Gingerol, a novel cardiotonic agent, activates the Ca2+-pumping ATPase in skeletal and cardiac sarcoplasmic reticulum.

Gingerol, isolated as a potent cardiotonic agent from the rhizome of ginger, stimulated the Ca2+-pumping activity of fragmented sarcoplasmic reticulum (SR) prepared from rabbit skeletal and dog cardiac muscles. The extravesicular Ca2+ concentrations of the heavy fraction of the fragmented SR (HSR) were measured directly with a Ca2+ electrode to examine the effect of gingerol on the SR. Gingerol (3-30 microM) accelerated the Ca2+-pumping rate of skeletal and cardiac SR in a concentration-dependent manner. The rate of 45Ca2+ uptake of HSR was also increased markedly by 30 microM gingerol without affecting the 45Ca2+ efflux from HSR. Furthermore, gingerol activated Ca2+-ATPase activities of skeletal and cardiac SR (EC50, 4 microM). The activation of SR Ca2+-ATPase activity by gingerol (30 microM) was completely reversed by 100-fold dilution with the fresh saline solution. Kinetic analysis of activating effects of gingerol suggests that the activation of SR Ca2+-ATPase is uncompetitive and competitive with respect to Mg . ATP at concentrations of 0.2-0.5 mM and above 1 mM, respectively. Kinetic analysis also suggests that the activation by gingerol is mixed-type with respect to free Ca2+ and this enzyme is activated probably due to the acceleration of enzyme-substrate complex breakdown. Gingerol had no significant effect on sarcolemmal Ca2+-ATPase, myosin Ca2+-ATPase, actin-activated myosin ATPase and cAMP-phosphodiesterase activities, indicating that the effect of gingerol is rather specific to SR Ca2+-ATPase activity. Gingerol may provide a valuable chemical tool for studies aimed at clarifying the regulatory mechanisms of SR Ca2+-pumping systems and the causal relationship between the Ca2+-pumping activity of SR and muscle contractility.

Enhancement of the actin-activated ATPase activity of myosin from canine cardiac ventricle by purealin.

The effects of purealin isolated from the sea sponge, Psammaplysilla purea, on the enzymatic properties of myosin and natural actomyosin (a complex of myosin, actin, tropomyosin and troponin) from canine cardiac ventricle were studied. Purealin increased the ATPase activity of natural actomyosin and the actin-activated ATPase activity of myosin, and accelerated the superprecipitation of natural actomyosin. The Ca2+- and Mg2+-ATPase activities of myosin were inhibited by purealin, whereas the K+-EDTA-ATPase activity was increased. These results suggest that purealin binds to the myosin portion involved in actin-myosin interaction and increases the actin-activated ATPase activity of myosin.

Identification of 30 kDa protein for Ca(2+) releasing action of myotoxin a with a mechanism common to DIDS in skeletal muscle sarcoplasmic reticulum.

The molecular mechanism of Ca(2+) release by myotoxin a (MTYX), a polypeptide toxin isolated from the venom of prairie rattlesnakes (Crotalus viridis viridis), was investigated in the heavy fraction of sarcoplasmic reticulum (HSR) of rabbit skeletal muscles. [(125)I]MYTX bound to four HSR proteins (106, 74, 53 and 30 kDa) on polyvinylidene difluoride (PVDF) membrane. DIDS, 4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid, bound predominantly to 30 kDa protein on the PVDF membrane, the molecular weight of which was similar to one of the MYTX binding proteins. The maximum (45)Ca(2+) release induced by caffeine (30 mM) was further increased in the presence of MYTX (10 microM) or DIDS (30 microM), whereas that induced by DIDS (30 microM) was not affected by MYTX (10 microM). MYTX inhibited [(3)H]DIDS binding to HSR in a concentration-dependent manner. Furthermore, [(125)I]MYTX binding to 30 kDa protein was inhibited by DIDS in a concentration-dependent manner. These results suggest that MYTX and DIDS release Ca(2+) from HSR in a common mechanism. The 30 kDa protein may be a target protein for the Ca(2+) releasing action of MYTX and DIDS.

Characteristics of 45Ca2+ release induced by quinolidomicin A1, a 60-membered macrolide from skeletal muscle sarcoplasmic reticulum.

Quinolidomicin A1, a 60-membered macrolide purified from an actinomycete Micromonospora sp. markedly induced 45Ca2+ release from the heavy fraction of skeletal muscle sarcoplasmic reticulum (HSR), but induced only slightly from the light fraction of sarcoplasmic reticulum (LSR), showing a lack of the ionophoretic activity even at a high concentration (300 microM). This was also confirmed by measuring the 45Ca2+ transport activity of quinolidomicin A1 across an organic solvent barrier. Quinolidomicin A1 (3-300 microM) increased 45Ca2+ release from HSR with an EC50 value of approx. 20 microM. The potency of quinolidomicin A1 was approx. 100-fold higher than that of caffeine. The bell-shaped profile of Ca2+ dependence for quinolidomicin A1 was different from that for caffeine. Blockers of Ca2+ release channels such as Mg2+ (10 mM), procaine (10 mM) and ruthenium red (10 microM) partially blocked quinolidomicin A1 (30 microM)-induced 45Ca2+ release from HSR. At 0 degrees C, quinolidomicin A1-induced 45Ca2+ release was ascertained not to be due to the inhibition of Ca2+ ATPase by the ATPase assay. Quinolidomicin A1 potentiated [3H]ryanodine binding to HSR with a decrease in KD but without a change in Bmax. These results suggest that quinolidomicin A1-induced Ca2+ release from HSR is consisted of two components, which are both sensitive and insensitive to blockers of Ca2+ release channels, and that the former component is associated with the ryanodine receptor.

Amphidinolide B, a powerful activator of actomyosin ATPase enhances skeletal muscle contraction.

Amphidinolide B caused a concentration-dependent increase in the contractile force of skeletal muscle skinned fibers. The concentration-contractile response curve for external Ca2+ was shifted to the left in a parallel manner, suggesting an increase in Ca2+ sensitivity. Amphidinolide B stimulated the superprecipitation of natural actomyosin. The maximum response of natural actomyosin to Ca2+ in superprecipitation was enhanced by it. Amphidinolide B increased the ATPase activity of myofibrils and natural actomyosin. The ATPase activity of actomyosin reconstituted from actin and myosin was enhanced in a concentration-dependent manner in the presence or absence of troponin-tropomyosin complex. Ca2+-, K+-EDTA- or Mg2+-ATPase of myosin was not affected by amphidinolide B. These results suggest that amphidinolide B enhances an interaction of actin and myosin directly and increases Ca2+ sensitivity of the contractile apparatus mediated through troponin-tropomyosin system, resulting in an increase in the ATPase activity of actomyosin and thus enhances the contractile response of myofilament.

Mastoparan elicits prostaglandin E2 generation and inhibits inositol phosphate accumulation via different mechanisms in rabbit astrocytes.

The effects of mastoparan on phosphoinositide hydrolysis and prostaglandin E2 (PGE2) generation were investigated in astrocytes cultured from rabbit brain. Mastoparan inhibited the accumulations of [3H]inositol phosphates induced by bradykinin (1 microM) in a time- and concentration-dependent manner. Mastoparan (3-30 microM) also released PGE2 in a time- and concentration-dependent manner. Mastoparan-induced release of PGE2 was inhibited by indomethacin, a cyclooxygenase inhibitor, by dexamethasone, a steroidal anti-inflammatory drug, and by pertussis toxin, an inactivator of some G proteins, such as Gi and Go. Mastoparan also caused [3H]arachidonic acid liberation, which was inhibited by dexamethasone or pertussis toxin. In contrast, indomethacin, dexamethasone and pertussis toxin failed to attenuate mastoparan-induced inhibition of [3H]inositol phosphate accumulation induced by bradykinin. Thus, mastoparan-induced inhibition of phosphoinositide hydrolysis does not involve pertussis toxin-sensitive G protein nor arachidonic acid metabolites. In addition to the inhibition of phospholipase C, mastoparan activates phospholipase A2 through pertussis toxin-sensitive G protein.

[3H]9-Methyl-7-bromoeudistomin D, a caffeine-like powerful Ca2+ releaser, binds to caffeine-binding sites distinct from the ryanodine receptors in brain microsomes.

[3H]9-Methyl-7-bromoeudistomin D ([3H]MBED), the most powerful Ca2+ releaser from sarcoplasmic reticulum, specifically bound to the brain microsomes. Caffeine competitively inhibited [3H]MBED binding. [3H]MBED binding was markedly blocked by procaine, whereas that was enhanced by adenosine-5'-(beta,gamma-methylene)triphosphate. The Bmax value was 170 times more than that of [3H]ryanodine binding. The profile of sucrose-density gradient centrifugation of solubilized microsomes indicated that [3H]MBED binding protein was different from [3H]ryanodine binding protein. These results suggest that there are MBED/caffeine-binding sites in brain that are distinct from the ryanodine receptor and that MBED becomes an essential molecular probe for characterizing caffeine-binding protein in the central nervous system.

Disulfide pairings in geographutoxin I, a peptide neurotoxin from Conus geographus.

The three intramolecular disulfide linkages of geographutoxin I, a peptide neurotoxin isolated from the venom of the marine snail Conus geographus, were examined by a novel method for determination of the positions of disulfide linkages in peptides [(1989) Bull. Chem. Soc. Jp. 62, 1986-1994]. The disulfide bridges were found to be between Cys3 and Cys15, Cys4 and Cys20, and Cys21, indicating that geographutoxin I has a rigid conformation consisting of three loops stabilized by these three disulfide linkages.

Contractions induced by grayanotoxin I in the guinea-pig vas deferens.

1 In the guinea-pig vas deferens, grayanotoxin I (G-I), a diterpenic toxin isolated from certain ericaceous plants caused rhythmic contractions which were dose-dependent (5 x 10(-5) M - 10(-3) M); these followed an initial transient contraction.2 The G-I (3 x 10(-4) M)-induced contraction was markedly inhibited or abolished by reserpine (2 mg/kg on 2 days), phentolamine (2 x 10(-7) M and 10(-6) M) or tetrodotoxin (TTX, 5 x 10(-7) M), but remained almost unaffected by atropine (10(-6) M) or mecamylamine (3 x 10(-5) M). This contraction was also abolished after storage at 4 degrees C for 7 days or incubation in Na-deficient or Ca-free medium.3 After treatment with G-I (3 x 10(-5) M), which did not alter the tension of the preparation, transmural stimulation (10-50 Hz, 0.5 ms, supramaximal voltage, for 3 s) induced a slower contraction (second contraction) following the first rapid contraction caused by stimulation.4 The second contraction was inhibited or abolished by reserpine (2 mg/kg on 2 days), phentolamine (2 x 10(-7) M and 10(-6) M) and TTX (2 x 10(-8) M), but was not affected by atropine (10(-6) M) and mecamylamine (3 x 10(-5) M).5 G-I (3 x 10(-5) M) shifted the dose-response curves for noradrenaline (NA), acetylcholine and high-K contractions to the left in a parallel manner and slightly increased the maximal response to these agonists.6 G-I (3 x 10(-4) M) caused a release of endogenous NA from the vas deferens which was approximately 120 times that of control preparations. This response was inhibited or abolished by TTX (5 x 10(-7) M) or incubation in Ca-free medium.7 These results suggest that the G-I-induced contraction of the vas deferens and the G-I-induced second contraction on electrical stimulation are the result of an indirect action mediated through the release of NA from the adrenergic nerve endings.

Possible mechanism of the dual action of the new polypeptide (anthopleurin-B) from sea anemone in the isolated ileum and taenia caeci of the guinea-pig.

1 Anthopleurin-B (AP-B), a newly isolated polypeptide from a sea anemone (Anthopleura xanthogrammica) caused relaxation of the guinea-pig isolated ileum following a transient contraction at concentrations greater than 3 x 10(-9) M. 2 AP-B caused a tonic contraction followed by rhythmic relaxation of the guinea-pig isolated taenia caeci at a concentration of 3 x 10(-9) M or more. 3 The other polypeptides, anthopleurin-A (AP-A) from the same species of anthopleurin-C (AP-C) from Anthopleura elegantissima elicited similar effects but higher concentrations were required in both tissues. 4 These responses induced by AP-B in both tissues were abolished by treatment of each tissue with tetrodotoxin or incubation in a low-Na+ medium. 5 In the ileum, the AP-B-induced contraction was markedly inhibited by atropine but not by mecamylamine, whereas the AP-B-induced relaxation was not affected by phentolamine of guanethidine. 6 The AP-B-induced contraction of the taenia caeci was also inhibited by atropine, but not mecamylamine. However, in contrast to the ileum, the spontaneous relaxation of this tissue was completely abolished in the presence of phentolamine or guanethidine. 7 These results suggest that the AP-B-induced contractions of both tissues are mainly caused by acetylcholine (ACh) release from the cholinergic nerve terminals and that the AP-B-induced relaxation of the taenia caeci is due to the excitation of adrenergic nerves, while the relaxation of the ileum is mediated through non-adrenergic inhibitory mechanisms.

Contraction and increase in tissue calcium content induced by maitotoxin, the most potent known marine toxin, in intestinal smooth muscle.

Maitotoxin (MTX), the most potent known marine toxin, isolated from toxic dinoflagellates and poisonous fish, caused a dose-dependent contraction of the guinea-pig isolated ileum and taenia caeci at concentrations of 100 pg to 30 ng/ml. These contractile responses to MTX (3 ng/ml) in both tissues were abolished by incubation in Ca2+-free solution and were markedly inhibited by treatment with methoxyverapamil (D600), but were not affected by tetrodotoxin and atropine. Furthermore, MTX markedly elevated tissue Ca2+ content of the taenia caeci. These results suggest that MTX activates Ca2+ channels in the smooth muscle membrane of both tissues to increase Ca2+ influx and thus induces contractions.

Maitotoxin-activated single calcium channels in guinea-pig cardiac cells.

1. In order to clarify the mechanism of Ca-dependent excitatory action of maitotoxin (MTX), the most potent marine toxin known, patch-clamp techniques were used to analyse electrophysiological effects of MTX on guinea-pig isolated cardiac myocytes. 2. The whole-cell recordings showed that MTX (0.3 ng ml-1) produced a sustained inward current that was enhanced by adrenaline (2 microM) and abolished by Cd2+ (1 mM). 3. This current was predominantly carried by Ca2+ or Ba2+ and has an almost linear current-voltage relationship. 4. In cell-attached patches, MTX added to the pipette solution activated Ca channels with novel properties. The opening events of these channels occurred as long bursts, and the channel gating showed little voltage-dependence. 5. The unitary conductance was 12 pS in the presence of 50 mM Ba2+. Within a burst, the distribution of opening times was a single exponential with a mean open time of 10.4 ms. 6. The channel described here represents either a new class of voltage-independent Ca channel or an entirely modified form of voltage-gated Ca channel. This channel may account for the mechanism of enhanced Ca2+ influx through the cell membrane induced by MTX, and presumably regulates some ionic movements in myocardial cells.

Thromboxane A2-mediated shape change: independent of Gq-phospholipase C--Ca2+ pathway in rabbit platelets.

1. Thromboxane A2 (TXA2) receptor-mediated signal transduction was investigated in washed rabbit platelets to clarify the mechanisms of induction of shape change and aggregation. 2. The TXA2 agonist, U46619 (1 nM to 10 microM) caused shape change and aggregation in a concentration-dependent manner. A forty-times higher concentration of U46619 was needed for aggregation (EC50 of 0.58 microM) than shape change (EC50 of 0.013 microM). The aggregation occurred only when external 1 mM Ca2+ was present, but the shape change could occur in the absence of Ca2+. 3. SQ29548 at 30 nM and GR32191B at 0.3 microM (TXA2 receptor antagonists) competitively inhibited U46619-induced shape change and aggregation with similar potency, showing that both aggregation and shape change induced by U46619 were TXA2 receptor-mediated events. However, ONO NT-126 at 1 nM, another TXA2 receptor antagonist, inhibited U46619-induced aggregation much more potently than the shape change, suggesting the possible existence of TXA2 receptor subtypes. 4. ONO NT-126 (2 nM to 3 microM) by itself caused a shape change without aggregation in a concentration-dependent manner, independent of external Ca2+. Therefore, ONO NT-126 is a partial agonist at the TXA2 receptor in rabbit platelets. 5. U46619 (10 nM to 10 microM) increased internal Ca2+ concentration ([Ca2+]i) and activated phosphoinositide (PI) hydrolysis in a concentration-dependent manner with a similar concentration-dependency. 6. U46619 (3 nM to 10 microM) also activated GTPase concentration-dependently in the membranes derived from platelets. U46619-induced activation of GTPase was partly inhibited by treatment of membranes with QL, an antibody against Gq/11. 7. The EC50 values of U46619 in Ca2+ mobilization (0.15 microM), PI hydrolysis (0.20 microM) and increase in GTPase activity (0.12 microM) were similar, but different from the EC50 value in shape change (0.013 microM), suggesting that activation of TXA2 receptors might cause shape change via an unknown mechanism. 8. U46619-induced shape change was unaffected by W-7 (30 microM), a calmodulin antagonist or ML-7 (30 microM), a myosin light-chain kinase inhibitor, indicating that an increase in [Ca2+]i might not be involved in the shape change. In fact, U46619 (10 nM) could cause shape change without affecting [Ca2+]i level, determined by simultaneous recordings. 9. [3H]-SQ29548 and [3H]-U46619 bound to platelets at a single site with a Kd value of 14.88 nM and Bmax of 106.1 fmol/10(8) platelets and a Kd value of 129.8 nM and Bmax of 170.4 fmol/10(8) platelets, respectively. The inhibitory constant Ki value for U46619 as an inhibitor of 3H-ligand binding was similar to the EC50 value of U46619 in GTPase activity, phosphoinositide hydrolysis and Ca2+ mobilization, but significantly different (P < 0.001 by Student's t test) from the effect on shape change. 10. Neither U46619 nor ONO NT-126 affected the adenosine 3',5'-cyclic monophosphate (cyclic AMP) level in the presence or absence of external Ca2+ and/or isobutyl methylxanthine. 11. The results indicate that TXA2 receptor stimulation causes phospholipase C activation and increase in [Ca2+]i via a G protein of the Gq/11 family leading to aggregation in the presence of external Ca2+, and that shape change induced by TXA2 receptor stimulation might occur without involvement of the Gq-phospholipase C-Ca2+ pathway.

Mastoparan-induced phosphatidylcholine hydrolysis by phospholipase D activation in human astrocytoma cells.

1. The effect of mastoparan on phosphatidylcholine hydrolysis was examined in 1321N1 human astrocytoma cells. Mastoparan (3-30 microM) caused an accumulation of diacylglycerol (DG) and phosphatidic acd (PA) accompanied by choline release in a concentration- and time-dependent manner. 2. In the presence of 2% n-butanol, mastoparan (3-100 microM) induced phosphatidylbutanol (PBut) accumulation in a concentration- and time-dependent manner, suggesting that mastoparan activates phospholipase D (PLD). Propranolol (30-300 microM), a phosphatidate phosphohydrolase inhibitor, inhibited DG accumulation induced by mastoparan, supporting this idea. 3. Depletion of extracellular free calcium ion did not alter the effect of mastoparan on PLD activity. 4. A protein kinase C (PKC) inhibitor, calphostin C (1 microM), did not inhibit mastoparan-induce PLD activation but the ability of mastoparan to stimulate phospholipase D activity was decreased in the PKC down regulated cells. 5. PLD activity stimulated by mastoparan was not prevented by pretreatment of the cells with pertussis toxin (PT) or C3 ADP-ribosyltransferase. Furthermore, guanine nucleotides did not affect PLD activity stimulation by mastoparan in membrane preparations. 6. Mastoparan stimulated PLD in several cell lines such as RBL-2H3, RBL-1, HL-60, P388, endothelial cells, as well as 1321N1 human astrocytoma cells. 7. These results suggest that mastoparan induces phosphatidylcholine (PC) hydrolysis by activation of PLD, not by activation of phosphatidylcholine-specific phospholipase C (PC-PLC); mastoparan-induced PLD activation is not mediated by G proteins.

The mechanism of action of maitotoxin in relation to Ca2+ movements in guinea-pig and rat cardiac muscles.

Maitotoxin (MTX), the most potent marine toxin known, produced a dose-dependent positive inotropic effect on guinea-pig isolated left atria and rat ventricle strips at concentrations of 0.1 ng to 4 ng ml-1. MTX (4 ng ml-1) also exhibited a positive chronotropic effect on guinea-pig right atria. The MTX-induced inotropic effect was almost abolished by Co2+ or verapamil, but was little affected by propranolol, reserpine or tetrodotoxin. The tissue Ca content of guinea-pig left atria was increased by MTX (2-30 ng ml-1) in a dose-dependent manner, and this increase was markedly inhibited by Co2+ or verapamil. Furthermore, on the rat isolated cardiac myocytes MTX (0.01-10 ng ml-1) caused an arrhythmogenic effect which was followed by their transformation into irreversibly rounded cells. The effects of MTX on the isolated cells were inhibited by verapamil or Ca2+-free solution. These results suggest that the excitatory effects of MTX on heart muscle are caused by a direct action on the cardiac muscle membrane mainly due to an increase in Ca2+ permeability.

Selective potentiation of noradrenaline in the guinea-pig vas deferens by 2-(4-methylaminobutoxy) diphenylmethane hydrochloride (MCI-2016), a new psychotropic drug.

In the isolated vas deferens of the guinea-pig, the effects of 2-(4-methylaminobutoxy) diphenylmethane hydrochloride (MCI-2016), a new psychotropic drug, on the contractile response to various agonists or transmural electrical stimulation and on the release of noradrenaline (NA) from the tissue were examined and compared with cocaine. MCI-2016 (3 X 10(-6)M) and cocaine (3 X 10(-5)M) produced a leftward shift (15 and 20 times, respectively) of the dose-response curves for the contractile effect of NA and increased the maximum contractile response to NA by approximately 7 and 14% respectively. MCI-2016 had no apparent effect on the dose-response curves for methoxamine, acetylcholine and high K, while cocaine markedly shifted those for these agents to the left and increased the maximal responses (10, 16 and 16%, respectively). MCI-2016 and cocaine abolished the tyramine (3 X 10(-4)M)-induced contraction and inhibited the twitch response to transmural electrical stimulation in a dose-dependent manner. The inhibitory effects of both drugs on the twitch were reversed by yohimbine (10(-5)M). The spontaneous outflow of NA from the vas deferens was unaffected by MCI-2016 (3 X 10(-6)M) and cocaine (10(-5)M), while the high-K-evoked release of NA was increased by both drugs. In the presence of cocaine (10(-5)M), the high-K-evoked release of NA was markedly increased by yohimbine (10(-5)M) and decreased by clonidine (3 X 10(-8)M), but only slightly increased by MCI-2016 (3 X 10(-6)M). 7 In phaeochromocytoma cells, both MCI-2016 and cocaine at concentrations of 10-7 to 10-5 M caused a dose-dependent inhibition of the [3H]-NA uptake. 8 These results suggest that MCI-2016-induced supersensitivity is specific for NA and is due to interference with the neuronal uptake process for NA.

Biphasic mechanical responses of the guinea-pig isolated ileum to the venom of the marine snail Conus striatus.

Venom extract of Conus striatus elicited a rhythmic, transient contraction of the guinea-pig isolated ileum followed by a relaxation at concentrations greater than 1 microgram/ml, which was abolished by tetrodotoxin and a low-Na medium. The contraction induced by the venom was inhibited by atropine but not mecamylamine, whereas the relaxation was not affected by bretylium, guanethidine or phentolamine. These results suggest that the contraction of the ileum induced by the venom is due to the excitation of cholinergic nerves, while the relaxation is mediated through non-adrenergic inhibitory nerves.

The mechanism of acidic pH-induced contraction in aortae from SHR and WKY rats enhanced by increasing blood pressure.

1. Effect of pH on vascular smooth muscle contraction was analyzed by use of biochemical and pharmacological techniques. 2. In the aorta isolated from spontaneously hypertensive rats (SHR) decreasing extracellular pH (pH0) caused a rapid acidification of intracellular pH accompanied by a pH0-dependent increase in tension. The contraction of the SHR aorta was remarkable compared with that of the Wistar Kyoto rat (WKY) aorta. 3. Removal of NH4Cl caused a transient decrease in intracellular pH followed by a marked increase in tension. 4. Both contraction and intracellular Ca2+ mobilization induced by acidic pH0 were markedly inhibited by removal of extracellular Ca2+, verapamil and adenosine, whereas these were not affected by tetrodotoxin or Gd3+, a stretch-activated cation channel blocker. Furthermore, cromakalim (a K+ channel opener) inhibited acidic pH0-induced contraction (APIC). 5. Acidic pH0 induced a depolarization of cultured smooth muscle cells from SHR aorta. 6. Blood pressure elevated with increasing age of WKY and SHR accompanied by an increase in APIC. Feeding WKY with NG-nitro-L-arginine, an inhibitor of nitric oxide synthases caused a marked elevation of their blood pressure followed by an increase in APIC. 7. These results suggest that APIC is caused by Ca2+ influx mediated through the activation of voltage-sensitive Ca2+ channels mainly due to acidic pH0-induced depolarization of the plasma membrane of smooth muscle cells. It is also suggested that APIC is strengthened by the elevation of blood pressure.

Excitatory effect of a new polypeptide (anthopleurin-B) from sea anemone on the guinea-pig vas deferens.

1 Anthopleurin-B (AP-B, greater than 3 x 10(-9) M), a newly isolated polypeptide from sea anemone (Anthopleura xanthogrammica), caused powerful rhythmic contractions in the guinea-pig isolated vas deferens. The other polypeptides anthopleurin-A from A. xanthogrammica and anthopleurin-C from A. elegantissima, elicited similar effects but in higher concentrations ( less than 5 x 10(-8) M). Toxin II (10(-6) M) isolated from the sea anemone, Anemonia sulcata, had no effect. 2 The rhythmic contractions induced by AP-B were inhibited by phentolamine, bretylium, guanethidine, reserpine, 6-hydroxydopamine, tetrodotoxin (TTX) and verapamil. Mecamylamine, atropine, methysergide, chlorpheniramine, and indomethacin had no effect. 3 AP-B (10(-8) M approximately 10(-5) M) caused a dose-dependent increase in the amount of endogenous noradrenaline (NA) released from the vas deferens. AP-B (10-5M) increased the amount of NA released to approximately 310 times (12 micrograms/g tissue) that of untreated tissues. 4 The AP-B-induced release of NA was inhibited or abolished by TTX, verapamil, or incubation in Ca-free medium. 5 These results suggest that the AP-B-induced rhythmic contraction of the vas deferens is mediated through the release of NA from adrenergic nerve endings; AP-B is one of the most potent substances in stimulating NA release from the vas deferens.

Maitotoxin-induced nerve growth factor production accompanied by the activation of a voltage-insensitive Ca2+ channel in C6-BU-1 glioma cells.

1. The aim of the present study was to determine the effects of maitotoxin on nerve growth factor production and the Ca2+ influx in clonal rat glioma cells (C6-BU-1). 2. Maitotoxin (1 - 10 ng ml-1) induced a profound increase in 45Ca2+ influx in an extracellular Ca2+-dependent manner. However, high KCl had no effect at all. These effects were supported by the results from the analysis of intracellular Ca2+ concentration using fura 2. 3. The maitotoxin-induced 45Ca2+ influx was inhibited by inorganic Ca2+ antagonists, such as Mg2+, Mn2+ and Co2+. The inhibitory effect of Co2+ was antagonized by increasing the extracellular Ca2+ concentrations. 4. Maitotoxin (3 ng ml-1) as well as A-23187 (1microM) and dibutyryl cyclic AMP (0.5 mM) caused an acceleration of nerve growth factor (NGF) production in C6-BU-1 cells, as determined by NGF enzyme immunoassay. 5. Reverse transcription polymerase chain reaction (RT - PCR) analysis showed that maitotoxin (10 ng ml-1) enhanced the expression of NGF mRNA, which was abolished by the removal of extracellular Ca2+. A-23187 also accelerated its expression. 6. These results suggest that maitotoxin activates a voltage-insensitive Ca2+ channel and accelerates NGF production mediated through a Ca2+ signalling pathway in C6-BU-1 glioma cells.