Effects of dapiprazole on contractile responses of guinea pig isolated ileum

The effects of dapiprazole, a relatively new alpha 1-adrenolytic agent, on contractile responses and on spontaneous mechanical activity were studied in guinea pig isolated ileum. Dapiprazole (10(-10) to 10(-4) M) produced a concentration-dependent inhibition of high K+ (80 mM) -induced contractions. These inhibitory effects were observed with dapiprazole added either before or after the induced contractions. The Ca2+-induced contractions of K+-depolarized ileum were also inhibited by dapiprazole. Dapiprazole inhibited in a non competitive manner the responses of the ileum to: carbachol, histamine, 5-hydroxytryptamine, pentagastrin, angiotensin II and cholecystokinin. In order to analize whether dapiprazole exerts an intracellular effect on Ca2+-store, skinned preparations were used. The results suggest that dapiprazole might inhibit Ca2+ entry through both voltage-and receptor-operated channels of the smooth muscle membrane.     

Mechanism of action of angiotensin II in human isolated subcutaneous resistance arteries

1. Human isolated subcutaneous arteries were mounted in a myograph and isometric tension measured. In some experiments, intracellular calcium [Ca(2+)]i was also measured using fura-2. 2. Angiotensin II (100 pM - 1 microM) increased [Ca(2+)]i and tone in a concentration-dependent manner. The effects of angiotensin II (100 nM) were inhibited by an AT1-receptor antagonist, candesartan (100 pM). 3. Ryanodine (10 microM), had no effect on angiotensin II-induced responses, but removal of extracellular Ca(2+) abolished angiotensin II-induced rise in [Ca(2+)]i and tone. Inhibition of Ca(2+) entry by Ni(2+) (2 mM), also inhibited angiotensin II responses. The dihydropyridine, L-type calcium channel antagonist, amlodipine (10 microM), only partially attenuated angiotensin II responses. 4. Inhibition of protein kinase C (PKC) by chelerythrine (1 microM), or by overnight exposure to a phorbol ester (PDBu; 500 nM) had no effect on angiotensin II-induced contraction. 5. Genistein (10 microM), a tyrosine kinase inhibitor, inhibited angiotensin II-induced contraction, but did not inhibit the rise in [Ca(2+)]i, suggesting that at this concentration it affected the calcium sensitivity of the contractile apparatus. Genistein did not affect responses to norepinephrine (NE) or high potassium (KPSS). 6. A selective MEK inhibitor, PD98059 (30 microM), inhibited both the angiotensin II-induced contraction and rise in [Ca(2+)]i, but had no effect on responses to NE or KPSS. 7. AT1 activation causes Ca(2+) influx via L-type calcium channels and a dihydropyridine-insensitive route, but does not release Ca(2+) from intracellular sites. Activation of tyrosine kinase(s) and the ERK 1/2 pathway, but not classical or novel PKC, also play a role in angiotensin II-induced contraction in human subcutaneous resistance arteries.     

Muscarinic M1- and M2-receptors mediating opposite effects on neuromuscular transmission in rabbit vas deferens

Twitch contractions of the rabbit vas deferens elicited by electrical field stimulation were inhibited by tetrodotoxin, guanethidine, bretylium and alpha,beta-methylene-ATP but were unaffected by hexamethonium, physostigmine, 1,1-dimethyl-4-phenylpiperazinium and prazosin, suggesting that they resulted from ATP released following postganglionic sympathetic nerve stimulation. McN-A-343 inhibited but carbachol and several other muscarinic agonists potentiated the twitch contractions; these effects were not modified by hexamethonium or physostigmine. Muscarinic agonists had no effect on the tension in unstimulated organs whereas contractions elicited by ATP, noradrenaline and KCl were potentiated by carbachol but remained unaffected by McN-A-343. The responses of the twitch contractions to McN-A-343 and carbachol were inhibited to different degrees by antimuscarinic drugs: the affinity (pA2) of atropine, secoverine and himbacine against McN-A-343 and carbachol was similar. However, pirenzepine, telenzepine, trihexyphenidyl, dicyclomine and hexahydro-sila-difenidol displayed preferential antagonism of the responses to McN-A-343 whereas the converse was true for AF-DX 116 and gallamine. The highly significant correlation between the pA2 values obtained for 10 antagonists against carbachol responses in rabbit vas deferens and rat left atrium suggests that the receptors may be similar. The data support the presence of a presynaptic M1-receptor mediating inhibition and a postsynaptic, cardiac-like M2-receptor responsible for enhancing neurogenic contractions in rabbit vas deferens.     

Differential calcium movements induced by agonists in guinea pig tracheal muscle

The effects of high potassium, carbachol and histamine on tension responses and 45Ca fluxes in tracheal smooth muscle were examined. Calcium depletion or nitrendipine (10(-8) M) inhibited potassium-induced contractile responses more than those obtained with either histamine or carbachol, whereas Sr2+ inhibited mainly responses to histamine or carbachol. The Ca2+ entry facilitator, CGP 28392 (3 X 10(-6) M), potentiated contractions induced only by potassium. Uptake of 45Ca in guinea pig tracheal muscle can be separated into high and low affinity components. The 45Ca efflux rate from tracheal muscle into a La3+-substituted solution was over four-fold higher than in other smooth muscles. Potassium, carbachol and histamine induced sustained increases in 45Ca efflux into solutions containing 1.5 mM Ca2+; only transient increases in 45Ca efflux with carbachol and histamine were obtained after Ca2+ depletion. These agonists elicit contractile responses in tracheal muscle by selectively mobilizing different cellular and extracellular Ca2+ components.     

Contribution of Ca2+ influx to carbachol-induced detrusor contraction is different in human urinary bladder compared to pig and mouse

Carbachol-induced detrusor contractions are mainly mediated via M3 receptor subtype and depend not only on Ca2+ release from the intracellular calcium stores but also on Ca2+ influx via L-type Ca2+ channels. The purpose of this study was to examine the different contributions of Ca2+ influx and Ca2+ release underlying muscarinic receptor-mediated contractions in human, porcine and murine urinary bladder. Detrusor contractions were measured in urothelium-denuded detrusor strips as responses to cumulatively increasing carbachol concentrations, release of intracellular Ca2+ was determined in Chinese hamster ovary cells stably transfected with human muscarinic M3 (hM3) receptors. In human tissue, 1 microM of the L-type Ca2+-channel blocker nifedipine reduced carbachol contractions to 74%, in pig to 18% and in mouse to 27% of pre-drug controls. 2-aminoethoxyphenyl borate (2-APB, 300 microM), which impairs inositol trisphosphate (IP3)-induced release of Ca2+, reduced carbachol responses in human detrusor to 60%, in pig to 35% and in mouse to 20%, whereas block of the Ca2+-induced Ca2+ release with ryanodine had no significant effect on carbachol contractions in all three species. Carbachol-induced release of intracellular Ca2+ in Chinese hamster ovary cells expressing muscarinic hM3 receptors was completely prevented by 100 microM 2-APB. The direct intracellular IP3 receptor antagonist xestospongin C (10 microM) reduced carbachol-stimulated intracellular Ca2+ to 41% of the control value. Blockade of ATP-dependent Ca2+ uptake into intracellular stores with thapsigargin was associated with a concentration-dependent increase of detrusor contraction, but limited on-top contractions with carbachol. In conclusion, carbachol-induced contractions in human, porcine and mouse detrusor depend differently on Ca2+ influx, since potency of nifedipine reducing muscarinic receptor-mediated detrusor contraction is lower in human bladder. On the other hand, slight species differences are also found when inhibiting IP3-induced Ca2+ release and Ca2+ reuptake into intracellular stores. Taken together, our data show considerable species differences between human, porcine and murine detrusor regarding the relative contributions of Ca2+ influx and maybe also carbachol-induced Ca2+ release that could be of relevance when using different animal models.     

Postjunctional alpha 1- and beta-adrenoceptor effects of noradrenaline on electrical slow waves and phasic contractions of cat colon circular muscle.

1. The postjunctional excitatory and inhibitory effects of noradrenaline and selective alpha 1- and beta-adrenoceptor agonists on electrical and mechanical activity of cat colon muscle strips were studied by microelectrode recordings and isometric force measurements. Experiments were performed in the presence of tetrodotoxin (0.5 microM) or atropine (0.5 microM). 2. Circular muscle cells near the submucosal border had a mean resting membrane potential of -76.1 +/- 1.2 mV and exhibited electrical slow waves at frequencies of 4-6 cycles min-1. The mean values of electrical slow wave components were: upstroke potential, -40.7 +/- 1.2 mV; plateau potential, -43.7 +/- 0.8 mV; and duration, 4.9 +/- 0.4 s. Electrical slow waves were in phase with rhythmic contractions of the circular muscle layer. Muscle cells near the myenteric border had a mean testing membrane potential of -51.1 +/- 5.5 mV and did not exhibit electrical slow waves. 3. Noradrenaline (1 microM) increased the duration of electrical slow waves. This effect was inhibited by prazosin (1 microM) and potentiated by propranolol (5 microM), indicating activation of alpha 1- and beta-adrenoceptors. Also, when alpha 1-adrenoceptors were irreversibly blocked by phenoxybenzamine (1 microM), noradrenaline decreased the duration of electrical slow waves. Phenylephrine (1 microM), a selective alpha 1-adrenoceptor agonist, and isoprenaline (1 microM), a beta-adrenoceptor agonist, increased or decreased the duration of electrical slow waves, respectively. 4. Phenylephrine (0.01-5 microM) caused a linear increase in the area of electrical slow waves and phasic contractions but did not affect resting membrane potential or resting muscle tension. Higher concentrations of phenylephrine (5-50 microM) depolarized the resting membrane potential (2-6 mV) and increased muscle tone. 5. Nitrendipine or verapamil (each at 5 microM) reduced the amplitude of the upstroke potential and nearly abolished the plateau phase of the electrical slow waves. In the presence of L-type Ca2+ antagonists, noradrenaline (1-10 microM) or phenylephrine (1-100 microM) had no effect on electrical slow waves and phasic contractions. This indicates that the effects of noradrenaline and phenylephrine involve the influx of extracellular Ca2+ through voltage-dependent L-type Ca2+ channels. 6. Ryanodine, an alkaloid that depletes intracellular Ca2+ stores nearly abolished phasic contractions. In muscle strips, pretreated with ryanodine (10 microM for 30 min), phenylephrine (1 microM) increased and isoprenaline (1 microM) decreased the duration of electrical slow waves but neither was able to reverse the ryanodine-suppressed phasic contractions. This suggests that adrenoceptor effects on electrical slow waves are coupled to contractions via Ca2+ release from ryanodine-sensitive intracellular stores. 7. In summary, noradrenaline activates postjunctional alpha 1- and beta-adrenoceptors. Activation of alpha 1-adrenoceptors increases the magnitude of electrical slow waves and phasic contractions, whereas activation of beta-adrenoceptors decreases them. The alpha 1-adrenoceptor mediated effects on electrical slow waves and phasic contractions require the influx of Ca2+ through voltage-gated L-type Ca2+ channels. Phasic contractions also involve Ca2+ release from ryanodine-sensitive intracellular stores.     

Receptor-responses in fresh human ciliary muscle.

The physiological and pharmacological properties of ciliary muscle isolated from fresh human eyes were investigated. The muscle exhibited no spontaneous activity. Concentration-dependent contractions in response to carbachol were competitively antagonized by atropine (pA2 = 8.95). The muscle, precontracted by carbachol (2.7 X 10(-4)M), responded to the application of isoprenaline by concentration-dependent relaxation blocked by propranolol (3.5 X 10(-9)M to 3.5 X 10(-8)M; pA2 = 9.15). Angiotensin-evoked contractions were antagonized by 8-Ala-angiotensin II (4.5 X 10(-8)M) in a competitive manner, but were not inhibited by phentolamine or propranolol. Contractions generated by electrical stimulation of the muscle (30 ms, 20 Hz, 60 pulses) were antagonized by atropine (10(-7) M) and tetrodotoxin (6.3 X 10(-7) M). Phentolamine and propranolol did not influence these responses. An increase of the external potassium concentration ([K+]o) from 5.4 to 158.8 mM produced a mechanical response, antagonized by atropine, but not influenced by tetrodotoxin, phentolamine or propranolol. The human ciliary muscle appears to carry muscarinic and angiotensin receptors and beta 2-adrenoceptors. The estimate of Katropine for muscarinic receptors mediating carbachol-induced contractions agrees with estimates of Katropine reported for human and rabbit iris.     

Cholinergic inositol phosphate formation in striatal neurons is mediated by distinct mechanisms

In murine striatal neurons devoid of functional synapses (6 days in vitro) the cholinergic agonists carbachol and arecoline evoked dose-dependent inositol phosphate (InsP) responses with mean log EC50s of -4.1 +/- 0.5 and -4.48 +/- 0.1, respectively. Carbachol (1 mM) and arecoline (1 mM) responses were insensitive to tetrodotoxin, a voltage-sensitive Na+ channel blocker, and were blocked by pirenzepine with relatively low affinity (logIC50 = -5.9 +/- 0.3 for the carbachol response and logIC50 = -5.8 +/- 0.3 for the arecoline response). After synaptogenesis (13 days in vitro) the maximal carbachol effect doubled whereas the arecoline response remained unchanged. This additional effect was sensitive to tetrodotoxin and the voltage-dependent Ca2+ channel blocker, omega-conotoxin. The tetrodotoxin-sensitive carbachol response was blocked by lower concentrations of pirenzepine than the tetrodotoxin-insensitive carbachol response. More than 75% of the InsP response evoked by low concentrations of muscarine (1 and 10 microM) was sensitive to tetrodotoxin whereas only 38% of the InsP response stimulated by 1 mM of muscarine could be blocked by tetrodotoxin. These results suggest that there are at least two different mechanisms (depending on the stage of development), activated most probably by two different muscarinic receptors responsible for the carbachol-induced InsP formation in striatal neurons.     

Modification by charybdotoxin and apamin of spontaneous electrical and mechanical activity of the circular smooth muscle of the guinea-pig stomach.

1. The effects of charybdotoxin and apamin, putative blockers of Ca(2+)-activated K+ channels, on spontaneous electrical and mechanical activity of circular smooth muscle of the guinea-pig stomach antrum were examined in the presence of 1 microM tetrodotoxin and 1 microM atropine. 2. Both charybdotoxin (> 3 nM) and apamin (> 3 nM) dose-dependently increased the amplitude of spontaneous contractions without altering their frequency. The maximum effect of charybdotoxin was much greater than that of apamin. Both toxins increased the amplitude of intracellular Ca2+ oscillations measured with fura-2. 3. When the extracellular Ca2+ concentration was lowered to 1.5 mM or less, apamin did not significantly potentiate the contractions whereas charybdotoxin still potentiated them but with less potency. 4. Charybdotoxin (30 nM) increased the amplitude of spikes and slow waves, and slightly decreased the resting membrane potential. On the other hand, apamin (100 nM) preferentially increased the slow wave amplitude with no effect on the resting membrane potential. 5. These results suggest that both toxins affect the spontaneous contraction by modifying the electrical activity and that charybdotoxin-sensitive K+ channels and apamin-sensitive ones are differently involved in the spontaneous electrical activity.     

Role of Rho-kinase in guinea-pig gallbladder smooth muscle contraction

Guinea-pig gallbladder smooth muscle contractions can be elicited pharmacologically by a range of mechanisms. The involvement of Rho-kinase in contractions mediated by receptor-dependent and receptor-independent mechanisms was investigated using the Rho-kinase inhibitor (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexane carboxamide (Y-27632). In a separate series of experiments, the role of Rho-kinase in the contractile response to Ca2+ entry through store-operated Ca2+ channels and to electrical field stimulation was also examined. Y-27632 (10 microM), which caused a significant decrease (P<0.0005) in basal resting tone, significantly inhibited gallbladder contractions evoked by cumulative additions of the G-protein-coupled agonists, carbachol (1 nM-100 microM; P<0.05) and cholecystokinin (10 nM-1 microM; P<0.005). Y-27632 also inhibited the contractions evoked by a single addition of the sarcoplasmic reticulum ATPase inhibitor, thapsigargin (1 microM; P<0.0005) and cumulative additions of KCl (10-85 mM; P<0.0005). The contractile response to Ca2+ entry through store-operated Ca2+ channels was significantly inhibited by Y-27632 (P<0.05) as were the contractile responses evoked by electrical field stimulation (2-25 Hz; P<0.0005). In contrast, Y-27632 had no significant effect on contractions evoked by phorbol 12,13-dibutyrate (0.1 nM-1 microM; a protein kinase C activator) or by the phosphatase inhibitor, cantharidin (100 microM). In conclusion, Rho-kinase contributes to the contractile response in guinea-pig gallbladder smooth muscle evoked by both G-protein-coupled and non-G-protein-coupled mechanisms in addition to contributing to the maintenance of basal tone. It also contributes to the contractile responses resulting from electrical field stimulation and store-operated Ca2+ channel entry.     

Angiotensin II type 1 receptor-mediated increase in cytosolic Ca(2+) and proliferation in mesothelial cells

We investigated the Ca(2+) signaling pathways of the response to angiotensin II in pleural mesothelial cells and the role of these Ca(2+) signaling pathways in mesothelial cell proliferation. Rat pleural mesothelial cells were maintained in vitro, and the Ca(2+) movement to angiotensin II was evaluated using the fluorescent Ca(2+) indicator fura 2. Furthermore, proliferation of mesothelial cells was assessed using a spectrophotometric 3-(4, 5-dimethylthazol-2-yl)-2,5-diphenyl-2H-tetrasodium bromide (MTT) assay. Angiotensin II (1 pM-100 microM) induced in mesothelial cells a biphasic elevation of intracellular Ca(2+) concentration ([Ca(2+)](i)) that consisted of a transient initial component, followed by a sustained component. Neither removal of extracellular Ca(2+) nor inhibition of Ca(2+) influx by 1 microM nifedipine affected the angiotensin II-induced initial transient elevation of [Ca(2+)](i) in mesothelial cells. Nifedipine did not block angiotensin II-induced sustained elevation of [Ca(2+)](i). Angiotensin II (1 pM-100 microM) had a proliferative effect on mesothelial cells in a dose-dependent manner. Angiotensin II type 1 (AT(1)) receptor antagonist ([Sar(1), Ile(8)]angiotensin II) inhibited both angiotensin II-induced elevation of [Ca(2+)](i) and proliferation of mesothelial cells. Pertussis toxin did not affect angiotensin II-induced responses. These results suggest that angiotensin II-induced responses to mesothelial cells are extremely dependent on the angiotensin AT(1) receptor coupled with pertussis toxin-insensitive G protein.     

Resveratrol prevents bradykinin-induced contraction of rat urinary bladders by decreasing prostaglandin production and calcium influx

Resveratrol, a polyphenol found in grapes and peanuts, exerts beneficial effects on a number of diseases of cardiovascular and central nervous system. However, effects of resveratrol on the urinary system have not been fully investigated. In the present study, we examined effects of resveratrol on bradykinin-induced contraction and release of prostaglandin E2 in isolated rat urinary bladders. The effects of resveratrol on contractions induced by several agonists (prostaglandin E2, prostaglandin F2α and carbachol) and high K+ were also examined. We found that resveratrol concentration-dependently reduced the bradykinin-induced contraction in the rat urinary bladder preparations. The higher concentration of resveratrol (100 μM) abolished the bradykinin-induced prostaglandin E2 release. Similar results were obtained when the cyclooxygenase inhibitor indomethacin (10 μM) was used instead of resveratrol. Resveratrol also attenuated the prostaglandin E2-, prostaglandin F2α-, and to a lesser extent carbachol-induced contractions. Contractile responses to bradykinin, prostaglandin E2 and carbachol were largely prevented by blockade of Ca2+ channels with diltiazem. Both resveratrol and diltiazem prevented contractions induced by an addition of Ca2+ (2.5- 10 mM) into Ca2+-free/50 mMK+ solution or by 50 mMK+ solution containing normal Ca2+ (2.5 mM). These results suggest that resveratrol prevents bradykinin-induced contractions by attenuating not only the production of prostaglandins but also actions of them. The effect of resveratrol on contractile actions seems to be in part due to inhibition of Ca2+ influx. Because bradykinin plays an important role in pathological conditions of urinary bladder function, resveratrol may exert beneficial effects on the urinary bladder diseases.     

Mechanism of relaxing action of the antiasthmatic drug, azelastine, in isolated porcine tracheal smooth muscle.

Azelastine (1-300 microM) inhibited contractions of isolated porcine trachea induced by high K+, carbachol and endothelin-1 (ET-1) with a decrease in [Ca2+]cyt (as measured by fura-2-fluorescence). Verapamil (0.1-10 microM) also inhibited the high K(+)-induced increases in [Ca2+]cyt and contraction, although it only partially inhibited the responses evoked by carbachol or ET-1. In the absence of extracellular Ca2+ (with 0.5 mM EGTA), carbachol induced a transient increase in [Ca2+]cyt and force by releasing Ca2+ from cellular stores. Azelastine (100 microns) completely inhibited these contransient changes. In the absence of extracellular Ca2+, carbachol and 12-deoxyphorbol 13-isobutyrate (DPB) induced small sustained contractions without increasing [Ca2+]cyt. Azelastine inhibited these contractions. In muscle permeabilized with alpha-toxin, Ca2+ (0.3-3 microM) induced contraction in a concentration-dependent manner. DPB (without GTP) and carbachol or ET-1 (with GTP) enhanced the Ca(2+)-induced contraction. Azelastine partially inhibited the contraction induced by 0.3 microM Ca2+ but not the contraction induced by 3 microM Ca2+, and strongly inhibited the potentiating effects of DPB, carbachol and ET-1. Azelastine had no effect on the content of cyclic AMP or cyclic GMP. These results suggest that azelastine inhibits smooth muscle contraction by (i) decreasing [Ca2+]cyt, by inhibition of Ca2+ channels, (ii) decreasing agonist-induced Ca2+ release, and (iii) direct inhibition of contractile elements.     

Desensitization of depolarization-mediated contractile pathways does not necessarily regulate receptor-mediated excitation-contraction coupling in longitudinal smooth muscle of guinea pig ileum

1. Activation of G(q) protein-coupled receptors, such as muscarinic M(3) and histamine H(1) receptors, induces smooth muscle contraction through activation of voltage-dependent Ca channels. 2. To evaluate roles of depolarization-mediated contractile pathways in the desensitization of receptor-mediated contraction, we compared the development of carbachol-induced desensitization to receptor agonists, carbachol and histamine, and to receptor-bypassed stimulation of voltage-dependent Ca channels with depolarizing high K in longitudinal smooth muscle of guinea pig ileum. 3. Under Ca-containing physiological conditions, pretreatment with 10(-4) mol/L carbachol for 15 s-30 min induced desensitization to carbachol as well as to high K, whereas contractile responses to histamine remained normal. 4. In contrast, under Ca-free conditions containing 0.2 mmol/L EGTA, carbachol pretreatment induced desensitization to high K in a manner similar to that induced under Ca-containing physiological conditions, whereas contractile responses to carbachol and histamine remained normal. 5. Thus, it was shown that contractile responses to carbachol and histamine were not necessarily desensitized, even under conditions where contractile responses to high K were desensitized. These results suggest that desensitization of depolarization-mediated contractile pathways might not necessarily regulate excitation-contraction coupling through muscarinic M(3) and histamine H(1) receptors in longitudinal smooth muscle of guinea pig ileum.     

Caffeine inhibits a low affinity but not a high affinity mechanism for cholecystokinin-evoked Ca2+ signalling and amylase release from guinea pig pancreatic acini

Caffeine has been found to inhibit the formation and action of Ca2+-mobilizing inositol 1,4,5-trisphosphate (IP3) in pancreatic acinar cells. The aim of the present study was to investigate the effects of caffeine on cytoplasmic Ca2+ concentrations ([Ca2+]i) and amylase release in response to different agonists. [Ca2+]i was determined by cytofluorometry using fura-2 as indicator and amylase release with a substrate reagent. Stimulation with low concentrations of carbachol or cholecystokinin octapeptide (CCK-8) induces [Ca2+]i oscillations whereas higher concentrations cause sustained elevation of [Ca2+]i. The less efficacious agonists pilocarpine and CCK-JMV-180 evoke oscillations only. Caffeine inhibited carbachol-induced elevation of [Ca2+]i and amylase responses in a competitive manner, abolishing the responses to low and incompletely inhibiting the responses to high concentrations of the agonist. Also, the [Ca2+]i elevations by pilocarpine were abolished by caffeine. The effects on CCK-8-induced elevation of [Ca2+]i and amylase secretion were paradoxical, the caffeine inhibition being more pronounced at high than at low concentrations of CCK-8. This enigma was further emphasized by moderate effects of caffeine on the responses to CCK-JMV-180. The results indicate that carbachol, pilocarpine and high concentrations of CCK-8 elicit IP3-mediated responses and that CCK-JMV-180 and low concentrations of CCK-8 elevate [Ca2+]i and stimulate amylase release by another signal transduction mechanism.     

Effects of the calcium antagonists diltiazem, verapamil and nitrendipine on the contractile responses of guinea-pig isolated ileum to electrical stimulation or carbachol.

The effects of the organic Ca2+ antagonists nitrendipine, verapamil and diltiazem on the cholinergic contractile responses induced by field electrical stimulation or carbachol (0.1 microM) and on contractions evoked by high concentration KCl (30 mM) were studied in isolated preparations from the guinea-pig ileum. The three Ca2+ antagonists dose-dependently suppressed the contractile responses showing the same order of potency (nitrendipine greater than verapamil greater than diltiazem) with the three different types of stimulation. Comparison of the IC50 values of the Ca2+ antagonists for carbachol-, KCl- and electrically-evoked contractions demonstrated that the carbachol-evoked contractions were most sensitive to the inhibitory action of the antagonists tested. The presynaptic inhibitory effect of (Met)enkephalin (10 nM) on the electrically-evoked cholinergic contractions was only slightly potentiated by high concentrations (1 or 10 microM) of nitrendipine and diltiazem and remained unchanged by verapamil. The results suggest that the Ca2+ antagonists tested block mainly the carbachol-activated L-type Ca2+ channels on the smooth muscle cells, while the effects on the N-type Ca2+ channels are insignificant, except for the high concentrations of nitrendipine and diltiazem.     

Inhibition of neuronal nicotinic acetylcholine receptors by La(3+)

We investigated the role of angiotensin II type 1 (AT(1)) receptors in angiotensin II-induced actin reorganization and the signaling pathways of the response in pleural mesothelial cells. The effects of angiotensin II on actin reorganization in pleural mesothelial cells were evaluated by dual fluorescence labeling of filamentous (F) and monomeric (G) actin with fluorescein isothiocyanate (FITC)-labeled phalloidin and Texas Red-labeled DNase I, respectively. Angiotensin II (10 microM) induced actin reorganization in the presence and the absence of extracellular Ca(2+). An angiotensin AT(1) receptor antagonist ([Sar(1),Ile(8)]angiotensin II) inhibited angiotensin II-induced actin reorganization. Pretreatment with C3 exoenzyme or tyrosine kinase inhibitors significantly reduced angiotensin II-induced actin reorganization. However, pertussis toxin, phosphatidylinositol-3-kinase and protein kinase C inhibitors had no effect on these responses. These results suggest that angiotensin II-induced actin reorganization in pleural mesothelial cells is extremely dependent on the angiotensin AT(1) receptor coupled with pertussis toxin-insensitive heterotrimeric G proteins, Rho GTPases and tyrosine phosphorylation pathways.     

Inhibition of neuromuscular transmission in the myenteric plexus of guinea-pig ileum by omega-conotoxins GVIA, MVIIA, MVIIC and SVIB.

1. The effects of a number of Ca2+ channel blockers on the transmural electrical stimulation or receptor agonist-elicited contractile responses of guinea-pig ileum were compared. 2. omega-Conotoxins (MVIIA, GVIA, SVIB and MVIIC), but not omega-agatoxin IVA, completely blocked the twitch responses evoked by low frequency (0.1 Hz) transmural stimulation without inhibition of the contractures evoked by exogenous acetylcholine. The concentration-inhibition curves were shifted by changes of external Ca2+. 3. The tetanic contractures produced by a high frequency (30 Hz) train of stimulation were inhibited by omega-conotoxins by only 25-30%, except for omega-conotoxin MVIIC, which produced about 55% inhibition, all significantly less than that produced by atropine (about 70%) or tetrodotoxin (about 85%). Combinations of omega-conotoxins did not produce additive inhibitory effects. 4. The four omega-conotoxins as well as atropine produced similar partial inhibition (53-62%) of the contractures evoked by dimethylphenylpiperazinium, while tetrodotoxin inhibited the contracture completely. 5. Nifedipine and Ni2+ depressed the nerve stimulation-evoked twitch response and tetanic contracture as well as acetylcholine contracture. 6. These observations suggest that, in the myenteric plexus, a subset of N-type Ca2+ channel dominates under low frequency stimulation, while high frequency stimulation may recruit additional channels and non-cholinergic pathways.     

Blockade of T-type voltage-dependent Ca2+ channels by benidipine, a dihydropyridine calcium channel blocker, inhibits aldosterone production in human adrenocortical cell line NCI-H295R

Benidipine, a long-lasting dihydropyridine calcium channel blocker, is used for treatment of hypertension and angina. Benidipine exerts pleiotropic pharmacological features, such as renoprotective and cardioprotective effects. In pathophysiological conditions, the antidiuretic hormone aldosterone causes development of renal and cardiovascular diseases. In adrenal glomerulosa cells, aldosterone is produced in response to extracellular potassium, which is mainly mediated by T-type voltage-dependent Ca2+ channels. More recently, it has been demonstrated that benidipine inhibits T-type Ca2+ channels in addition to L-type Ca2+ channels. Therefore, effect of calcium channel blockers, including benidipine, on aldosterone production and T-type Ca2+ channels using human adrenocortical cell line NCI-H295R was investigated. Benidipine efficiently inhibited KCl-induced aldosterone production at low concentration (3 and 10 nM), with inhibitory activity more potent than other calcium channel blockers. Patch clamp analysis indicated that benidipine concentration-dependently inhibited T-type Ca2+ currents at 10, 100 and 1000 nM. As for examined calcium channel blockers, inhibitory activity for T-type Ca2+ currents was well correlated with aldosterone production. L-type specific calcium channel blockers calciseptine and nifedipine showed no effect in both assays. These results indicate that inhibition of T-type Ca2+ channels is responsible for inhibition of aldosterone production in NCI-H295R cells. Benidipine efficiently inhibited KCl-induced upregulation of 11-beta-hydroxylase mRNA and aldosterone synthase mRNA as well as KCl-induced Ca2+ influx, indicating it as the most likely inhibition mechanism. Benidipine partially inhibited angiotensin II-induced aldosterone production, plus showed additive effects when used in combination with the angiotensin II type I receptor blocker valsartan. Benidipine also partially inhibited angiotensin II-induced upregulation of the above mRNAs and Ca2+ influx inhibitory activities of benidipine for aldosterone production. T-type Ca2+ channels may contribute to additional benefits of this drug for treating renal and cardiovascular diseases, beyond its primary anti-hypertensive effects from blocking L-type Ca2+ channels.     

Angiotensin II-mediated cellular responses: a role for the 3'-untranslated region of the angiotensin AT1 receptor

We have previously demonstrated that Chinese hamster ovary (CHO) cells transfected with the angiotensin II AT1 receptor gene containing only the coding region, presented tachyphylaxis to the total inositol phosphate (InsPs) and Ca2+ responses mediated by angiotensin II and [2-lysine]angiotensin II ([Lys2]angiotensin II). Now we have evaluated the possible role of the 3'-untranslated region of the angiotensin AT1 receptor mRNA in modulating the angiotensin AT1 receptor-mediated cellular responses. The binding parameters, as well as the Ca2+ and InsPs responses induced by angiotensin II and [Lys2]angiotensin II were similar in cells transfected with the angiotensin AT1 receptor with or without the 3'-untranslated region sequence. In cells transfected with the receptor containing the 3'-untranslated region sequence, angiotensin II-induced Ca2+ and InsPs responses were desensitized by repeated stimulations, whereas [Lys2]angiotensin II caused desensitization of InsPs production but not of Ca2+ uptake in these cells. Our results suggest that the 3'-untranslated region plays a role in modulating cell signalling involved in the tachyphylaxis of angiotensin AT1 receptor-mediated Ca2+ responses.