin-induced rises in intracellular cGMP levels which reflect Ca(2+)-dependent formation of EDRF(NO) were clearly attenuated in the presence of TBA (10 mM). Our results suggest that bradykinin hyperpolarizes pig aortic endothelial cells by activation of small-conductance Ca(2+)-activated K+ channels. Opening of these K+ channels results in membrane hyperpolarization which promotes Ca2+ entry, and consequently, NO synthesis.     

Role of superoxide and angiotensin II suppression in salt-induced changes in endothelial Ca2+ signaling and NO production in rat aorta

Male Sprague-Dawley rats were maintained on a low-salt (LS) diet (0.4% NaCl) or changed to a high-salt (HS) diet (4% NaCl) for 3 days. Increases in intracellular Ca2+ ([Ca2+]i) in response to methacholine (10 microM) and histamine (10 microM) were significantly attenuated in aortic endothelial cells from rats fed a HS diet, whereas thapsigargin (10 microM)-induced increases in [Ca2+]i were unaffected. Methacholine-induced nitric oxide (NO) production was eliminated in endothelial cells of aortas from rats fed a HS diet. Low-dose ANG II infusion (5 ng x kg(-1) x min(-1) iv) for 3 days prevented impaired [Ca2+]i signaling response to methacholine and histamine and restored methacholine-induced NO production in aortas from rats on a HS diet. Adding Tempol (500 microM) to the tissue bath to scavenge superoxide anions increased NO release and caused N(omega)-nitro-L-arginine methyl ester-sensitive vascular relaxation in aortas from rats fed a HS diet but had no effect on methacholine-induced Ca2+ responses. Chronic treatment with Tempol (1 mM) in the drinking water restored NO release, augmented vessel relaxation, and increased methacholine-induced Ca2+ responses significantly in aortas from rats on a HS diet but not in aortas from rats on a LS diet. These findings suggest that 1) agonist-induced Ca2+ responses and NO levels are reduced in aortas of rats on a HS diet; 2) increased vascular superoxide levels contribute to NO destruction, and, eventually, to impaired Ca2+ signaling in the vascular endothelial cells; and 3) reduced circulating ANG II levels during elevated dietary salt lead to elevated superoxide levels, impaired endothelial Ca2+ signaling, and reduced NO production in the endothelium.     

Participation of transient-type Ca2+ channels in the sustained increase of Ca2+ level in GH3 cells

Participation of two types of Ca2+ channels (T- and L-types) in the sustained increase of cytosolic-free Ca2+ concentration [( Ca2+]i) was studied in thyrotropin-releasing hormone (TRH)-stimulated clonal GH3 pituitary cells. The effects of Ca2+ channel blockers were analyzed by measuring Ca2+ channel current and [Ca2+]i, using whole-cell voltage-clamp and Fura-2 fluorometry, respectively. Phenytoin (100 microM) and Ni2+ (100 microM) selectively blocked T-type Ca2+ channels and suppressed the TRH-induced sustained [Ca2+]i increase in single cells. Synthetic omega-conotoxin (omega-CgTX, 2 microM) preferentially blocked L-type Ca2+ channels, but it did not suppress the TRH-induced sustained [Ca2+]i increase. The present results suggest that the sustained elevations of [Ca2+]i triggered by TRH may be mediated by T-type Ca2+ channels in GH3 cells.     

The isoflavone Equol mediates rapid vascular relaxation: Ca2+-independent activation of endothelial nitric-oxide synthase/Hsp90 involving ERK1/2 and Akt phosphorylation in human endothelial cells

We recently reported that soy isoflavones increase gene expression of endothelial nitric-oxide synthase (eNOS) and antioxidant defense enzymes, resulting in improved endothelial function and lower blood pressure in vivo. In this study, we establish that equol (1-100 nM) causes acute endothelium- and nitric oxide (NO)-dependent relaxation of aortic rings and rapidly (2 min) activates eNOS in human aortic and umbilical vein endothelial cells. Intracellular Ca2+ and cyclic AMP levels were unaffected by treatment (100 nM, 2 min) with equol, daidzein, or genistein. Rapid phosphorylation of ERK1/2, protein kinase B/Akt, and eNOS serine 1177 by equol was paralleled by association of eNOS with heat shock protein 90 (Hsp90) and NO synthesis in human umbilical vein endothelial cells, expressing estrogen receptors (ER)alpha and ERbeta. Inhibition of phosphatidylinositol 3-kinase and ERK1/2 inhibited eNOS activity, whereas pertussis toxin and the ER antagonists ICI 182,750 and tamoxifen had negligible effects. Our findings provide the first evidence that nutritionally relevant plasma concentrations of equol (and other soy protein isoflavones) rapidly stimulate phosphorylation of ERK1/2 and phosphatidylinositol 3-kinase/Akt, leading to the activation of NOS and increased NO production at resting cytosolic Ca2+ levels. Identification of the nongenomic mechanisms by which equol mediates vascular relaxation provides a basis for evaluating potential benefits of equol in the treatment of postmenopausal women and patients at risk of cardiovascular disease.     

Cross-linking of surface immunoglobulin on B lymphocytes induces both intracellular Ca2+ release and Ca2+ influx: analysis with indo-1

The new Ca2+-probe indo-1 has a high fluorescence intensity, which allows low intracellular dye loadings. Stimulation of indo-1-loaded mouse B cells with anti-Ig antibodies provoked rapid rise of free cytoplasmic Ca2+ from 100 nM to greater than 1 microM, followed by a decline to a plateau at 300-400 nM. The initial rapid rise was not detected in quin2-loaded cells, presumably due to the Ca2+-buffering effects of the dye. The sustained Ca2+ increase was due to influx, whereas the initial rise was caused by release from intracellular stores. The magnitudes of Ca2+ release and inositol trisphosphate release were closely correlated. Concanavalin A does not provoke inositol trisphosphate release in mouse B cells. It did not induce a rapid initial Ca2+ rise in indo-1-loaded B cells either, but only a sustained increase to 200-300 nM. Finally, Ca2+ influx induced by both anti-Ig and concanavalin A were not affected by membrane depolarization.     

The effect of oxidative stress on Ca2+ release and capacitative Ca2+ entry in vascular endothelial cells

Oxidative stress imposed by the accumulation of oxygen free radicals (reactive oxygen species, ROS) has profound effects on Ca2+ homeostasis in the vascular endothelium, leading to endothelial dysfunctions and the development of cardiovascular pathologies. We tested the effect of the oxidant and ROS generator tert-butyl-hydroperoxide (tBuOOH) on Ca2+ signaling in single cultured calf pulmonary artery endothelial (CPAE) cells loaded with the fluorescent Ca2+ indicator indo-1. Acute brief (5 min) exposures to tBuOOH had no effect on basal cytosolic free Ca2+ ([Ca2+](i)), agonist (ATP)-induced Ca2+ release from the endoplasmic reticulum (ER) and on Ca(2+) store depletion-dependent capacitative Ca2+ entry (CCE). Prolonged (60 min) exposure to tBuOOH did not affect intracellular Ca2+ release, but caused a profound inhibition of CCE. After 120 min of treatment with tBuOOH not only was CCE further reduced, but also ATP-induced Ca2+ release due to a slow depletion of the stores that resulted from CCE inhibition. The antioxidant Trolox (synthetic vitamin E analog) prevented the inhibition of CCE by tBuOOH and attenuated the increase of [ROS](i), indicating that inhibition of CCE was due to the oxidant effects of tBuOOH. The data suggest that in vascular endothelial cells oxidative stress primarily affects Ca2+ influx in response to Ca2+ loss from internal stores. [Ca2+](i) is an important signal for the production and release of endothelium-derived factors such as nitric oxide (NO). Since CCE is the preferential Ca2+ source for NO synthase activation, the finding that oxidative stress inhibits CCE may explain how oxidative stress contributes to endothelial dysfunction-related cardiovascular pathologies.     

ATP-dependent Ca2+ transport in the rat parotid basolateral plasma membrane is regulated by calmodulin

Calmodulin regulation of ATP-dependent Ca2+ transport activity was assessed in inverted basolateral plasma membrane vesicles (BLMV) isolated from rat parotid glands. The initial rate of Ca2+ transport in media containing 100 nM Ca2+ was stimulated by approximately 60% at maximal concentrations (300 nM) of exogenously added calmodulin (CAM). Half-maximal activation was obtained at 50 and 175 nM CAM in KCl and mannitol containing assay media, respectively. In the KCl medium, addition of 300 nM CAM increased the affinity of the BLMV Ca2+ transport activity for Ca2+ from approximately 70 nM, in the absence of added CAM, to approximately 50 nM. Vmax was consistently increased by approximately 20% under these conditions. When BLMV were treated with ethylene glycol bis(beta-aminoethylether) N,N'-tetraacetic acid (EGTA) (200 microM), the affinity of the transporter for Ca2+ decreased by 50% to approximately 150 nM, with no change in Vmax. When CAM was added to the EGTA-treated membranes, Ca2+ transport activity was comparable to that obtained when CAM was added directly to control, untreated BLMV. The CAM antagonists, trifluoperazine (TFP), W-7, and calmidazolium, inhibited Ca2+ transport in the presence of CAM. Half-maximal inhibition of transport was achieved by 12 microM TFP and 20 microM W-7. Calmidazolium (1 microM) inhibited Ca2+ transport by 75%. The inhibitory effects on ATP-dependent Ca2+ transport exerted by these agents were not due to an increase in the passive permeability of the membranes to Ca2+. Furthermore, in the absence of added CAM, the inhibitory effects of these agents on initial Ca2+ transport rate was decreased. The data presented suggest that the Ca2+-dependent interaction of CAM with the ATP-dependent Ca2+ transporter in rat parotid BLMV modifies the kinetic properties of this Ca2+ transporting mechanism.     

Ca2+-independent PLA2 controls endothelial store-operated Ca2+ entry and vascular tone in intact aorta

During an agonist stimulation of endothelial cells, the sustained Ca2+ entry occurring through store-operated channels has been shown to significantly contribute to smooth muscle relaxation through the release of relaxing factors such as nitric oxide (NO). However, the mechanisms linking Ca2+ stores depletion to the opening of such channels are still elusive. We have used Ca2+ and tension measurements in intact aortic strips to investigate the role of the Ca2+-independent isoform of phospholipase A2 (iPLA2) in endothelial store-operated Ca2+ entry and endothelium-dependent relaxation of smooth muscle. We provide evidence that iPLA2 is involved in the activation of endothelial store-operated Ca2+ entry when Ca2+ stores are artificially depleted. We also show that the sustained store-operated Ca2+ entry occurring during physiological stimulation of endothelial cells with the circulating hormone ATP is due to iPLA2 activation and significantly contributes to the amplitude and duration of ATP-induced endothelium-dependent relaxation. Consistently, both iPLA2 metabolites arachidonic acid and lysophosphatidylcholine were found to stimulate Ca2+ entry in native endothelial cells. However, only the latter triggered endothelium-dependent relaxation through NO release, suggesting that lysophosphatidylcholine produced by iPLA2 upon Ca2+ stores depletion may act as an intracellular messenger that stimulates store-operated Ca2+ entry and subsequent NO production in endothelial cells. Finally, we found that ACh-induced endothelium relaxation also depends on iPLA2 activation, suggesting that the iPLA2-dependent control of endothelial store-operated Ca2+ entry is a key physiological mechanism regulating arterial tone.     

Inhibition of Ca2+-activated K+ channels in pig pancreatic acinar cells by Ba2+, Ca2+, quinine and quinidine

Patch-clamp whole-cell and single-channel current recordings were made from pig pancreatic acinar cells to test the effects of quinine, quinidine, Ba2+ and Ca2+. Voltage-clamp current recordings from single isolated cells showed that high external concentrations of Ba2+ or Ca2+ (88 mM) abolished the outward K+ currents normally associated with depolarizing voltage steps. Lower concentrations of Ca2+ only had small inhibitory effects whereas 11 mM Ba2+ almost blocked the K+ current. 5.5 mM Ba2+ reduced the outward K+ current to less than 30% of the control value. Both external quinine and quinidine (200-500 microM) markedly reduced whole-cell outward K+ currents. In single-channel current studies it was shown that external Ba2+ (1-5 mM) markedly reduced the probability of opening of high-conductance Ca2+ and voltage-activated K+ channels whereas internal Ba2+ (6 X 10(-6) to 3 X 10(-5) M) caused activation at negative membrane potentials and inhibition at positive potentials. Quinidine (200-400 microM) evoked rapid chopping of single K+ channel openings acting both from the outside and inside of the membrane and in this way markedly reduced the total current passing through the channels.     

Molecular requirements for L-type Ca2+ channel blockade by testosterone

Despite being generally perceived as detrimental to the cardiovascular system, testosterone has marked beneficial vascular effects; most notably it acutely and directly causes vasodilatation. Indeed, men with hypotestosteronaemia can present with myocardial ischemia and angina which can be rapidly alleviated by infusion of testosterone. To date, however, in vitro studies have failed to provide a convincing mechanism to account for this clinically important effect. Here, using whole-cell patch-clamp recordings to measure current flow through recombinant human L-type Ca2+ channel alpha(1C) subunits (Ca(v)1.2), we demonstrate that testosterone inhibits such currents in a concentration-dependent manner. Importantly, this occurs over the physiological range of testosterone concentrations (IC50 34 nM), and is not mimicked by the metabolite 5alpha-androstan-17beta-ol-3-one (DHT), nor by progesterone or estradiol, even at high (10 microM) concentration. L-type Ca2+ channels in the vasculature are also important clinical targets for vasodilatory dihydropyridines. A single point mutation (T1007Y) almost completely abolishes nifedipine sensitivity in our recombinant expression system. Crucially, the same mutation renders the channels insensitive to testosterone. Our data strongly suggest, for the first time, the molecular requirements for testosterone binding to L-type Ca2+ channels, thereby supporting its beneficial role as an endogenous Ca2+ channel antagonist in the treatment of cardiovascular disease.     

Thapsigargin increases cytoplasmic free Ca2+ without influencing steroidogenesis in chicken granulosa cells.

The effects of thapsigargin on intracellular Ca2+ concentration ([Ca2+]i) and progesterone production were determined in granulosa cells from the two largest preovulatory follicles of laying hens. [Ca2+]i was measured in cells loaded with the Ca(2+)-responsive fluorescent dye Fura-2. Thapsigargin stimulated a 4.6 +/- 0.2-fold increase in [Ca2+]i from a resting level of 55 +/- 6 nM up to 233 +/- 23 nM (n = 8) in 100% of the cells tested (n = 86). However, two different response patterns were observed. Dependent on the cell populations, a maximally effective concentration of thapsigargin (100 nM) stimulated either a rapid (within 16 +/- 2 s) transient increase in [Ca2+]i or a slowly (99 +/- 20 s) developing and sustained increase in [Ca2+]i. Both [Ca2+]i responses were concentration (0.001-1 microM)-dependent with an EC50 around 40 nM. The transient [Ca2+]i response occurred in the absence of extracellular Ca2+ and was unaffected by pretreating the cells with the Ca2+ channel blockers methoxyverapamil (50 microM) or lanthanum (1 mM). The plateau phase of the sustained [Ca2+]i response returned to resting level in the absence of extracellular Ca2+, but remained elevated in the presence of methoxyverapamil (50 microM) or lanthanum (1 mM). Despite its ability to cause transient or prolonged increases in [Ca2+]i, thapsigargin (0.001-1 microM) did not affect basal or luteinizing hormone-stimulated progesterone production by chicken granulosa cells.     

Dissociation of Ca2+ entry and Ca2+ mobilization responses to angiotensin II in bovine adrenal chromaffin cells

In fura-2-loaded bovine adrenal chromaffin cells, 0.5 microM angiotensin II (AII) stimulated a 185 +/- 19 nM increase of intracellular-free calcium [( Ca2+]i) approximately 3 s after addition. The time from the onset of the response until achieving 50% recovery (t 1/2) was 67 +/- 10 s. Concomitantly, AII stimulated both the release of 45Ca2+ from prelabeled cells, and a 4-5-fold increase of [3H]inositol 1,4,5-trisphosphate [( 3H]Ins(1,4,5)P3) levels. In the presence of 50 microM LaCl3, or when extracellular-free Ca2+ [( Ca2+]o) was less than 100 nM, AII still rapidly increased [Ca2+]i by 95-135 nM, but the t 1/2 for recovery was then only 23-27 s. In medium with 1 mM MnCl2 present, AII also stimulated a small amount of Mn2+ influx, as judged by quenching of the fura-2 signal. When [Ca2+]o was normal (1.1 mM) or low (less than 60 nM), 1-2 microM ionomycin caused [Ca2+]i to increase 204 +/- 26 nM, while also releasing 45-55% of bound 45Ca2+. With low [Ca2+]o, ionomycin pretreatment abolished both the [Ca2+]i increase and 45Ca2+ release stimulated by AII. However, after ionomycin pretreatment in normal medium, AII produced a La3+-inhibitable increase of [Ca2+]i (103 +/- 13 nM) with a t 1/2 of 89 +/- 8 s, but no 45Ca2+ release. No pretreatment condition altered AII-induced formation of [3H]Ins(1,4,5)P3. We conclude that AII increased [Ca2+]i via rapid and transient Ca2+ mobilization from Ins(1,4,5)P3- and ionomycin-sensitive stores, accompanied (and/or followed) by Ca2+ entry through a La3+-inhibitable divalent cation pathway. Furthermore, the ability of AII to activate Ca2+ entry in the absence of Ca2+ mobilization (i.e. after ionomycin pretreatment) suggests a receptor-linked stimulus other than Ca2+ mobilization initiates Ca2+ entry.     

Ca2+-dependent K+ channels from rat olfactory cilia characterized in planar lipid bilayers

Olfactory cilia contain cyclic nucleotide-gated and Ca2+-dependent Cl- conductances that underlie excitatory chemotransduction, and a Ca2+-dependent K+ (KCa) conductance, apparently involved in inhibitory transduction. Previous single-channel patch-clamp studies on olfactory cilia revealed four different KCas, with different conductances and kinetics. Here, we further characterized these channels in planar bilayers, where blockers could be properly tested. All four ciliary KCas were observed: The 16 pS channel, K0.5,Ca=40 microM and apamin-sensitive; the 30 and 50 pS channel, K0.5,Ca=59 microM, clotrimazole-sensitive and charybdotoxin-insensitive; the 60 pS channel, clotrimazole-sensitive and charybdotoxin-insensitive; and the 210 pS channel, K0.5,Ca=63 microM, blocked by charybdotoxin and iberiotoxin. The presence of the 16 and 210 pS channels was confirmed by immunoblotting.     

Beta-adrenergic inhibition of AGEPC-stimulated Na+/Ca2+ exchange and AGEPC-induced platelet activation

Recently, AGEPC (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) was found to initiate contraction of ileal smooth muscle strips and to enhance Na+/Ca2+ exchange in ileal plasmalemmal vesicles. In the present study, the effects of the smooth muscle relaxant, isoproterenol, on Na+/Ca2+ exchange in rat ileal plasmalemmal vesicles was examined. In this preparation, Na+/Ca2+ exchange was stimulated 131 +/- 8% and 264 +/- 19% by addition of 50 nM and 100 nM AGEPC, respectively. Isoproterenol, a beta-adrenergic agonist, inhibited AGEPC stimulation of Na+/Ca2+ exchange in a dose- and time-dependent manner but had no effect on basal rates of Na+/Ca2+ antiport. At 1 microM, isoproterenol inhibited 86% of the Na+/Ca2+ exchange stimulated by 50 nM AGEPC. Vesicular cAMP levels were increased over 100% following the addition of 1 microM isoproterenol for 30 s. Inhibition of AGEPC-stimulated vesicular Na+/Ca2+ exchange and elevation of vesicular cAMP levels by isoproterenol was prevented by the beta-receptor antagonist propranolol (5 microM), demonstrating that these effects of isoproterenol were mediated by interaction with vesicular beta-adrenergic receptors. Additional studies with washed rabbit platelets demonstrated that isoproterenol inhibited AGEPC-induced aggregation and serotonin release. These effects of isoproterenol were dose- and time-dependent and were antagonized by propranolol. Isoproterenol had no effect on thrombin-induced aggregation and did not change appreciably platelet cAMP levels. Moreover, dibutyryl cAMP could not mimic the effect of isoproterenol to inhibit an AGEPC-induced aggregation. On a molar basis, the inhibitory effects of isoproterenol toward AGEPC action were greater in the ileal preparation than in the platelets. It is suggested that beta-adrenergic agonists may modulate AGEPC-induced ileal Na+/Ca2+ exchange and AGEPC-induced platelet aggregation through cAMP-dependent and-independent mechanisms, respectively.     

Effect of L-alpha-phosphatidylinositol on a vascular smooth muscle Ca2+-dependent protease. Reduction of the Ca2+ requirement for autolysis

Vascular smooth muscle contains large amounts of a Ca2+-dependent protease. Similar to a Ca2+-dependent protease previously purified from chicken gizzard smooth muscle (Hathaway, D. R., Werth, D. K., and Haeberle, J. R. (1982) J. Biol. Chem. 257, 9072-9077), the mammalian vascular muscle protease is a heterodimer consisting of 76,000- and 30,000-dalton subunits (IIa). The enzyme can undergo autolysis in the presence of Ca2+ to produce a smaller species consisting of 76,000- and 18,000-dalton subunits (IIb). Autolysis greatly reduces the Ca2+ dependence of catalytic activity. The autolytic species, IIb, was approximately 23-fold more sensitive to Ca2+ (K0.5 = 39 microM) than the native enzyme, IIa (K0.5 = 891 microM). In this communication, we report that phosphatidylinositol and to a lesser extent one metabolic derivative, dioleoylglycerol, stimulate autolysis of the vascular Ca2+-dependent protease by reducing the Ca2+ for autolysis from K0.5 = 680 microM in the absence of lipid to K0.5 = 87 microM in the presence of both phosphatidylinositol and dioleoylglycerol. Moreover, the reduction in the Ca2+ requirement for autolysis produced by the phosphatidylinositol was antagonized by the phospholipid-binding drug, trifluoperazine. In addition, the effect of phosphatidylinositol was specific for autolysis, and none of several phospholipids or derivatives tested altered the Ca2+ dependence or maximal rate for protein degradation of the autolytic product, IIb. Our results suggest that autolysis may be an important initial step in the activation of the Ca2+-dependent protease in vascular smooth muscle and that this step may be regulated by a combination of Ca2+ and phosphatidylinositol.     

Large-conductance Ca2+-activated K+ currents blocked and impaired by homocysteine in human and rat mesenteric artery smooth muscle cells

Plenty of evidence suggests that increased blood levels of homocysteine (Hcy) are an independent risk factor for the development of vascular diseases, but the underlying mechanisms are not well understood. It is well known that the larger conductance Ca(2+)-activated K(+) channels (BK(Ca)) play an essential role in vascular function, so the present study was conducted to determine direct effects of Hcy on BK(Ca) channel properties of smooth muscle cells. Whole-cell patch-clamp recordings were made in mesenteric artery smooth muscle cells isolated from normal rat and patients to investigate effects of 5, 50 and 500 microM Hcy on BK(Ca), the main current mediating vascular responses in these cells. In human artery smooth muscle cells, maximum BK(Ca) density (measured at +60 mV) was inhibited by about 24% (n=6, P<0.05). In rat artery smooth muscle cells, maximum BK(Ca) density was decreased by approximately 27% in the presence of 50 microM Hcy (n=8, P<0.05). In addition, when rat artery smooth muscle cells was treated with 50 microM Hcy for 24 h, maximum BK(Ca) density decreased by 58% (n=5, P<0.05). These data suggest that Hcy significantly inhibited BK(Ca) currents in isolated human and rat artery smooth muscle cells. BK(Ca) reduced and impaired by elevated Hcy levels might contribute to abnormal vascular diseases.