Mechanism of nitric oxide-induced vasodilatation: refilling of intracellular stores by sarcoplasmic reticulum Ca2+ ATPase and inhibition of store-operated Ca2+ influx

The precise mechanisms by which nitric oxide (NO) decreases free [Ca2+]i, inhibits Ca2+ influx, and relaxes vascular smooth muscle are poorly understood. In rabbit and mouse aorta, agonist-induced contractions and increases in [Ca2+]i were resistant to nifedipine, suggesting Ca2+ entry through non-L-type Ca2+ channels. Relaxations to NO were inhibited by thapsigargin (TG) or cyclopiazonic acid (CPA) indicating the involvement of sarcoplasmic reticulum ATPase (SERCA). Studies of the effect of NO on [Ca2+]i and the rate of Mn2+ influx with fura-2 fluorometry in rabbit aortic smooth muscle cells in primary culture were designed to test how SERCA is involved in mediating the response to NO. When cells were stimulated with angiotensin II (AII), NO accelerated the removal of Ca2+ from the cytoplasm, decreased [Ca2+]i, and inhibited Ca2+ and Mn2+ influx. Inhibition of SERCA abolished all the effects of NO. In contrast, inhibition of the Na+/Ca2+exchanger or the plasma membrane Ca2+ ATPase had no influence on the ability of NO to decrease [Ca2+]i. NO maximally decreased [Ca2+]i within 5 s, whereas significant inhibition of AII-induced Ca2+ and Mn2+ influx required more than 15 s. The inhibition of cation influx strictly depended on [Ca2+]o and functional SERCA, suggesting that during the delay before NO inhibits Ca2+ influx, the influx of Ca2+ and the uptake into intracellular stores are required. In the absence of [Ca2+]o, NO diminished the AII-induced [Ca2+]i transient by a SERCA-dependent mechanism and increased the amount of Ca2+ in the stores subsequently released by ionomycin. The present study indicates that the initial rapid decrease in [Ca2+]i caused by NO in vascular smooth muscle is accounted for by the uptake of Ca2+ by SERCA into intracellular stores. It is proposed that the refilling of the stores inhibits store-operated Ca2+ influx through non-L-type Ca2+ conducting ion channels and that this maintains the decrease in [Ca2+]i and NO-induced relaxation.     

Effect of 5-hydroxytryptamine on intracellular calcium dynamics in cultured rat vascular smooth muscle cells

The present study elucidated the precise mechanism of 5-hydroxytryptamine (5-HT)-induced increase of intracellular Ca2+ concentration ([Ca2+]i) in cultured vascular smooth muscle cells isolated from rat aortic media. [Ca2+]i was measured using fluorescent Ca2+ indicator, fura-2. 5-HT caused a dose-dependent increase in [Ca2+]i, which was completely inhibited by ketanserin. alpha-Methyl-5-HT had an equipotent effect to 5-HT. Diltiazem at 10 microM partially suppressed the 5-HT-induced increase in [Ca2+]i. 5-HT also augmented Mn2+ influx, when monitored by Mn2+ quenching of fura-2 fluorescence. When extracellular Ca2+ (1.3 mM) was removed, a decrease in resting level and a small, transient increase in [Ca2+]i were observed. 5-HT stimulation also induced an increase in the production of inositol triphosphate. 5-HT-induced increase in [Ca2+]i was significantly, but partially inhibited by staurosporin and H-7. Phorbol 12-myristate 13-acetate induced an increase in [Ca2+]i, which was abolished by removal of extracellular Ca2+. 5-HT-induced increase in [Ca2+]i was not affected by the pretreatment with pertussis toxin (PTX), and was not accompanied by a change in cyclic AMP content. These results suggest that, in cultured rat aortic smooth muscle cells, 5-HT increases [Ca2+]i via 5-HT2 receptor subtype by inducing influx of extracellular Ca2+ partially through L-type voltage-dependent Ca2+ channel, as well as by mobilizing Ca2+ from its intracellular stores. Activation of protein kinase C may be positively involved in the regulatory mechanism of Ca2+ influx, but PTX-sensitive G protein and cyclic AMP seem to be not involved.     

Chronic amitriptyline exposure reduces 5-HT3 receptor-mediated cyclic GMP formation in NG 108-15 cells

In the present study, we investigated the effects of chronic in vitro administration of amitriptyline, a tricyclic antidepressant, on cyclic GMP formation stimulated by 5-hydroxytryptamine (5-HT) in the neuroblastoma x glioma hybrid cell line, NG 108-15, 5-HT (0.01-100 microM)-stimulated cyclic GMP formation was concentration-dependent and was sensitive to ICS 205-930, a 5-HT3 receptor antagonist. Exposure of NG 108-15 cells to 5 microM amitriptyline for 3 days significantly reduced 5-HT-stimulated cyclic GMP formation. Acute treatment with amitriptyline had no effect on 5-HT-stimulated cyclic GMP formation. The reduction by chronic amitriptyline exposure of 10 microM 5-HT-stimulated cyclic GMP formation was concentration-dependent over the concentration range examined (0.5 to 10 microM). The IC50 of amitriptyline was 1.9 microM. In contrast, amitriptyline exposure, even at a concentration of 8 microM, failed to modify cyclic GMP formation stimulated by bradykinin, sodium nitroprusside, or atrial natriuretic peptide. Increases in intracellular Ca2+ concentration ([Ca2+]i) evoked by 10 microM 5-HT were attenuated in amitriptyline-exposed cells, while 100 nM bradykinin-induced [Ca2+]i increases were not affected. In addition, chronic exposure to 5 microM amitriptyline caused a decrease in affinity (Kd) of [3H]zacopride specific binding to 5-HT3 recognition sites. The Bmax for the labelled ligand remained unchanged. These results suggest that chronic amitriptyline exposure reduces 5-HT-stimulated cyclic GMP formation and [Ca2+]i increases, and this may reflect the functional changes of 5-HT3 receptors.     

Allelic loss on chromosome 18q as a prognostic marker in stage II colorectal cancer

The aim of the study was to elucidate the vasodilatory mechanism due to Cu2+ by assessing nitric oxide (NO) production as determined by NOx (NO, NO2-, and NO3-) that is released from human pulmonary arterial endothelial cell (HPAEC) monolayers using a NO chemiluminescence analyzer, and also to assess Ca2+ movement using 45Ca and fura 2 in HPAEC. Cu2+ (10(-6)-10(-4) M) significantly increased NO production in a dose-dependent manner when extracellular Ca2+ was present. 45Ca influx into the adherent cells was dose-dependently enhanced by Cu(2+) (10(-6)-10(-4) M), but not by Mn(2+), Zn(2+) or Fe(2+). [Ca2+]i, measured by monitoring the fluorescence changes of fura 2, was significantly elevated in the presence of Cu2+. The increase in [Ca2+]i induced by Cu2+ was inhibited by either diethyldithiocarbamate (DDC) or the depletion of extracellular Ca2+. The dihydropyridine receptor agonist, BayK8644, significantly attenuated the Cu2+-induced increase in [Ca2+]i in a dose dependent manner and nitrendipine or nifedipine, the dihydropyridine receptor antagonists, dose-dependently inhibited a Cu2+-induced increase in [Ca2+]i. These results suggest that Cu2+ activates eNOS through the mechanism of [Ca2+]i elevation due to Ca2+ influx into HPAEC and that the Cu2+-induced [Ca2+]i elevation in HPAEC is likely due to activation of the dihydropyridine-like receptors.     

The Rho GTPases in macrophage motility and chemotaxis

The present study was carried out to clarify the role of nonselective cation channels as a Ca2+ entry pathway in the contraction and the increase in [Ca2+]i induced by endothelin- in endothelium-denuded rat thoracic aorta rings, and their suppression by nitric oxide (NO). In Ca2+-free medium, the endothelin-1-induced contraction was suppressed to about 20% of control values, although the increase in [Ca2+]i became negligible. The contraction and the increase in [Ca2+]i monitored by fura 2 fluorescence were unaffected by a blocker of L-type voltage-operated Ca2+ channels nifedipine. A blocker of nonselective cation channels 1-[beta-[3-(4-methoxyphenyl)propoxyl]-4-methoxyphenethyl]-1H-imida zole . HCl(SK&F 96365) suppressed the endothelin-1-induced contraction and increase in [Ca2+]i to the level similar to that after removal of extracellular Ca2+. SK&F 96365 had no further effect on the endothelin-1-induced contraction in the absence of extracellular Ca2+. The endothelin-1-induced contraction and increase in [Ca2+]i were abolished by a donor of NO sodium nitroprusside. The effects of another NO donor 3-morpholinosydnonimine (SIN-1) were also tested and yielded essentially similar results to those for sodium nitroprusside on the endothelin-1-induced contraction. Furthermore, the inhibitory effects of sodium nitroprusside could be blocked with a guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ) at 30 microM. These findings suggest that Ca2+ entry through nonselective cation channels but not voltage-operated Ca2+ channels plays a critical role in the endothelin-1-induced increase in [Ca2+]i and the resulting contraction and that inhibition by NO of the endothelin-1-induced contraction is mainly the result of blockade of Ca2+ entry through these channels.     

Sensitivity of G-protein involved in endothelin-1-induced Ca2+ influx to pertussis toxin in porcine endothelial cells in situ

1. We designed a new method to determine quantitatively the intracellular Ca2+ concentration ([Ca2+]i) in endothelial cells in situ, using front-surface fluorometry and fura-2-loaded porcine aortic valvular strips. Using this method, we investigated the characteristics of the G-protein involved in endothelin-1 (ET-1)-induced changes in [Ca2+]i of endothelial cells in situ. 2. Endothelial cells were identified by specific uptake of acetylated-low density lipoprotein labelled with 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (DiI-Ac-LDL). Double staining with DiI-Ac-LDL and fura-2 showed that the valvular strip was covered with a monolayer of endothelial cells and that the cellular component which contributed to the fura-2 fluorescence, [Ca2+]i signal, was exclusively endothelial cells. 3. ET-1 (10(-7) M) induced an elevation of [Ca2+]i consisting of two components: the first was a rapid and transient elevation to reach a peak, followed by a second, sustained elevation (the second phase). The first phase was composed of extracellular Ca(2+)-independent and -dependent components, while the second phase was exclusively extracellular Ca(2+)-dependent. The extracellular Ca(2+)-independent component of the first phase was due to the release of Ca2+ from intracellular storage sites. The second phase and part of the first phase of [Ca2+]i elevation were attributed to the influx of extracellular Ca2+. The Ca2+ influx component was completely inhibited by 10(-3) M Ni2+ but was not affected by 10(-5) M diltiazem. 4. Pertussis toxin (IAP) markedly inhibited the extracellular Ca2+-dependent elevation of [Ca2+]j, but had no effect on the extracellular Ca2+-independent elevation of [Ca2+], caused by ET-1 (10-7M).5. Bradykinin (10-7 M) or ATP (10- 5M) elevated [Ca2+]i and these responses also consisted of extracellular Ca2+-independent and extracellular Ca2+-dependent components. IAP had no effect on either component of the [Ca2+]i elevation induced by bradykinin or ATP.6. From these findings we conclude that, in porcine endotheliel cells in situ, ET-1 elevates [Ca2+]i as are result of a Ca2+ influx component from the extracellular space and release of intracelluarly stored Ca2+ .The Ca2+ influx is regulated by an IAP-sensitive G-protein, while the release of Ca2+ from the intracellular store is not.     

Nicotine administration stimulates the in vivo N-methyl-D-aspartate receptor/nitric oxide/cyclic GMP pathway in rat hippocampus through glutamate release

1. The in vivo effects of nicotine on the nitric oxide (NO) synthase/cyclic GMP pathway of the adult rat hippocampus have been investigated by monitoring the levels of extracellular cyclic GMP during microdialysis in conscious unrestrained animals. 2. Intraperitoneal (i.p.) administration of nicotine caused elevation of cyclic GMP levels which was prevented by mecamylamine. The effect of nicotine was abolished by local infusion of the NO synthase inhibitor N(G)-nitro-L-arginine (L-NOARG) or by the soluble guanylyl cyclase blocker 1H-[1,2,4]oxadiazolo[4.3-a]quinoxaline-1-one (ODQ). 3. Local administration of the NMDA receptor antagonists cis-4-(phosphonomethyl)-2-piperidinecarboxylic acid (CGS19755) and dizocilpine (MK-801) inhibited by about 60% the nicotine-induced elevation of cyclic GMP. Nicotine was able to stimulate cyclic GMP outflow also when administered directly into the hippocampus; the effect was sensitive to mecamylamine, L-NOARG, ODQ or MK-801. 4. Nicotine, either administered i.p. or infused locally, produced augmentation of glutamate and aspartate extracellular levels, whereas the outflows of gamma-aminobutyric acid (GABA) and glycine remained unaffected. Following local administration of high concentrations of nicotine, animals displayed symptoms of mild excitation (sniffing, increased motor and exploratory activity) during the first 20-40 min of infusion, followed by wet dog shake episodes; these behavioural effects were prevented by mecamylamine or MK-801, but not by L-NOARG or by ODQ. 5. It is concluded that (a) nicotine stimulates the production of NO and cyclic GMP in the hippocampus; (b) this occurs, at least in part, through release of glutamate/aspartate and activation of NMDA receptors. Modulation of the NMDA receptor/NO synthase/cyclic GMP pathway may be involved in the cognitive activities of nicotine.     

In vivo microdialysis study of a specific inhibitor of soluble guanylyl cyclase on the glutamate receptor/nitric oxide/cyclic GMP pathway

1. Nitric oxide (NO) is known to stimulate soluble guanylyl cyclase, thereby eliciting an elevation of guanosine 3':5'-cyclic monophosphate (cyclic GMP) in target cells. Recently, a selective inhibitor of soluble guanylyl cyclase, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), has been identified and characterized in vitro. We have investigated the in vivo effects of ODQ on the glutamate receptor/NO/ cyclic GMP pathway by monitoring extracellular cyclic GMP during microdialysis of the cerebellum or the hippocampus of freely-moving adult rats. 2. Intracerebellar administration of ODQ (1-100 microM) via the microdialysis probe inhibited, in a concentration-dependent manner, the basal extracellular level of cyclic GMP. The maximal inhibition, measured after a 20 min perfusion with 100 microM ODQ, amounted to 80% and persisted unchanged as long as ODQ was perfused. When ODQ was removed from the perfusion stream after 20 min, the levels of cyclic GMP started to recover, suggesting reversibility of guanylyl cyclase inhibition by ODQ. 3. The cyclic GMP response evoked in the cerebellum by NMDA (200 microM) or by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA; 100 microM) was largely attenuated by 100 microM ODQ. The pattern of the inhibition curves suggests competition for guanylyl cyclase between ODQ and the NO generated by NMDA or AMPA receptor activation. 4. ODQ (100 microM) prevented the elevation of extracellular cyclic GMP levels provoked by intracerebellar infusion of the NO generator S-nitroso-N-acetylpenicillamine (SNAP; 1 mM). The inhibition of the SNAP effect was rapidly relieved when ODQ was removed from the perfusion fluid. However, ODQ (100 microM) was unable to affect the cyclic GMP response elicited by 5 mM SNAP, in keeping with the proposed idea that ODQ binds to the "NO receptor' in a reversible and competitive manner. 5. Infusion of ODQ (10, 100 or 300 microM) into the hippocampus of freely-moving rats diminished the basal extracellular level of cyclic GMP. The maximal inhibition amounted to 50% and was produced by 100 microM ODQ. 6. The cyclic GMP response observed when 1 mM SNAP was perfused in the hippocampus, similar in percentage terms to that seen in cerebellum, was dramatically reduced during co-infusion of 100 microM ODQ. 7. ODQ appears to act in vivo as a selective, reversible and possibly competitive inhibitor of the soluble guanylyl cyclase targeted by NO. This enzyme may generate most (about 80%) of the cyclic GMP found under basal conditions in the extracellular space of the cerebellum. In the hippocampus, about 50% of the basal cyclic GMP does not seem to originate from the ODQ-sensitive soluble guanylyl cyclase.     

Evidence that potassium channels make a major contribution to SIN-1-evoked relaxation of rat isolated mesenteric artery

1. The NO donor 3-morpholino-sydnonimine (SIN-1; 0.01-10 microM) evoked concentration-dependent relaxation of rat isolated mesenteric arteries pre-constricted with phenylephrine (1-3 microM). The relaxation to SIN-1 was not significantly different between endothelium-intact or denuded arterial segments or segments in which basal nitric oxide (NO) synthesis was inhibited (n = 8; P > 0.05). In contrast, the membrane permeable analogue of guanosine 3':5'-cyclic monophosphate (cyclic GMP), 8-Br-cyclic GMP (0.01-1 mM), was much less effective in relaxing intact than denuded arterial segments or intact arterial segments pre-incubated with NO synthase blockers (n = 4; P < 0.01). 2. 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10 microM; 10 min) alone, did not alter SIN-1-evoked relaxation in any tissues (n = 5; P > 0.05). However, in parallel experiments, ODQ almost completely inhibited both basal and SIN-1-stimulated production of cyclic GMP in both the presence and absence of NO synthase blockers (n = 6; P < 0.01) indicating that full relaxation to SIN-1 can be achieved in the absence of an increase in cyclic GMP. 3. Exposure of endothelium-intact arterial segments to the potassium channel blocker charybdotoxin (50 nM; 10 min), significantly inhibited SIN-1-evoked relaxation, reducing the maximum response by around 90% (n = 5; P < 0.01). In contrast, in arterial segments in which either the endothelial cell layer had been removed or basal NO synthesis inhibited, relaxation to SIN-1 was not reduced in the presence of charybdotoxin (n = 6; P > 0.05). However, in the presence of NO synthase blockers and L-arginine (300 microM) together, charybdotoxin did significantly inhibit SIN-1-evoked relaxation to a similar extent as intact tissues (maximum response induced by around 80%; n = 4; P < 0.01). 4. Pre-incubation with apamin (30 nM; 10 min) or glibenclamide (10 microM; 10 min) did not alter SIN-1-evoked relaxation of phenylephrine-induced tone in any tissues (n = 4 and n = 6, respectively; P > 0.05). However, in the presence of either ODQ and apamin, or ODQ and glibenclamide, SIN-1-evoked relaxation was significantly attenuated in intact arterial segments and segments in which NO synthesis was blocked. 5. Exposure of intact arterial segments to charybdotoxin and apamin, in the presence of NO synthase blockers, also significantly inhibited SIN-1-evoked relaxation, reducing the maximum response by around 80% (n = 4; P < 0.01). 6. Addition of superoxide dismutase (SOD; 30 u ml-1), potentiated relaxations to SIN-1 in all tissues, but did not alter the effects of charybdotoxin and ODQ and SIN-1-evoked relaxation. 7. These data show that although relaxation to the NO-donor SIN-1 is not significantly different between endothelium-intact and denuded arterial segments, the mechanisms which mediate SIN-1-evoked relaxation in the rat isolated mesenteric artery appear to be modulated by the basal release of endothelium-derived NO. In the presence of an intact endothelial cell layer, the major mechanism for SIN-1-evoked relaxation appears to be the activation of charybdotoxin-sensitive potassium channels. In contrast, when basal NO synthesis is inhibited, SIN-1 appears to cause full relaxation by both the activation of a charybdotoxin-sensitive pathway and the stimulation of soluble guanylyl cyclase.     

Mechanism of nitric oxide-induced contraction in the rat isolated small intestine

1. The contractile response to nitric oxide (NO) in ral ileal myenteric plexus-longitudinal muscle strips was pharmacologically analysed. 2. NO (10(-7) M) induced only contraction while 10(-6) M NO induced contraction followed by relaxation. Methylene blue (up to 10(-4) M) did not affect the NO-induced contractions but significantly reduced the relaxation evoked by 10(-6) M NO. Administration of 8-bromo-cyclic GMP (10(-6)-10(-4) M) only induced relaxation. 3. Sodium nitroprusside (SNP; 10(-7)-10(-5) M) induced concentration-dependent contractions per se; the contractile response to NO, administered within 10 min after SNP, was concentration-dependently reduced. The guanosine 3':5'-cyclic monophosphate (cyclic GMP) content of the tissues was not increased during contractions with 10(-8) M NO and 10(-6) M SNP; it was increased by a factor of 2 during contraction with 10(-7) M NO, and by a factor of 12 during relaxation with 3 x 10(-6) M NO. 4. The NO-induced contractions were not affected by ryanodine (3 x 10(-5) M) but were concentration-dependently reduced by nifedipine (10(-8)-10(-7) M) and apamin (3 x 10(-9)-3 x 10(-8) M). 5. These results suggest that cyclic GMP is not involved in the NO-induced contraction in the rat small intestine. The NO-induced contraction is related to extracellular Ca2+ influx through L-type Ca2+ channels, that might be activated in response to the closure of Ca(2+)-dependent K+ channels.     

Possible mechanism of the potent vasoconstrictor actions of ryanodine on femoral arteries from spontaneously hypertensive rats

1. The Ca2+ buffering function of sarcoplasmic reticulum (SR) in the resting state of arteries from spontaneously hypertensive rats (SHR) was examined. Differences in the effects of ryanodine that removes the function of SR, on tension and cellular Ca2+ level were assessed in endothelium-denuded strips of femoral arteries from 13-week-old SHR and normotensive Wistar-Kyoto rats (WKY). 2. The addition of ryanodine to the resting strips caused a concentration-dependent contraction in SHR. This contraction was extremely small in WKY. In the presence of 10(-5) M ryanodine, caffeine (20 mM) failed to cause a further contraction in SHR, but it caused a small contraction in WKY. After washout of the strips with a Krebs solution, the resting tone was greatly elevated in SHR when compared with WKY. 3. The elevated resting tone in SHR strips was abolished by 10(-7) M nifedipine. The ryanodine-induced contraction was also abolished by 10(-7) M nifedipine. Nifedipine itself caused a relaxation from the resting tone of SHR strips, suggesting the maintenance of myogenic tone. 4. In strips preloaded with fura-PE3, the addition of 10(-5) M ryanodine caused a large and moderate elevation of cytosolic Ca2+ level ([Ca2+]i) in SHR and WKY, respectively. After washout, the resting [Ca2+]i was greatly elevated in SHR. The ryanodine-induced elevation of [Ca2+]i was decreased by 5 x 10(-6) M verapamil in SHR. Verapamil itself caused a decrease in resting [Ca2+]i which was significantly greater in SHR than in WKY, and caused a relaxation only in SHR. 5. The resting Ca2+ influx in arteries measured by a 5 min incubation with 45Ca was significantly increased in SHR when compared with WKY. The resting Ca2+ influx was not increased by 10(-5) M ryanodine in both SHR and WKY. The net cellular Ca2+ uptake in arteries measured by a 30 min incubation with 45Ca was decreased by 10(-5) M ryanodine in both strains. 6. The resting Ca2+ influx was decreased by 10(-7) M nifedipine in the SHR artery, but it was unchanged in the WKY artery. 7. These results suggest that (1) the Ca2+ influx via L-type voltage-dependent Ca2+ channels was increased in the resting state of the SHR femoral artery, (2) the greater part of the increased Ca2+ influx was buffered by Ca2+ uptake into the SR and some Ca2+ reached the myofilaments resulting in the maintenance of the myogenic tone, and (3) therefore the functional removal of SR by ryanodine caused a potent contraction in this artery.     

Glucose activates both K(ATP) channel-dependent and K(ATP) channel-independent signaling pathways in human islets

We investigated the effects of platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) on intracellular Ca2+ concentration ([Ca2+]i) and cell length in isolated and field-stimulated rat cardiomyocytes. [Ca2+]i and cell length of field-stimulated cells were determined simultaneously by confocal laser scan microscopy by using the fluorescent Ca2+ dye Fluo-3. PAF (10(-12)-10(-8) M) inhibited systolic [Ca2+]i increase in a time- and concentration-dependent manner. Maximal effects were observed after an incubation time of 6-8 min, resulting in a 17% (10(-12) M), 41% (10(-10) M), and 52% (10(-8) M PAF) inhibition of systolic [Ca2+]i increase. A time- and concentration-dependent decrease in simultaneously measured cell shortening also was demonstrated. Cell shortening was inhibited by 10% (10(-12) M), 32% (10(-10) M), and 50% (10(-8) M) after an incubation time of 8 min. The effects of PAF could be antagonized by the PAF-receptor antagonist WEB 2170. These data demonstrate that PAF receptor-dependently induces a negative inotropic effect, which is correlated with a decrease in systolic [Ca2+]i and is most likely not due to a decrease in myofilament sensitivity.     

Lysophosphatidic acid-induced proliferation-related signals in astrocytes

Lysophosphatidic acid (LPA) is a potent lipid biomediator that is likely to have diverse roles in the brain. Thus, LPA-induced events in astrocytes were defined. As little as 1 nM LPA induced a rapid increase in the concentration of intracellular free calcium ([Ca2+]i) in astrocytes from neonatal rat brains. This increase was followed by a slow return to the basal level. Intracellular calcium stores were important for the initial rise in [Ca2+]i, whereas the influx of extracellular calcium contributed significantly to the extended elevation of [Ca2+]i. LPA treatment also resulted in increases in lipid peroxidation and DNA synthesis. These increases in [Ca2+]i, lipid peroxidation, and DNA synthesis were inhibited by pretreatment of cells with pertussis toxin or H7, a serine/threonine protein kinase inhibitor. Moreover, the LPA-induced increase in [Ca2+]i was inhibited by a protein kinase C inhibitor, Ro 31-8220, and a calcium-dependent protein kinase C inhibitor, G? 6976. The increase in [Ca2+]i was important for the LPA-induced increase in lipid peroxidation, whereas the antioxidant, propyl gallate, inhibited the LPA-stimulated increases in lipid peroxidation and DNA synthesis. In contrast, pertussis toxin, H7, and propyl gallate had no effect on LPA-induced inhibition of glutamate uptake. Thus, LPA appears to signal via at least two distinctive mechanisms in astrocytes. One is a novel pathway, namely, activation of a pertussis toxin-sensitive G protein and participation of a protein kinase, leading to sequential increases in [Ca2+]i, lipid peroxidation, and DNA synthesis.     

Intracellular sodium concentration in cultured cerebellar granule cells challenged with glutamate

We monitored simultaneously the changes in the intracellular sodium concentration ([Na+]i) and intracellular calcium concentration ([Ca2+]i) in individual neurons from primary cultures of cerebellar granule cells loaded with sodium-binding benzofuran isophthalate and fluo-3. An application of glutamate (50 microM) in Mg(2+)-free medium containing 10 microM glycine evoked [Na+]i and [Ca2+]i increases that exceeded 60 mM and 1 microM, respectively. The kinetics of [Na+]i and [Ca2+]i decreases after the termination of the glutamate pulse were different. [Na+]i failed to decrease immediately after glutamate withdrawal and the delay in the onset of [Na+]i decrease after the glutamate pulse termination was proportional to the glutamate dose, the glutamate pulse duration, and the extent of [Ca2+]i elevation elicited by glutamate. The kinetics of [Ca2+]i decrease were biphasic, with the first phase occurring immediately after glutamate withdrawal and the second phase being correlated in time with a [Na+]i value lower than 15-20 mM. These results were interpreted to indicate that the glutamate-evoked calcium influx may lead to sodium homeostasis destabilization. The delay in the restoration of the sodium gradient may in turn prolong the neuronal exposure to toxic [Ca2+]i values, due to the decrease in the efficiency of the Na+/Ca2+ exchanger to extrude calcium. The glutamate effects on [Na+]i and [Ca2+]i were potentiated by glycine. Glycine (10 microM) added alone also evoked [Na+]i and [Ca2+]i increases; this effect was inhibited by a competitive inhibitor of the N-methyl-D-aspartate receptor, 3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid, indicating an involvement of endogenous glutamate.     

Intracellular Ca2+ elevation and contraction due to prostaglandin F2alpha in rat aorta

Prostaglandin F2alpha was tested to determine (a) whether its effect on intracellular Ca2+ levels ([Ca2+]i) and force in vascular smooth muscle was mediated through activation of the thromboxane A2 and/or prostaglandin receptor, and (b) the relative roles of Ca2+ influx via L-type and non-L-type Ca2+ channels in prostaglandin receptor-mediated contraction. [Ca2+]i and force were measured simultaneously in fura-2-loaded rat aortic strips. The thromboxane A2 receptor antagonist, SQ29548 ([1S]-1a,2b(5Z),3b,4a-7-(3-[2-[(phenylamino)carbonyl] hydrazinomethyl)-7-oxobicyclo-[2.2.1]hept-2-yl-5-heptenoic acid), prevented the prostaglandin F2alpha-induced plateau [Ca2+]i elevation and force by 80-90%, while abolishing these responses due to the thromboxane A2 receptor agonist, U46619 (9,11-dideoxy-9alpha,11alpha-methanoepoxy prostaglandin F2alpha). Prostaglandin F2alpha (+ SQ29548)-induced plateau [Ca2+]i elevation and force were not inhibited by verapamil. Ni2+, a non-selective cation channel blocker, in the presence of verapamil, abolished the prostaglandin F2alpha (+ SQ29548)-elevated [Ca2+]i, while the contraction was only partially inhibited. These results suggest that, in rat aorta, (1) elevated [Ca2+]i and force due to high prostaglandin F2alpha concentrations largely results from thromboxane A2 receptor activation, and (2) the prostaglandin component of the prostaglandin F2alpha-induced contraction is dependent on Ca2+ influx via non-L-type channels.     

Agonist-induced intracellular Ca2+ transients in HT29 cells

In the present study we have investigated the mechanism of intracellular Ca2+ activity ([Ca2+]i) changes in HT29 cells induced by adenosine triphosphate (ATP), carbachol (CCH), and neurotensin (NT). [Ca2+]i was measured with the fluorescent Ca2+ indicator fura-2 at the single-cell level or in small cell plaques with high time resolution (1-40Hz). ATP and CCH induced not only a dose-dependent [Ca2+]i peak response, but also changes of the plateau phase. The [Ca2+]i plateau was inversely dependent on the ATP concentration, whereas the CCH-induced [Ca2+]i plateau increased at higher CCH concentrations. NT showed (from 10(-10) to 10(-7) mol/l) in most cases only a [Ca2+]i spike lasting 2-3 min. The [Ca2+]i plateau induced by ATP (10(-6) mol/l) and CCH (10(-5) mol/l) was abolished by reducing the Ca2+ activity in the bath from 10(-3) to 10(-4) mol/l (n = 7). In Ca(2+)-free bathing solution the [Ca2+]i peak value for all three agonists was not altered. Using fura-2 quenching by Mn2+ as an indicator of Ca2+ influx the [Ca2+]i peak was always reached before Mn2+ influx started. Every agonist showed this delayed stimulation of the Ca2+ influx with a lag time of 23 +/- 1.5 s (n = 15) indicating a similar mechanism in each case. Verapamil (10(-6)-10(-4) mol/l) blocked dose dependently both phases (peak and plateau) of the CCH-induced [Ca2+]i increase. Short pre-incubation with verapamil augmented the effect on the [Ca2+]i peak, whereas no further influence on the plateau was observed. Ni2+ (10(-3) mol/l) reduced the plateau value by 70%.     

Effect of cilostazol, a phosphodiesterase type III inhibitor, on histamine-induced increase in [Ca2+]i and force in middle cerebral artery of the rabbit

1. The effect of cilostazol, an inhibitor of phosphodiesterase type III (PDE III), on the contraction induced by histamine was studied by making simultaneous measurements of isometric force and the intracellular concentration of Ca2+ ([Ca2+]i) in endothelium-denuded muscle strips from the peripheral part of the middle cerebral artery of the rabbit. 2. High K+ (80 mM) produced a phasic, followed by a tonic increase in both [Ca2+]i and force. Cilostazol (10 microM) did not modify the resting [Ca2+]i, but it did significantly decrease the tonic contraction induced by high K+ without a corresponding change in the [Ca2+]i response. 3. Histamine (3 microM) produced a phasic, followed by a tonic increase in both [Ca2+]i and force. Cilostazol (3 and 10 microM) significantly reduced both the phasic and tonic increases in [Ca2+]i and force induced by histamine, in a concentration-dependent manner. 4. Rp-adenosine-3':5'-cyclic monophosphorothioate (Rp-cAMPS, 0.1 mM), a PDE-resistant inhibitor of protein kinase A (and as such a cyclic AMP antagonist), did not modify the increases in [Ca2+]i and force induced by histamine alone, but it did significantly decrease the cilostazol-induced inhibition of the histamine-induced responses. 5. In Ca2+-free solution containing 2 mM EGTA, both histamine (3 microM) and caffeine (10 mM) transiently increased [Ca2+]i and force. Cilostazol (1-10 microM) (i) significantly reduced the increases in [Ca2+]i and force induced by histamine, and (ii) significantly reduced the increase in force but not the increase in [Ca2+]i induced by caffeine. 6. In ryanodine-treated strips, which had functionally lost the histamine-sensitive Ca2+ storage sites, histamine (3 microM) slowly increased [Ca2+]i and force. Cilostazol (3 and 10 microM) lowered the resting [Ca2+]i, but did not modify the histamine-induced increase in [Ca2+]i, suggesting that functional Ca2+ storage sites are required for the cilostazol-induced inhibition of histamine-induced Ca2+ mobilization. 7. The [Ca2+]i-force relationship was obtained in ryanodine-treated strips by applying ascending concentrations of Ca2+ (0.16-2.6 mM) in Ca2+-free solution containing 100 mM K+. Histamine (3 microM) shifted the [Ca2+]i-force relationship to the left and increased the maximum Ca2+-induced force. Under the same conditions, whether in the presence or absence of 3 microM histamine, cilostazol (3-10 microM) shifted the [Ca2+]i-force relationship to the right without producing a change in the maximum Ca2+-induced force. 8. It is concluded that, in smooth muscle of the peripheral part of the rabbit middle cerebral artery, cilostazol attenuates the histamine-induced contraction both by inhibiting histamine-induced Ca2+ mobilization and by reducing the myofilament Ca2+ sensitivity. It is suggested that the increase in the cellular concentration of cyclic AMP that will follow the inhibition of PDE III may play an important role in the cilostazol-induced inhibition of the histamine-contraction.     

Mastoparan stimulates exocytosis at a Ca(2+)-independent late site in stimulus-secretion coupling. Studies with the RINm5F beta-cell line

Mastoparan, a tetradecapeptide from wasp venom, stimulated exocytosis in a concentration-dependent manner, which was enhanced by pertussis toxin pre-treatment, in the insulin secreting beta-cell line RINm5F. Mastoparan (3-20 microM) also elevated cytosolic free calcium concentration ([Ca2+]i), a rise that was not attenuated by nitrendipine. Divalent cation-free Krebs-Ringer bicarbonate (KRB) medium with 0.1 mM EGTA nullified the mastoparan-induced increase in [Ca2+]i, suggesting that the peptide increased Ca2+ influx but not through the L-type voltage-dependent Ca2+ channel. Depletion of the intracellular Ca2+ pool did not affect the mastoparan-induced elevation of [Ca2+]i. Remarkably, in divalent cation-free KRB medium with 0.1 mM EGTA and 2 microM thapsigargin in which mastoparan reduced [Ca2+]i, the mastoparan-stimulated insulin release was similar to that in normal Ca(2+)-containing KRB medium. Inhibitors of protein kinase C, such as bisindolylmaleimide, staurosporine, and 1-O-hexadecyl-2-O-methyl-rac-glycerol did not suppress the mastoparan-stimulated insulin release. Mastoparan at 10-20 microM did not increase cellular cAMP levels, nor did mastoparan at 5-10 microM affect [3H]arachidonic acid release. In conclusion, although mastoparan increased [Ca2+]i, this increase was not involved in the stimulation of insulin release. Rather, the data suggest that mastoparan directly stimulates exocytosis in a Ca(2+)-independent manner. As GTP-binding proteins (G proteins) are thought to be involved in the process of exocytosis and as mastoparan is known to exert at least some of its effects by activation of G proteins, an action of mastoparan to activate the putative stimulatory Ge (exocytosis) protein is likely.     

EDG1 is a functional sphingosine-1-phosphate receptor that is linked via a Gi/o to multiple signaling pathways, including phospholipase C activation, Ca2+ mobilization, Ras-mitogen-activated protein kinase activation, and adenylate cyclase inhibition

In Chinese hamster ovary (CHO) cells transiently transfected with an expression vector for EDG1, but not an empty vector, sphingosine-1-phosphate (SP) at a concentration as low as 10(-10) M caused an increase in the intracellular free Ca2+ concentration ([Ca2+]i) as a result of mobilization of Ca2+ from both intracellular and extracellular pools. In a CHO clone stably expressing EDG1 receptor (CHO-EDG1 cells), SP induced increases in the production of inositol phosphates and the [Ca2+]i and inhibited forskolin-induced increase in the cellular cAMP content, all in a manner sensitive to pertussis toxin. SP also activated mitogen-activated protein kinase in CHO-EDG1 cells in pertussis toxin-sensitive and Ras-dependent manners. To evaluate the spectrum of agonists for EDG1, we used human erythroleukemia (HEL) cells, which at naive state do not respond to SP or structurally related lipids with an increase in the [Ca2+]i. In HEL cells stably expressing EDG1 receptor (HEL-EDG1 cells), SP dose-dependently increased the [Ca2+]i with half-maximal and maximal concentration values of 10(-9) and 3 x 10(-7) M, respectively; sphingosylphosphorylcholine at exclusively high concentrations, but not sphingosine at all, also increased the [Ca2+]i. HEL-EDG1 cells bound 32P-labeled SP, which was displaced dose dependently by unlabeled SP. These results indicate that EDG1, a member of the EDG family G protein-coupled receptors, is a specific, high-affinity SP receptor.     

The role of immune complex in otitis media with effusion

Lead characteristically perturbs processes linked to the calcium messenger system. This study was undertaken to determine the role of PKC in the Pb2+ induced rise of [Ca2+]i. [Ca2+]i was measured using the divalent cation indicator, 1,2-bis(2-amino-5-fluorophenoxy) ethane N, N,N',N'-tetraacetic acid (5F-BAPTA) and 19F-NMR in the osteoblast cell line, ROS 17/2.8. Treatment of cells with Pb2+ at 1 and 5 microM produced a rise in [Ca2+]i from a basal level of 125 nM to 170 nM and 230 nM, respectively, while treatment with phorbol 12-myristate 13-acetate (PMA) (10 microM), an activator of PKC, produced a rise in [Ca2+]i to 210 nM. Pretreatment with calphostin C, a potent and highly selective inhibitor of PKC activation failed to produce a change in basal [Ca2+]i and prevented any rise in [Ca2+]i in response to Pb2+. To determine whether Pb2+ acts directly on PKC, we measured the Pb2(+)-dependent activation of phosphatidylserine/diolein-dependent incorporation of 32P from ATP into histone and endogenous TCA precipitable proteins in the 100,000 X g supernatant from homogenized ROS 17/2.8 cells. The free concentrations of Pb2+ and Ca2+ were set using 5F-BAPTA; and [Ca2+] and [Pb2+] in the PKC reaction mixtures were confirmed by 19F-NMR. We found that Pb2+ activates PKC in the range of 10(-11)-10(-7) M, with an activation constant of 1.1 X 10(-10) M, whereas Ca2+ activates PKC in the range from 10(-8) to 10(-3) M, with an activation constant of 3.6 X 10(-7) M. These data suggest that Pb2+ activates PKC in ROS 17/2.8 cells and that Pb2+ activation of PKC mediates the documented rise in [Ca2+]i and, perhaps, other toxic effects of Pb2+.