The contractile action of platelet-activating factor on gallbladder smooth muscle

Platelet-activating factor (PAF) may be a mediator of some sequelae of cholecystitis, a disorder with gallbladder motor dysfunction. The aims of this study were to determine the effect and mechanism of PAF on gallbladder muscle. Exogenous administration of PAF-16 or PAF-18 caused dose-dependent contractions of gallbladder muscle strips in vitro with threshold doses of 1 ng/ml and 10 ng/ml, respectively. The PAF-induced contractions were not significantly reduced by TTX, atropine, or hexamethonium but were significantly inhibited with the PAF receptor antagonists ginkolide B and CV-3988. The PAF-induced contraction was reduced by indomethacin. Preventing influx of extracellular calcium with a calcium-free solution nearly abolished the PAF contractile response. Nifedipine inhibited the PAF contractile response, whereas ryanodine had no effect. Pertussis toxin reduced the PAF contractile response. In conclusion, PAF causes gallbladder contraction through specific PAF receptors on gallbladder muscle. These PAF receptors appear to be linked to a prostaglandin-mediated mechanism and to pertussis toxin-sensitive G proteins. The contractile response is largely mediated through the utilization of extracellular calcium influx through voltage-dependent calcium channels.     

Phorbol ester contracts rabbit thoracic aorta by increasing intracellular calcium and by activating calcium influx

The ability of the phorbol ester tumor promoter, PDB, to activate contraction and stimulate calcium influx was investigated in rabbit thoracic aorta. PDB caused a strong, slowly-developing sustained contraction in physiological salt solution which was concentration-related (0.01 to 10.0 microM). PDB-induced contractions (0.1 microM) in calcium-free medium were attenuated but not prevented. PDB (1.0 microM) maximally stimulated Ca influx above basal control, vehicle = 39.2 +/- 2.2; PDB 1.0 microM = 70.7 +/- 6.7 mumoles Ca/kg tissue; N = 16, p less than 0.01). These data suggest that PDB activates rabbit thoracic aorta by a combination of intracellular and extracellular calcium dependent mechanisms.     

Investigations into the mechanism by which sodium fluoride stimulates prostaglandin production in guinea-pig uterus

Sodium fluoride (10 mM) caused a slow increase in the outputs of PGF-2 alpha, 6-keto-PGF-1 alpha and, to a lesser extent, PGE-2 from the Day-7 and Day-15 guinea-pig uterus superfused in vitro. This stimulatory action of sodium fluoride was not prevented by using calcium-free Krebs' solution. There was also a faster stimulation of 6-keto-PGF-1 alpha output from the Day-7 guinea-pig uterus produced by sodium fluoride, and this quicker response was abolished by using calcium-free Krebs' solution. TMB-8 (an intracellular calcium antagonist) inhibited the stimulatory action of sodium fluoride on the outputs of PGF-2 alpha, PGE-2 and 6-keto-PGF-1 alpha from the Day-7 guinea-pig uterus. W-7 and trifluoperazine (calmodulin antagonists) and neomycin (an inhibitor of phospholipase C) had no inhibitory effect on the increases in outputs of PGF-2 alpha, PGE-2 and 6-keto-PGF-1 alpha from the Day-7 guinea-pig uterus produced by sodium fluoride. These results indicate that sodium fluoride slowly stimulates uterine PGF-2 alpha, PGE-2 and 6-keto-PGF-1 alpha synthesis in the guinea-pig uterus by mobilizing intracellular calcium by a mechanism which apparently does not involve the activation of phospholipase C or the participation of calmodulin (or a related compound). The initial, faster stimulation of 6-keto-PGF-1 alpha synthesis in the Day-7 guinea-pig uterus by sodium fluoride is dependent upon extracellular calcium.     

In vitro effect of calcitonin on guinea pig gallbladder

Calcitonin (CT) is a 32 amino acidic polypeptide hormone which has been found in almost all species and whose effects are mainly concerned with calcium and phosphorous homeostasis. Three preparations are employed for therapeutic uses: salmon (sCT), porcine (pCT) and human CT (hCT). The sCT is the most powerful one and in human volunteers a strong relaxing effect has been shown on gallbladder (GB) basal volume and emptying in response to a meal, intraduodenal instillation of a liquid meal and i.v. cholecystokinin (CCK) infusion. Our study was aimed at investigating if a direct sCT effect could be demonstrated on smooth muscle strips from guinea pig GBs "in vitro" (organ bath). Isometric contractions were measured in response to maximal doses of acetylcholine (ACh: 10(-4) M), KCl (80 mM) and cholecystokinin octapeptide (CCK-OP: 10(-6) M), in absence and in presence of four doses of sCT (1 x 10(-9), 1 x 10(-8), 1 x 10(-7) and 1 x 10(-6) M). sCT did not affect the initial strip basal tone. ACh, CCK-OP and KCl caused, as expected, a powerful contraction of the strips, but no effect was shown when each of the sCT doses was administered before ACh (1.28+ 0.69 SEM without sCT vs 1.28g+ 0.69 with sCT; n = 6) and CCK-OP (1.46g+ 0.19 without sCT vs 1.46g+ 0.19 with sCT; n = 8) or 5 min after the induced KCl contraction. On the basis of these preliminary results, we conclude that no evidence of a direct sCT effect was found on guinea pig GBs when considering either basal smooth muscle tone or isometric contraction in response to ACh, KCl and CCK-OP. Further studies are therefore required to clarify the influence of CT on GB dynamics in vivo and to elucidate its the physiological significance.     

Dissociation of the metabolic from the contractile response to muscarinic stimulation in the rabbit urinary bladder

The calcium dependence of contraction and NADH flurorescence was investigated in rabbit bladder stimulated with bethanechol or KCl. The absence of calcium in the bathing solution induced a rightward shift in the dose response to bethanechol for both contraction and NADH flurorescence. The contractile response was shifted to a greater degree than the fluorescence response and the maximal response to bethanechol was reduced by 80% for contraction but only 20% for NADH fluorescence. This rightward shift was also induced by the benzothiazepine calcium antagonist diltiazem (200 M) and again the contractile response was shifted significantly more than the fluorescence response. The combination of zero calcium and 200 M diltiazem virtually abolished contractions but only inhibited the NADH fluorescence by 65% at maximally effective bethanechol concentrations. Unlike the effect of diltiazem on the response to bethanechol, diltiazem (200 M) shifted both the contraction and fluorescence curves to the right equally in response to KCl stimulation. These results indicate that a metabolic response to muscarinic stimulation (decreased NADH) can occur in the absence of any observable contractile response. This metabolic response may be due to post receptor signal processing events. For KCl stimulation, the NADH response is probably secondary to and a result of the contractile response.Abbreviations ATP Adenosine Triphosphate - KCl Potassium Chloride - HPLC High Performance Liquid Chromatography - NADH reduced nicotinamide Adenine Dinucleotide - NAD Oxidized Nicotinamide Adenine Dinucleotide     

Effect of verapamil on the contractions produced by high potassium and by noradrenaline in human isolated saphenous vein

The effect of a calcium channel blocker, e.g. verapamil, on the contractions produced by high potassium (K+) and noradrenalne (NA), was studied in the isolated saphenous vein in man. The aim of the present experiments was to see which of the two types of contractions was more sensitive to blockade by a calcium channel blocker, e.g. verapamil, and if verapamil had a differential effect on KCl and NA, whether this could be interpreted in terms of the presence of two calcium activation mechanisms in human saphenous vein. The results of the present investigation showed that KCl and NA contracted whereas verapamil relaxed the human saphenous vein. NA produced larger contraction (3.4 g tension) than did KCl (1.3 g tension). Lowering the calcium concentration in the external medium, from 2.5 mM to 1 mM, resulted in a reduced contraction in both NA and KCl responses, indicating dependence on influx of calcium. However, verapamil (1 microM) produced greater reduction in the KCl than NA-induced contraction, indicating that the NA contraction may involve additional mechanism, i.e. dependence on the release of calcium from intracellular Ca2+ stores. These results are in favour of the suggestion that the KCl-induced contraction was due to depolarization and voltage-dependent activation of calcium channels, whereas the NA-induced contraction was due to both depolarization and receptor-activation of the calcium channels, the latter being less sensitive to calcium channel blockers, e.g. verapamil. Thus, the KCl and NA-induced contractions in human saphenous vein may be due to two different calcium activation mechanisms; one is more sensitive (KCl) than the other (NA) to the presence of the calcium antagonist, verapamil.     

Study of mechanisms mediating contraction to leukotriene C4, D4 and other bronchoconstrictors on guinea pig trachea

Contractions of guinea pig trachea in the absence and presence of indomethacin to LTD4 greater than LTC4 greater than K+ greater than histamine greater than acetylcholine were reduced following a 45 minute exposure of the tissues to calcium-free Krebs' solution (Ca2+-free Krebs' solution), were further reduced by a transient exposure to EGTA (1.25 mM) in Ca2+-free Krebs' solution and were virtually abolished when tested in the presence of EGTA (0.125 mM) in Ca2+-free Krebs' solution. In normal Krebs' solution (2.5 mM Ca2+) the Ca2+ entry blockers nifedipine (N) much greater than D-600 greater than verapamil (V) greater than diltiazem (D) almost completely abolished the contractions to K+ but blocked only a component of the maximum response to the other agonists. After exposure to Ca2+-free Krebs' solution for 45 minutes, any residual contractions to LTC4 & LTD4, were reversed by low concentrations of N (0.3 microM) or D-600 (2.1 microM). Leukotrienes appear to mobilize a superficial and a bound store of Ca2+ which gains entry through at least two types of Ca2+ channels (or mechanisms), one of which is blocked by N and D600. K+-induced contractions appear to be dependent on superficial and tightly bound Ca2+ but entry is solely through channels which are blocked by the Ca2+ entry blockers studied. Contraction to histamine and acetylcholine persisted following exposure of the tissues to Ca2+ free Krebs' solution but contractile activity was virtually abolished in Ca2+ free Krebs' solution containing EGTA. Residual contractions to histamine and part of the residual contractions to acetylcholine in Ca2+-free Krebs' solution were blocked by low dose N (0.3 microM) or D600 (2.1 microM). These findings suggest a major role for extracellular Ca2+ during spasmogen-induced contraction in this tissue.     

Levobupivacaine-induced contraction of isolated rat aorta is calcium dependent

Levobupivacaine is a long-acting local anesthetic that intrinsically produces vasoconstriction in isolated vessels. The goals of this study were to investigate the calcium-dependent mechanism underlying levobupivacaine-induced contraction of isolated rat aorta in vitro and to elucidate the pathway responsible for the endothelium-dependent attenuation of levobupivacaine-induced contraction. Isolated rat aortic rings were suspended to record isometric tension. Cumulative levobupivacaine concentration-response curves were generated in either the presence or absence of the antagonists verapamil, nifedipine, SKF-96365, 2-aminoethoxydiphenylborate, Gd(3+), N(W)-nitro-l-arginine methyl ester (L-NAME), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), and methylene blue, either alone or in combination. Verapamil, nifedipine, SKF-96365, 2-aminoethoxydiphenylborate, low calcium concentrations, and calcium-free Krebs solution attenuated levobupivacaine-induced contraction. Gd(3+) had no effect on levobupivacaine-induced contraction. Levobupivacaine increased intracellular calcium levels in vascular smooth muscle cells. L-NAME, ODQ, and methylene blue increased levobupivacaine-induced contraction in endothelium-intact aorta. SKF-96365 attenuated calcium-induced contraction in a previously calcium-free isotonic depolarizing solution containing 100?mmol/L KCl. Levobupivacaine-induced contraction of rat aortic smooth muscle is mediated primarily by calcium influx from the extracellular space mainly via voltage-operated calcium channels and, in part, by inositol 1,4,5-trisphosphate receptor-mediated release of calcium from the sarcoplasmic reticulum. The nitric oxide - cyclic guanosine monophosphate pathway is involved in the endothelium-dependent attenuation of levobupivacaine-induced contraction.     

Action of cholecystokinin, cholinergic agents, and A-23187 on accumulation of guanosine 3':5'-monophosphate in dispersed guinea pig pancreatic acinar cells.

The COOH-terminal octapeptide of cholecystokinin (CCK-OP) and carbamylcholine each increased calcium outflux, cellular cyclic GMP and amylase secretion in dispersed guinea pig pancreatic acinar cells. Following addition of CCK-OP or carbamylcholine, cellular cyclic GMP increased as early as 15 s, became maximal after 1 to 2 min, and then decreased steadily during the subsequent incubation. For both CCK-OP and carbamylcholine there was close agreement between the dose-response curve for stimulation of calcium outflux and that for increase of cellular cyclic GMP. With CCK-OP an effect on both functions could be detected at 10(-10) M and maximal stimulation occurred at 3 X 10(-8) M. With carbamylcholine an effect on both functions could be detected at 10(-5) M and maximal stimulation occurred at 3 X 10(-3) M. Atropine inhibited stimulation of both cyclic GMP and calcium outflux by carbamylcholine but not by CCK-OP. Stimulation of calcium outflux or cellular cyclic GMP by CCK-OP or carbamylcholine did not require extracellular calcium since stimulation occurred in a calcium-free, ethylene glycol bis(beta, beta-aminoethyl ether) N,N'-tetraacetic acid (EGTA)-containing solution. The divalent cation ionophore A-23187 increased bidirectional fluxes of calcium, cellular cyclic GMP and secretion of amylase from dispersed pancreatic acinar cells. Like CCK-OP and carbamylcholine, the ionophore stimulated calcium outflux and cellular cyclic GMP in a calcium-free, EGTA-containing solution. These results suggest that in pancreatic acinar cells the initial step in the sequence of events mediating the action of ionophore as well as that of CCK-OP and carbamylcholine is stimulation of calcium outflux, and that this stimulation then increases cellular cyclic GMP.     

Effect of progesterone on the contractile response of isolated pulmonary artery in rabbits.

The purpose of this study was to assess the direct effect of progesterone on rabbit pulmonary arteries and to examine the mechanism of its action. Rings of pulmonary artery from male rabbits were suspended in organ baths containing Krebs solution, and isometric tension was measured. The response to progesterone was investigated in arterial rings contracted with noradrenaline (NA), KCl, and CaCl2. The effects of endothelium, nitric oxide (NO), prostaglandins, cyclic GMP (cGMP), and the adrenergic beta-receptor on progesterone-induced relaxation were also assessed. Progesterone inhibited the vasocontractivity to NA, KCl, and CaCl2, and relaxed rabbit pulmonary artery. The relaxing response of progesterone in pulmonary artery was significantly reduced by removal of endothelium, inhibitors of nitric oxide synthase and guanylate cyclase, but not by prostaglandin synthase inhibitor and blockage of the adrenergic beta-receptor. In Ca2+-free (0.1 mM EGTA) Krebs solution, progesterone inhibited NA-induced contraction that was intracellular Ca2+-dependent, but didn't affect the contraction of extracellular Ca2+-dependent component. Our results suggest that progesterone induces relaxation of isolated rabbit pulmonary arteries partially via NO and cGMP. Progesterone may also inhibit Ca2+ influx through potential-dependent calcium channels (PDCs) and Ca2+ release from intracellular stores.     

Comparative effects of endothelin and phorbol 12-13 dibutyrate in rat aorta

The vasoconstrictive properties of endothelin (ET-1) and the protein kinase C activator, phorbol 12-13 dibutyrate (PDB) were comparatively investigated in isolated rat aorta. ET-1 (0.3-100 nM) and PDB (10 nM-3 microM) induced a slowly developing sustained contraction in endothelium denuded aorta. Maximal contractions induced by ET-1 and PDB were unaffected by diltiazem (10 microM). Substantial contraction to ET-1 (30 nM) and PDB (0.1 microM) remained in calcium-free medium. Contractions of ET-1 and PDB in calcium-free medium were unaffected by intracellular calcium depletion induced by phenylephrine. Following the response to ET-1 and PDB in a calcium-free medium, an additional sustained contraction was observed after calcium (2.5 mM) was added to the bath. The protein kinase C inhibitor, H7 (100 microM) was more potent in inhibiting contractions induced by phenylephrine and KCl than the ones elicited by ET-1 and PDB. The other protein kinase C inhibitors i.e. staurosporine (50 nM) and phloretin (100 microM) inhibited to a similar extent all the agonists tested. These results suggest that protein kinase C may play an important role in mediating the contraction to ET-1 in rat aorta.     

Role of calcium in the potentiating effect of phorbol ester on KCl-induced vasocontraction

The mechanism of the potentiating effect of phorbol ester on potassium-induced contraction in rat aorta was investigated. The contractile response to KCl in the medium containing 0.5 mM CaCl2 was significantly increased by pretreatment with 10(-8) M phorbol 12-myristate 13-acetate (PMA), but not with 10(-7) M 4 alpha-phorbol. The dose-response curve to calcium in 30 mM KCl-induced contraction was shifted to the left by PMA pretreatment and the EC50 value (the concentration producing a half maximal response) of calcium was significantly lower in aorta pretreated with PMA than in the control. On the other hand, calcium influx stimulated by 30 mM KCl was not changed by PMA pretreatment. Both the contractile response and the corresponding calcium influx induced by 30 mM KCl were abolished by preincubation with 10(-6) M verapamil for 45 min. These results suggest that activation of protein kinase C potentiates the contractile response to KCl by increasing the sensitivity of the intracellular contractile apparatus for calcium.     

Calcium Dependence of Muscarinic Receptor-Mediated Catecholamine Secretion from the Perfused Rat Adrenal Medulla

It had previously been thought that muscarinic cholinergic receptors utilize an influx of extracellular calcium for activation of adrenomedullary catecholamine secretion. However, it has recently been demonstrated that muscarinic receptors on isolated adrenal chromaffin cells can elevate cytosolic free calcium levels in a manner independent of extracellular calcium, presumably by mobilizing intracellular calcium stores. We now demonstrate that muscarinic receptor-mediated catecholamine secretion from perfused rat adrenal glands can occur under conditions of extracellular calcium deprivation that are sufficient to block both nicotine- and electrically stimulated release. Three independent conditions of extracellular calcium deprivation were used: nominally calcium-free perfusion solution (no calcium added), EGTA-containing calcium-free perfusion solution, and perfusion solution containing the calcium channel blocker verapamil. Secretion was evoked from the perfused glands by either transmural electrical stimulation or injection of nicotine or muscarine into the perfusion stream. Each condition of calcium deprivation was able to block nicotine- and electrically stimulated catecholamine release in an interval that left muscarine-evoked release largely unaffected. The above results demonstrate that muscarine-evoked catecholamine secretion from perfused rat adrenal glands can occur in the absence of extracellular calcium, presumably by mobilization of intracellular calcium. The latter may be due to muscarinic receptor-mediated generation of inositol trisphosphate.     

Factors involved in the generation of tension during contraction to high potassium in the rat vas deferens

A large number of studies indicate that K⁺-induced contractions of smooth muscle depend on extracellular calcium. If these contractions depend exclusively on extracellular calcium then contractile responses to 140 mM K⁺, which are larger than the response to 35 mM K⁺, should be associated with a larger influx of ⁴⁵Ca. This is not the case in the vas deferens from reserpine pretreated rats. During a 2 min interval, ⁴⁵Ca influx induced by 140 mMK⁺ was identical to that produced by 35 mM K⁺. This suggests that a second mechanism may be involved in responses to high K⁺. Indeed, 140 mM K⁺ caused an approximately 300% increase above control in the formation of inositol trisphosphate (IP₃) in tissues prelabelled with ³H-myoionositol whereas 35 mM K⁺ did not increase IP₃. IP₃ is thought to cause the release of calcium from internal stores which is consistent with our finding of an increase in ⁴⁵Ca efflux into calcium-free medium from tissues prelabelled with ⁴⁵Ca and stimulated with 140 mM K⁺. Stimulation with 35 mM K⁺ did not influence ⁴⁵Ca efflux. We conclude that in the rat vas deferens high K⁺ promotes tension development by smooth muscle by a dual mechanism: influx of extracellular calcium and release of calcium from internal stores via a IP₃ mechanism.     

Impairment of Ca(2+) mobilization in circular muscle cells of the inflamed colon

This study investigated whether inflammation modulates the mobilization of Ca(2+) in canine colonic circular muscle cells. The contractile response of single cells from the inflamed colon was significantly suppressed in response to ACh, KCl, and BAY K8644. Methoxyverapamil and reduction in extracellular Ca(2+) concentration dose-dependently blocked the response in both normal and inflamed cells. The increase in intracellular Ca(2+) concentration in response to ACh and KCl was significantly reduced in the inflamed cells. However, Ca(2+) efflux from the ryanodine- and inositol 1,4, 5-trisphosphate (IP(3))-sensitive stores, as well as the decrease of cell length in response to ryanodine and IP(3), were not affected. Heparin significantly blocked Ca(2+) efflux and contraction in response to ACh in both conditions. ACh-stimulated accumulation of IP(3) and the binding of [(3)H]ryanodine to its receptors were not altered by inflammation. Ruthenium red partially inhibited the response to ACh in normal and inflamed states. We conclude that the canine colonic circular muscle cells utilize Ca(2+) influx through L-type channels as well as Ca(2+) release from the ryanodine- and IP(3)-sensitive stores to contract. Inflammation impairs Ca(2+) influx through L-type channels, but it may not affect intracellular Ca(2+) release. The impairment of Ca(2+) influx may contribute to the suppression of circular muscle contractility in the inflamed state.     

Calcium signaling in lymphocytes and ELF fields. Evidence for an electric field metric and a site of interaction involving the calcium ion channel.

Calcium influx increased during mitogen-activated signal transduction in thymic lymphocytes exposed to a 22 mT, 60 Hz magnetic field (E induced = 1.7 mV/cm, 37 degrees C, 60 min). To distinguish between an electric or a magnetic field dependence a special multi-ring annular cell culture plate based on Faraday's Law of Induction was employed. Studies show a dependence on the strength of the induced electric field at constant magnetic flux density. Moreover, exposure to a pure 60 Hz electric field or to a magnetically-induced electric field of identical strength resulted in similar changes in calcium transport. The first real-time monitoring of [Ca2+]i during application of a 60 Hz electric field revealed an increase in [Ca2+]i observed 100 s after mitogen stimulation; this suggests that the plateau phase rather than the early phase of calcium signaling was influenced. The hypothesis was tested by separating, in time, the early release of calcium from intracellular stores from the influx of extracellular calcium. In calcium-free buffer, 60 Hz field exerted little influence on the early release of calcium from intracellular stores. In contrast, addition of extracellular calcium during exposure enhanced calcium influx through the plasma membrane. Alteration of the plateau phase of calcium signaling implicates the calcium channel as a site of field interaction. In addition, an electric field exposure metric is mechanistically consistent with a cell-surface interaction site.     

Postsynaptic alpha adrenoceptors on vascular smooth muscle

A heterogeneous population of alpha adrenoceptors mediates vasoconstriction in the canine saphenous vein (CSV). Studies with isolated strips of venous smooth muscle incubated with selective alpha-adrenoceptor agonists and antagonists revealed that both alpha 1 and alpha 2 adrenoceptors exist independently in this tissue and both subtypes mediate a contractile response. Measurement of contractile responses in reduced or zero external calcium conditions indicates that stimulation of alpha 1 adrenoceptors induces contractions by influx of extracellular calcium and release of calcium from internal stores. In contrast, 45Ca uptake studies suggest that activation of the postsynaptic alpha 2 adrenoceptor produces vasoconstriction dependent only on influx of extracellular calcium. The influx of calcium produced by the selective alpha 2-adrenoceptor agonist BHT-920 is inhibited by calcium entry blockers. Measurements of transmembrane potentials from smooth muscle cells of the CSV suggest that alpha 1-adrenoceptor activation produces depolarization and contraction (electromechanical coupling) whereas alpha 2-adrenoceptor stimulation does not result in concentration-dependent depolarization of the smooth muscle cells (pharmacomechanical coupling).     

Hypoxic contractile response in isolated human pulmonary artery: role of calcium ion

The mechanism for hypoxic pulmonary vasoconstriction (HPVC) was investigated in human pulmonary arterial strips. Hypoxia in the presence of histamine (10(-6) M) caused marked pulmonary arterial contraction, which was reversed by O2. The hypoxic contraction in the presence of histamine was inhibited by diphenhydramine, but not by cimetidine. The hypoxic histamine-mediated contraction was attenuated but still present in the absence of extracellular Ca2+, or by the inhibitors of voltage-dependent Ca2+ influx. However, it was inhibited significantly by a further depletion of intracellular Ca2+, or by HA 1004, an intracellular calcium antagonist. A low concentration (10(-7) M) of a calcium ionophore, A23187, enhanced the hypoxic contraction in the presence of histamine, whereas procaine completely inhibited it. W-7, a calmodulin inhibitor, significantly decreased the hypoxic histamine-mediated contraction, but 12-O-tetradecanoylphorbol-13-acetate (TPA), a C-kinase promotor, had no effect. The hypoxic contractile response was also observed in the presence of both A23187 and KCl instead of histamine, but the hypoxia-induced contraction with KCl alone was much smaller than that. These results indicate that hypoxia in the presence of certain other vasoactive agents has a potent contractile effect on the human pulmonary artery and that the response is dependent on Ca2+. Enhancement of both Ca2+ influx and Ca2+ release from intracellular storage sites by hypoxia, which interacts with calmodulin, were suggested to be involved in the mechanism of HPVC.     

Involvement of protein kinase C in the regulation of Ca2+ exit from intracellular stores of prolactin stimulated pig oocytes

Involvement of protein kinase C in the regulation of Ca2+ exit from intracellular stores of pig oocytes activated by prolactin was investigated, using the fluorescent dye chlortetracycline. In the presence of extracellular calcium, the inhibitor of protein kinase C Ro 31-8220 increased calcium exit from intracellular stores in pig oocytes after prolactin treatment. In calcium-free medium, Ro 31-8220 exerted effect on calcium release from intracellular stores. In calcium-free medium, prolactin did not stimulate calcium release from intracellular stores of oocytes in the presence of thimerosal, while in the presence of protein kinase C inhibitor, prolactin increased Ca2+ content from intracellular stores in such oocytes. These data suggest a direct involvement of protein kinase C in the processes of regulation of Ca2+ exit from intracellular stores of pig oocytes stimulated by prolactin.     

Regulation of calcium influx into buccal muscles of Aplysia by acetylcholine and serotonin

Acetylcholine (ACh) causes contraction of Aplysia buccal muscles E1 and I5, and serotonin (5-hydroxytryptamine, 5-HT) enhances ACh-elicited contractions of these muscles. Possible roles of calcium influx in mediating these responses were examined by studying influx of 45Ca++. 5-HT increased calcium influx into both I5 and E1. Maximal influx occurred at 10(-6) M 5-HT and the increased influx could be sustained in the presence of 5-HT for at least 10 min. ACh also caused calcium influx, and calcium influx increased approximately in proportion to log[ACh] from 10(-5) M to 10(-3) M ACh. 5-HT and ACh probably bring about calcium influx by different mechanisms since the effect of ACh was additive to a maximal 5-HT response, and 10(-4) M hexamethonium bromide inhibited the increased influx caused by ACh but did not affect influx caused by 5-HT. Cyclic AMP analogues and forskolin neither caused an increase in calcium influx nor an increase in the influx caused by ACh. The data support a model in which ACh-elicited contractions of I5 and E1 are due primarily to calcium entry across the extracellular membrane, and 5-HT can "load" an intracellular site by a mechanism different from that activated by ACh. The data do not support a role for cyclic AMP in mediating the calcium influx response to 5-HT.