Participation of cystein proteinase in the high affinity ca2+-dependent binding of glutamate to hippocampal synaptic membranes

A dose of 25 μM Ca²⁺ causes half-maximal stimulation of high affinity binding (100 nM) of glutamate to crude synaptic membranes prepared from hippocampus. This facilitation is antagonized by leupeptin (IC₅₀ about 40 μg/ml), an inhibitor of Ca²⁺-dependent cystein proteinase (E.C. 3.4.22).     

Mechanisms of butylidenephthalide for twitch facilitation in electrically stimulated mouse vas deferens

Context: The rhizome of Ligusticum chuaxiong Hort. (Umbelliferae) has been used by Chinese for several thousand years. Its main constituent, butylidenephthalide (Bdph), was proved to be active in inhibiting rat uterine contractions induced by prostaglandin F_2α and was reported to be a nonspecific antispamodic and a blocker of voltage-dependent Ca²⁺ channels (VDCCs).

Objectives: The present study investigates the mechanisms of Bdph for twitch facilitation in ICR mouse vas deferens (MVD).

Materials and methods: Electrical field stimulation (EFS, supramaximal voltage ranging from 60–90 V, 1?ms, 0.2?Hz) was applied to the isolated MVD in Krebs solution. Interactions between Bdph (50 µM) and calcium antagonist (verapamil, diltiazem or aspaminol) on the EFS-evoked twitch responses were determined. The number of experiments was 3–18.

Results: Bdph (50 µM)-induced twitch facilitations from 100 to 391.9% were unrelated to activation of postjunctional cholinergic or adrenergic receptors. Verapamil and Bdph unabolished the twitch facilitation each other. Diltiazem unabolished the Bdph-induced twitch facilitation. In contrast, Bdph abolished those induced by diltiazem. Aspaminol at 20?μM abolished the Bdph-induced twitch facilitation. In contrast, Bdph abolished those induced by aspaminol. Tetraethylammonium and 4-aminopyridine, the K⁺ channel blockers, significantly augmented the Bdph-induced twitch facilitation.

Discussion and conclusions: Bdph may bind to the different, more and same subtypes of VDCCs from verapamil, than diltiazem, and as aspaminol does on prejunctional membrane, respectively. Besides a blocker of VDCCs, Bdph may be a blocker of K⁺ channels on prejunctional membrane. Thus, Bdph depolarized the membrane and facilitated the cumulative Ca²⁺-induced twitch responses.     

Investigation of the differential transport mechanism of cinnabar and mercury containing compounds

BackgroundCinnabar has a long history of uses in Chinese traditional medicines as an ingredient in various remedies. However, the detailed mechanism of cinnabar in medication remains unclear, and the toxicity of cinnabar has been a debate due to its containing mercury sulfide. This study was designed to investigate the differential transport mechanism of cinnabar and other Hg-containing compounds HgCl₂, MeHg and HgS, and to determine if organic anion transporters OAT1 and OAT3 were involved in the differential transport mechanism.Materials and methodsThe 293T cells were employed to investigate and compare the differential transport mechanism of cinnabar and HgCl₂, MeHg and HgS. Cells were incubated with a low dose (5 μM HgCl₂ and MeHg, 200 μM HgS and cinnabar), medium dose (10 μM HgCl₂ and MeHg, 400 μM HgS and cinnabar), and high dose (20 μM HgCl₂ and MeHg, 800 μM HgS and cinnabar) of HgCl₂, MeHg, HgS and cinnabar for 24 h. Following treatment, the cells were collected and the cell viability was determined by MTT assay. The intracellular mercury content was measured at 1, 4, and 24 h after treatment with 10 μM of the tested agents by an atomic fluorescence spectrophotometer. The effect of these tested agents on mitochondrial respiration was determined in a high-resolution oxygraphyat 24 h following treatment. Furthermore, the effect of modulation of expression of transporters OAT1 and OAT3 on the transport and cytotoxicity of the tested agents was evaluated. The up and down regulation of OAT1 and OAT3 were achieved by overexpression and siRNA transfection, respectively.ResultsCompared with HgCl₂ and MeHg, the cytotoxicity of cinnabar and HgS was lower, with cell viability at the high dose cinnabar and HgS being about 65%, while MeHg and HgCl₂ were 40% and 20%, respectively. The intracellular mercury accumulation was time-dependent. At 24 h the intracellular concentrations of HgCl₂ and MeHg were about 7 and 5 times higher, respectively, than that of cinnabar. No significant difference was found in the intracellular mercury content in cells treated with cinnabar compared to HgS. The knockdown and overexpression of the transporter OAT1 resulted in significant reduction and increase, respectively, in mercury accumulation in HgCl₂ -treated cells in relative to control cells, while no significant changes were observed in cells treated with cinnabar, MeHg, and HgS. In addition, the knockdown and overexpression of the transporter OAT3 caused significant reduction and increase, respectively, in mercury accumulation in both HgCl₂ and MeHg-treated cells in relative to control cells, while no significant changes were observed in cells treated with cinnabar and HgS. Furthermore, it was found that cells transfected with siOAT1 caused significant resistance to the cytotoxicity induced by HgCl₂, while no noticeable changes in cell viability were observed in cells treated with other tested agents. Additionally, cells transfected with OAT3 did not change cell sensitivity to cytotoxicity induced by all of the four tested agents.ConclusionThis study demonstrates that differential transport and accumulation of mercury in 293T cells exists among cinnabar and the three mercury-containing compounds HgCl₂, MeHg and HgS, leading to distinct sensitivity to mercury induced cytotoxicity. The kidney organic anion transporters OAT1 and OAT3 are partially involved in the regulation of the transport of HgCl₂ and MeHg, but not in the regulation of the transport of cinnabar.     

Effects of androctonus crassicauda scorpion venom on endothelium-dependent and -independent vascular responses of rabbit aorta

The effects of Androctonus crassicauda scorpion venom on acetycholine (ACh)-induced relaxations and contractions of rabbit thoracic aorta were studied. Endothelium-dependent relaxations induced by ACh in phenylephrine-precontracted arteries were enhanced by the scorpion venom. ACh-induced contractions in endothelium-intact open aortic rings were less than those obtained in denuded preparations (n=6, P<0.05, ANOVA). Venom (5, 10 and 30 μg/ml) potentiated ACh.induced contractions in intact and denuded segments. In the denuded segments, this potentiation was inhibited by indomethacin (10 μM). Thromboxane synthase inhibitor, BW 149H (100 μM) and thromboxane A₂ (TXA₂) receptor antagonist, R 68070 (10 μM) partly inhibited venom-induced potentiation. N^G-nitro-L-arginine (100 μM) increased venom-potentiated ACh responses in intact arterial segments. Venom increased the basal tone by 25–35% at 30 μg/ml. These results suggest that A. crassicauda venom may release a relaxing factor from endothelium and contracting factor from the smooth muscle of rabbit isolated thoracic aorta. The contracting factor may be a cyclooxygenase-like product, most likely TXA₂. The increase in basal tone by 30 μg/ml venom was inhibited by phentolamine (10 μM) and guanethidine (10 μM), indicating a venom-induced release of a neurotransmitter from adrenergic nerve endings.     

Effects of imidazoline derivatives on cholinergic motility in guinea-pig ileum: involvement of presynaptic α2-adrenoceptors or imidazoline receptors?

The present study investigates the possibility that imidazoline receptors mediate modulation of cholinergic motor functions of the guinea-pig ileum. For this purpose, the effects of a series of compounds with known affinity for α₂-adrenoceptors and/or imidazoline recognition sites were examined on the cholinergic twitch contractions evoked by electrical field stimulation (0.1 Hz) of longitudinal muscle-myenteric plexus preparations. Additional experiments were carried out on ileal strips preincubated with [³H]choline, superfused with physiological salt solution containing hemicholinium-3, and subjected to electrical field stimulation (1 Hz). The stimulation-induced outflow of radioactivity was taken as an index of endogenous acetylcholine release. α-Methyl-noradrenaline, noradrenaline, clonidine, medetomidine, oxymetazoline and xylazine caused a concentration-dependent inhibition of twitch responses (IC₅₀ from 0.13 to 1.05 μM; E_max from 85.9 to 92.5%). Rilmenidine and agmatine were less potent in reducing the twitch activity, and the latter compound acted also with low intrinsic activity (IC₅₀=44.9 μM; E_max=35.5%). In interaction experiments, the inhibitory action of clonidine on twitch responses was competitively antagonized by RX 821002 (2-(2-methoxy-1,4-benzodioxan-2-yl)-2-imidazoline), idazoxan, rauwolscine, yohimbine and BRL 44408 (2-[2H-(1-methyl-1,3-dihydroisoindole)-methyl]-4,5-dihydroimidazoline), whereas prazosin (10 μM), ARC 239 (2-(2,4-(O-methoxy-phenyl)-piperazin-1-yl)-ethyl-4,4-dimethyl-1,3-(2H,4H)-isoquinolindione; 10 μM) and BRL 41992 (1,2-dimethyl-2,3,9,13b-tetrahydro-1H-dibenzo[c,f]imidazol[1,5-a]azepi-ne; 10 μM) were without effect. Rauwolscine antagonized the inhibitory effects of various agonists on ileal twitch activity in a competitive manner and with similar potency. Agmatine and idazoxan did not significantly modify the twitch contractions when tested in the presence of α₂-adrenoceptor blockade by rauwolscine (3 μM) or RX 821002 (1 μM). Linear regression analysis showed that the affinity values of antagonists correlated with their affinity at the α_2A and α_2D binding sites as well as at previously classified α_2A/D adrenoceptor subtypes, whereas no significant correlation was obtained when comparing the potency estimates of agonists and antagonists with the affinity at I₁ or I₂ binding sites. When tested on the electrically induced outflow of tritium, α-methyl-noradrenaline, noradrenaline, clonidine, medetomidine, oxymetazoline, xylazine and rilmenidine yielded inhibitory concentration-response curves which were shifted rightward to a similar extent in the presence of rauwolscine (3 μM). In the absence of further drugs, agmatine significantly reduced the evoked tritium outflow at the highest concentrations tested (10 and 100 μM), whereas idazoxan (up to 100 μM) was without effect. When RX 821002 (1 μM) was added to the superfusion medium, neither agmatine nor idazoxan modified the evoked outflow of radioactivity. The results argue against modulation by imidazoline receptors of acetylcholine release from myenteric plexus nerve terminals. They provide evidence that compounds endowed with imidazoline-like structures affect the cholinergic motor activity of the guinea-pig ileum by interacting with presynaptic α₂-adrenoceptors belonging to the α_2D subtype. Received: 10 October 1997 / Accepted: 14 March 1998     

Modulating effects of opioids,purine compounds, 5-hydroxytryptamine and prostaglandin E2 on cholinergic neurotransmission in a guinea-pig oesophagus preparation

The submucous plexus-longitudinal muscularis mucosae preparation of the guinea-pig oesophagus was used to study the actions of morphine, opioid peptides, purine compounds, 5-hydroxytryptamine (5-HT) and prostaglandin E₂ (PGE₂) on electrically-induced twitch contractions which are probably mediated by cholinergic nerve stimulation. The twitch contractions were inhibited by morphine (1–100 μM), methionine-enkephalin (1–100 μM) and β-endorphin (0.1–1 μM), but increased by adenosine (1–30 μM), adenosine 5′-triphosphate (1–30 μM), 5-HT (0.01–3 μM) and PGE² (1–10 nM). The submaximal contraction induced by acetylcholine (12 or 20 nM) which is nearly equivalent to the twitch contractions was unaffected by morphine, methionine-enkephalin, β-endorphin and 5-HT, but augmented by purine compounds and PGE₂. It is concluded that cholinergic neurotransmission in the submucous plexus-longitudinal muscularis mucosae of the guinea-pig oesophagus is inhibited by morphine and opioid peptides acting at prejunctional opiate receptors, and facilitated by 5-HT, purine compounds and PGE₂ via prejunctional or postjunctional mechanisms.     

Effects of ω-conotoxin GVIA on electrical field stimulation- and agonist-induced changes in cytosolic Ca2+ and tension in isolated rat anococcygeus muscle

1 It has been reported that ω -conotoxin GVIA (ω-CgTx) blocks L- and N-type voltage-sensitive Ca²⁺ channels (VSCCs) in neurones and inhibits neurotransmitter release in various tissues. The present study investigates the effects of ω-CgTx on electrical field stimulation (EFS)- and agonist-induced changes in free cytosolic Ca²⁺ ([Ca²⁺]_cyt) levels and tension in isolated fura-2 loaded rat anococcygeus muscle. 2 EFS produced frequency-dependent increases in [Ca⁺]_cyt levels and contractions. Phentolamine (1 μM) and ω-CgTx (0.1 μM) significantly inhibited EFS-induced responses and shifted the frequency-response curves to the right. 3 α-adrenoceptor agonists (noradrenaline and clonidine) and carbachol (in the presence of phentolamine) produced concentration-dependent increases in [Ca²⁺]_cyt levels and contractions. Though ω-CgTx (0.1 μM) significantly inhibited the increases in [Ca²⁺]_cyt levels induced by low doses of noradrenaline, the overall concentration–response curves of [Ca²⁺]_cyt and contractions for noradrenaline, clonidine, and carbachol were not affected by ω-CgTx. 4 When the tone of rat anococcygeus muscle was raised with either clonidine (0.1 μM) or carbachol (30 μM, in the presence of 3 μM phentolamine), EFS (2 Hz) produced reproducible decreases in [Ca²⁺]_cyt levels and relaxations. These responses were significantly inhibited by ω-CgTx when the tissue was precontracted with clonidine, but not when it was precontracted with carbachol. 5 The results of the present study suggest that in rat anococcygeus muscle, ω-CgTx inhibits the EFS-induced release of both excitatory and inhibitory neurotransmitters, probably by blocking Ca²⁺ channels on nerve terminals. Furthermore, the Ca²⁺ channels present on the smooth muscle cell membrane, which are involved in the agonist-induced Ca²⁺ influx and contractions, may not be sensitive to ω-CgTx.     

Effects of methoxamine and barium on 45Ca2+ fluxes in the rat vas deferens

The aim of this study was to determine whether methoxamine and barium stimulate ⁴⁵Ca²⁺ uptake or efflux in the rat vas deferens in a manner that correlates with their contractile activity, and whether ⁴⁵Ca²⁺ movements are inhibited by verapamil or nifedipine. Basal La³⁺-resistant (cellular) ⁴⁵Ca²⁺ uptake was significantly greater in the epididymal half (791 ± 27 nmol g⁻¹) than in the prostatic half (654 ± 14 nmol g⁻¹) of the rat vas deferens and was unaffected by verapamil (61 μM) or nifedipine (14 μM). Methoxamine (8 μM) was without effect on ⁴⁵Ca²⁺ uptake in either half but BaCl₂ (1 mM) increased ⁴⁵Ca²⁺ uptake by 31% in the prostatic half and by 22% in the epididymal half. The barium-induced increases in ⁴⁵Ca²⁺ uptake were markedly reduced or abolished by verapamil (2 μM) or nifedipine (0.3 μM), which at these concentrations have no effect on the rhythmic contractions but abolish the initial small phasic contraction induced by barium. The basal rate of ⁴⁵Ca²⁺ efflux from the intact vas deferens (into Ca²⁺ containing Krebs-Henseleit solution or into Ca-free Krebs-Henseleit solution ± EGTA 0.05 mM) was not affected by verapamil (61 μM) or nifedipine (14 μM). Methoxamine (8 μM) produced a marked, transient and reversible increase in ⁴⁵Ca²⁺ efflux into 2.5 mM CaCl₂ Krebs-Henseleit in 50% of the intact vasa deferentia examined which was augmented by verapamil (61 μM). BaCl₂ (1 mM) produced a small increase in ⁴⁵Ca²⁺ efflux into Ca²⁺-containing and Ca²⁺-free Krebs-Henseleit solutions from some intact vasa deferentia and this was not inhibited by nifedipine (14 μM). It is concluded that the BaCl₂-induced increase in ⁴⁵Ca²⁺ uptake is likely to be associated with the initial phase of the contractile response, rather than the secondary rhythmic contractions. Rhythmic contractions initiated by methoxamine and barium may be initiated by ‘trigger Ca’ entering through a population of calcium channels that have low affinity for calcium antagonist drugs.     

Effects of Ro 5-4864 and PK 11195 in rat duodenum and vas deferens

Ro 5-4864 and PK 11195 inhibit in a concentration-dependent manner carbachol-induced contractions in rat duodenum (IC₅₀: 1.56 ± 0.07 × 10⁻⁵ M and 1.18 ± 0.07 × 10⁻⁵ M respectively). The antagonism is non-competitive and is not mediated by peripheral-type benzodiazepine receptors. The Ro 5-4864 effect is modulated by the calcium concentration of the Tyrode-Ringer solution. In the presence of 1 mM NaF/10 μM AlCl₃, Ro 5-4864 and PK 11195 do not inhibit carbachol-induced contractions. Moreover, Ro 5-4864 and PK 11195 significantly relax AlF₄⁻-induced contractions, with IC₅₀ values of 2.01±0.12×10⁻⁵ M and 1.28 ± 0.11 × 10⁻⁵ M respectively. This effect is also modulated by the calcium concentration of the medium. Pertussis toxin potentiates the antagonist effects of Ro 5-4864 and PK 11195 on carbachol-induced contractions, but cholera toxin does not affect them. Ro 5-4864 and PK 11195 inhibit ⁴⁵Ca²⁺ uptake induced by KCl (120 mM) in rat vas deferens, but do not affect either basal ⁴⁵Ca efflux or noradrenaline-induced ⁴⁵Ca²⁺ efflux. Only high doses of PK 11195 (above 5 × 10⁻⁵ M) are able to produce a slight reduction of the accumulation of inositol phosphates induced by methoxamine in rat vas deferens, while Ro 5-4864 has no significant effect. Finally, Ro 5-4864 and PK 11195 reduce calcium influx, but do not seen to be the only mechanism of the antagonistic effect on carbachol-induced contractions. An alteration of other second messengers. probably cyclic monophosphate nucleotides, may be involved.     

Stereoselectivity of Butylidenephthalide on Voltage-dependent Calcium Channels in Guinea-pig Isolated Ileum

Two geometric isomers, the Z- and the E- forms, can be separated from synthetic mixtures of butylidenephthalide (Bdph). Z-Bdph (50–100 μm ) non-competitively inhibited Ca²⁺-induced contractions in depolarized (K⁺, 60 mm ) guinea-pig ileum longitudinal smooth muscle, with a pD₂' value of 3.88 ± 0.20 (n = 5). However, E-Bdph (20–100 μm ) competitively inhibited these contractions with a pA₂ value of 4.56 ± 0.18 (n = 5) which was significantly (P < 0.05) greater than the pD₂' value of Z-Bdph. In contrast, the two isomers had no stereoselective inhibitory action on Ca²⁺ influx through pre- or post-junctional membranes of cholinergic nerve endings from which the transmitter acetylcholine is released or on Ca²⁺ release from intracellular stores. Therefore, the trans-Z and cis-E forms of Bdph might have geometric stereoselectivity for voltage-dependent calcium channels (VDC) in guinea-pig longitudinal smooth muscle. Both isomers might inhibit more selectively the contractile twitch responses evoked by electrical stimulation than by cumulative acetylcholine-or carbachol-induced transient contractions in guinea-pig ileum longitudinal smooth muscle.     

Effects of Alkylxanthines on Contractility of Diaphragm Fibres Isolated from Normal and Sensitized Guinea-pigs

Abstract— This study investigates the effects of alkylxanthines on twitch tension generated by electrical stimulation (supramaximal pulses, 0·2 ms duration, 1 Hz) of diaphragm muscle fibres isolated from normal and actively-sensitized guinea-pigs. Caffeine, theophylline and theobromine increased, in a concentration-dependent manner (50–500 μm ), twitch tension in normal and sensitized diaphragm. Caffeine (500 μm ) enhanced contractility to a greater extent than theophylline or theobromine. Twitch potentiation by caffeine (500 μm ) was significantly greater in sensitized diaphragm. Verapamil (0·1–100 μm ) did not alter twitch contractions in the absence or presence of alkylxanthines in normal or sensitized strips. Dantrolene (0·01–100 μm ) depressed, in a concentration-dependent fashion, twitch contractions of normal and sensitized diaphragm. The inhibitory concentration 50% (expressed as —log IC50) was 6·78 ± 0·13 in normal tissues and 6·15 ± 0·11 in sensitized tissues (n = 6 in each group; P < 0·05). Exposure to Ca²⁺-free, EGTA (0·1 Mm )-containing medium, depressed twitch contraction of normal diaphragm to a lesser extent than that of sensitized diaphragm. Methylxanthines reversed depression of twitch contractions produced by exposure to dantrolene (5 μm ) or a Ca²⁺ -free medium. Adenosine (1–1000 μm ) was without effect whereas enprofylline (50–500 μm ) enhanced diaphragm contractility in normal tissues. This indicates that blockade of adenosine receptors is not involved in the inotropic effect of alkylxanthines in guinea-pig diaphragm. Results from this study suggest that alkylxanthines enhance diaphragm contractility in the guinea-pig by releasing intracellular Ca²⁺ and promoting extracellular Ca²⁺ entry through verapamil-insensitive pathways. An alteration of Ca²⁺ movements and stores may be present in the sensitized diaphragm.     

Neomycin blocks dihydropyridine-insensitive Ca2+ influx in bovine adrenal chromaffin cells

There is evidence that bovine adrenal chromaffin cells are provided with both dihydropyridine-sensitive and -resistant voltage-sensitive Ca²⁺ influx pathways. Although recent electrophysiological work indicates that the dihydropyridine-resistant pathway is partially mediated by w-conotoxin-sensitive and -insensitive Ca²⁺ channels, the pharmacological sensitivity of the latter channels remains elusive. We have now found that combined incubations with nitrendipine (1 μM) and neomycin (0.5 mM) reduced high K⁺ (50 mM)-evoked intracellular Ca²⁺ concentration ([Ca²⁺]_i) transients to a larger extent than each drug separately. [Ca²⁺]_i was measured using the fluorescent intracellular Ca²⁺ indicator fura-2. Neomycin (0.05−2 mM) reduced high K⁺-evoked ⁴⁵Ca²⁺ uptake in a dose-dependent manner (IC₅₀ = 0.09 mM). In the presence of nitrendipine (1 μM), the minimal neomycin concentration necessary for total blockade of ⁴⁵Ca²⁺ uptake was reduced to 0.3 mM. Moreover, in the absence of nitrendipine the ⁴⁵Ca²⁺ uptake remaining in 0.3 mM neomycin (26% of maximum) was similar to the fractional inhibition by nitrendipine alone (29%). Neomycin (0.05−2 mM) inhibited the [Ca²⁺]_i transient induced by the L-type Ca²⁺ channel agonist Bay K 8644 (1 μM) much more extensively at 2 mM than at 0.3 mM (percent inhibition = 59% and 15%, respectively). Neomycin (0.05−2 mM) blocked high K⁺-evoked noradrenaline and adrenaline release in a dose-dependent fashion (IC₅₀ = 0.8−1.1 mM), the blockade efficiency being enhanced in the presence of 1 μM nitrendipine (IC₅₀ = 0.17−0.19 mM). It is concluded that neomycin (≤ 0.3 mM) blocks preferentially the dihydropyridine-insensitive Ca²⁺ influx pathway of the chromaffin cell. Moreover, both the dihydropyridine-sensitive and the dihydropyridine-resistant, neomycin-sensitive Ca²⁺ influx pathways contribute strongly to depolarization-evoked catecholamine secretion.     

Effects of Terbutaline on α-Adrenergic Responses and Ca2+ Influx in Isolated Rabbit Aorta

Effects of terbutaline applied in vivo or in vitro on α-adrenergic receptors in the rabbit aorta in normal and Ca²⁺–free solution, and on basal, high potassium-, and phenylephrine-stimulated Ca²⁺ uptake into aorta were investigated. Three day terbutaline administration (25 mg/kg, subcutaneously three times daily) to rabbits increased the pK_B for phentolamine in aorta rings (control 7.3 + 0.2, n = 9; terbutaline 7.8 +0.2, n = 15). It also depressed phenylephrine-stimulated contractions of aorta rings in Ca²⁺–free but not those in normal Krebs solution. It did not significantly depress the basal, or phenylephrine-evoked Ca²⁺ influx into aorta rings, but decreased high potassium-induced Ca²⁺–influx (control 0.58 + 0.05 umoles/g aorta; n = 3, terbutaline 0.41 +0.06 umoles/g aorta, n = 3). In vitro application of 50 μM terbutaline did not significantly alter phenylephrine-stimulated contractions of aorta rings in Ca²⁺–free Krebs solution or significantly depress basal or phenylephrine-induced Ca²⁺ influx into aortas, but did decrease high potassium-stimulated Ca²⁺–influx. Thus, 3-day terbutaline administration increased the affinity of α-adrenergic receptors for phentolamine and had a tendency to increase contractions of aorta rings to phenylephrine. It also decreased high potassium-stimulated Ca²⁺ influx, and depressed phenylephrine-induced contractions in Ca²⁺–free Krebs solution, while in vitro terbutaline application also decreased potassium-induced Ca²⁺ influx.     

In vitro vasodilator mechanisms of the indole alkaloids rhynchophylline and isorhynchophylline,isolated from the hook of<Emphasis Type="Italic"> Uncaria rhynchophylla</Emphasis> (Miquel)

Rhynchophylline (Rhy) and isorhynchophylline (Isorhy), indole alkaloids from Uncaria hooks, reportedly exert hypotensive and vasodilatory effects, but the mechanism of action is unclear. We therefore investigated the relaxant effects of these two isomeric alkaloids in rat arteries in vitro, in particular in respect of the various functional Ca²⁺ pathways. Both Rhy and Isorhy relaxed aortic rings precontracted with phenylephrine (PE, 1 µM) in a dose-dependent manner (3–300 µM). Removal of endothelium and preincubation with L-NAME (300 µM) slightly inhibited but did not prevent the relaxant response. These results indicate that Rhy and Isorhy act largely in an endothelium-independent manner. Unlike nicardipine, both alkaloids not only inhibited the contraction induced by 60 mM KCl (IC₅₀ 20–30 µM), but also that induced by PE and U46619, albeit to a lesser extent (IC₅₀ 100 and 200 µM, respectively). These results suggest that Rhy and Isorhy may act via multiple Ca²⁺ pathways. In contrast to their inhibitory effects on KCl-induced and receptor-mediated contractions, where both isomers were comparably potent, Rhy was more potent than Isorhy at higher concentrations (>100 µM) in inhibiting both caffeine (25 mM)- and cyclopiazonic acid (CPA, 30 µM)-induced contractions. Similar results observed with caffeine in Ca²⁺-containing medium were also observed in Ca²⁺-free medium. However, 0.1–0.3 µM nicardipine (which completely inhibited KCl-induced contraction) had no significant inhibitory effect on CPA-induced contractions. Taken together, these results indicate discrimination between these two isomers with respect to Ca²⁺-induced Ca²⁺ release and non-L-type Ca²⁺ channel, but not for IP₃-induced Ca²⁺ release and L-type Ca²⁺ channels. Similar relaxant responses to KCl- and caffeine-induced contractions were seen when these two alkaloids were tested on the smaller mesenteric and renal arteries. In conclusion, the vasodilatory effects of Rhy and Isorhy are largely endothelium independent and are mediated by L-type Ca²⁺ channels. At higher concentrations, they also affect other Ca²⁺-handling pathways, although to a lesser extent. While there is no discrimination between the two isomers with respect to the contraction induced by KCl or agonists (PE and U46619), differential effects between Rhy and Isorhy were seen on caffeine- and CPA-induced contractions.     

Influence of oligopeptide aldehydes on intracellular Ca2+ concentration in rat pituitary cells

We investigated the effects of some synthetic tripeptide aldehydes, earlier shown to influence pituitary hormone secretion and ⁴⁵Ca²⁺ uptake, on the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) of rat anterior pituitary cells in suspension. Boc-D-Phen- Leu-Phenylalaninal or Boc-D-Phe-Leu-Prolinal in the tested range of 1–100 or 200 μM, respectively, were ineffective in influencing basal [Ca²⁺]_i but caused a concentration-dependent inhibition in K⁺ (25 mM)-induced [Ca²⁺]_i elevation. The IC₅₀ of both effects was about 50 μM. In contrast, they did not interfere with the stimulation caused by the calcium channel agonist BAY K 8644 and were also ineffective in influencing the receptor-mediated stimulus of thyrotropin-releasing hormone on [Ca²⁺]_i. On the basis of the present and foregoing results the possible involvement of calcium channels is discussed, but different mechanisms mediating the tripeptide aldehyde inhibition are also considered. A third tripeptide aldehyde. Boc-Gln-Leu-Lysinal (Boc-GLL), showed ionophore-like properties. This nontoxic substance caused a dose-dependent rise up to 400% (at 100 μM) in [Ca²⁺]_i. Its effect is not mediated by voltage-dependent calcium channels, as it cannot be inhibited either by the classicalp calcium channel antagonists verapamil and nifedipine, or by the above-mentioned inhibitory tripeptide aldehydes. When we decreased the extracellular Ca²⁺ concentration by the addition of 4 mM EGTA, the effect was inverted and Boc-GLL caused a large fall in [Ca²⁺]_i. We suggest that Boc-GLL may open cell membrane pores through which Ca²⁺ moves along the concentration gradient. The calcium flux can be inhibited by 20 mM Mg²⁺ and 100 μM Co²⁺ but not by 500 μM La³⁺. Thus, tripeptide aldehydes. depending on their structure, may decrease or increase [Ca²⁺]_i via uncoventional mechanisms and may serve as tools for dissecting details of cell calcium homeostasis.     

Capsaicin inhibits catecholamine secretion and synthesis by blocking Na+ and Ca2+ influx through a vanilloid receptor-independent pathway in bovine adrenal medullary cells

We report here the effects of capsaicin, a flavoring ingredient in the hot pepper Capsicum family, on catecholamine secretion and synthesis in cultured bovine adrenal medullary cells. Capsaicin inhibited catecholamine secretion (IC₅₀=9.5, 11.8, and 62 μM) stimulated by carbachol, an agonist of the nicotinic acetylcholine receptor, by veratridine, an activator of voltage-dependent Na⁺ channels, and by high K⁺, an activator of voltage-dependent Ca²⁺ channels, respectively. Capsaicin also suppressed carbachol-induced ²²Na⁺ influx (IC₅₀=5.0 μM) and ⁴⁵Ca²⁺ influx (IC₅₀=24.4 μM), veratridine-induced ²²Na⁺ influx (IC₅₀=2.4 μM) and ⁴⁵Ca²⁺ influx (IC₅₀=1.1 μM), and high K⁺-induced ⁴⁵Ca²⁺ influx (IC₅₀=5.8 μM). The reduction in catecholamine secretion caused by capsaicin was not overcome by increasing the concentration of carbachol. Furthermore, capsazepine (10 μM), a competitive antagonist for the transient receptor potential vanilloid 1, and ruthenium red (30 μM), a nonselective cation channel antagonist, did not block the inhibition by capsaicin of catecholamine secretion. Capsaicin also suppressed both basal and carbachol-stimulated ¹⁴C-catecholamine synthesis (IC₅₀=10.6 and 26.4 μM, respectively) from [¹⁴C] tyrosine but not from l-3, 4-dihydroxyphenyl [3-¹⁴C] alanine ([¹⁴C] DOPA) as well as tyrosine hydroxylase activity (IC₅₀=8.4 and 39.0 μM, respectively). The present findings suggest that capsaicin inhibits catecholamine secretion and synthesis via suppression of Na⁺ and Ca²⁺ influx through a vanilloid receptor-independent pathway.     

Effects of GS 386, a novel dihydroisoquinoline compound,on rabbit atrial myocytes and rat aorta

The effects of GS 386, a novel benzylisoquinoline derivative, on Ca²⁺ current and vasodilatation were investigated using the single rabbit atrial myocyte and rat thoracic aorta preparations. In rabbit myocytes, the Ca²⁺ current was recorded during various depolarizations for 200 ms from a holding potential of ?40 mV using the whole patch-clamp technique. Superfusion of GS 386 led to a reduction of the Ca²⁺ current amplitude concentration-dependently, in which the IC₅₀ value was 0.25 μM. However, the dependence of the Ca²⁺ current on the membrane potential was not altered by GS 386. In rat aorta, GS 386 inhibited high K⁺-induced contractions more strongly than that induced by phenylephrine. These results indicate that GS 386 has Ca²⁺ antagonistic actions in rat aorta and rabbit heart. © 1994 Wiley-Liss, Inc.     

Facilitation and inhibition by capsaicin of cholinergic neurotransmission in the guinea-pig small intestine

The effects of capsaicin on [³H]acetylcholine release and muscle contraction were studied on the myenteric plexus-longitudinal muscle preparation of the guinea-pig ileum preincubated with [³H]choline. Capsaicin concentration-dependently increased both basal [³H]acetylcholine release (pEC₅₀ 7.0) and muscle tone (pEC₅₀ 6.1). The facilitatory effects of capsaicin were antagonized by 1 μM capsazepine (pK _B 7.0 and 7.6), and by the combined blockade of NK₁ and NK₃ tachykinin receptors with the antagonists CP99994 plus SR142801 (each 0.1 μM). This suggests that stimulation by capsaicin of TRPV1 receptors on primary afferent fibres causes a release of tachykinins which, in turn, mediate via NK₁ and NK₃ receptors an increase in acetylcholine release. The capsaicin-induced acetylcholine release was significantly enhanced by the NO synthase inhibitor L-N^G-nitroarginine (100 μM). This indicates that tachykinins released from sensory neurons also stimulate nitrergic neurons and thus lead, via NO release, to inhibition of acetylcholine release. Capsaicin concentration-dependently reduced the electrically-evoked [³H]acetylcholine release (pEC₅₀ 6.4) and twitch contractions (pEC₅₀ 5.9). The inhibitory effects were not affected by either capsazepine, NK₁ and NK₃ receptor antagonists, the cannabinoid CB₁ antagonist SR141716A or by L-N^G-nitroarginine. Desensitization of TRPV1 receptors by a short exposure to 3 μM capsaicin abolished the facilitatory responses to a subsequent administration, but did not modify the inhibitory effects. In summary, capsaicin has a dual effect on cholinergic neurotransmission. The facilitatory effect is indirect and involves tachykinin release and excitation of NK₁ and NK₃ receptors on cholinergic neurons. The inhibition of acetylcholine release may be due to a decrease of Ca²⁺ influx into cholinergic neurons.     

Calcium Entry Blockade as a Mechanism for Chlordimeform-Induced Inhibition of Motor Activity in the Isolated Guinea-Pig Ileum

Abstract: Central and peripheral α₂-adrenoceptors, including those of the gastrointestinal tract, have been indicated as a toxicity target of formamidine pesticides in mammals. In this study, the inhibitory effect of chlordimeform on twitch contractions from electrically-stimulated longitudinal muscle-myenteric plexus preparations (LMMPs) of the guinea-pig ileum was found to be resistant to the action of the α₂-adrenoceptor antagonist idazoxan. This drug was also ineffective on chlordimeform-induced inhibition of peristalsis recorded in whole ileal segments. As expected, idazoxan antagonized the inhibitory effect of the α₂-adrenoceptor agonist clonidine on twitch contractions and peristaltic activity. Chlordimeform reduced the amplitude of direct mechanical responses to a variety of spasmogens such as acetylcholine, histamine and substance P, suggesting a muscular site of action. Moreover, in Ca²⁺-free, K⁺-depolarized LMMPs, chlordimeform inhibited submaximal contractions caused by addition of exogenous calcium, through an action apparently similar to that of the Ca²⁺ entry blocker nifedipine. Both chlordimeform- and nifedipine-induced inhibition of calcium contractions were reversed by the calcium channel activator BAY K 8644. This compound also partially prevented the inhibitory action of chlordimeform on peristaltic activity. On the whole, these results indicate that chlordimeform-induced depression of motor activity in the guinea-pig ileum is, at least in part, related to inhibition of transmembrane Ca²⁺ fluxes responsible for smooth muscle contraction.     

Genotoxicity of inorganic mercury salts based on disturbed microtubule function

This study investigated the hypothesis that the chromosomal genotoxicity of inorganic mercury results from interaction(s) with cytoskeletal proteins. Effects of Hg²⁺ salts on functional activities of tubulin and kinesin were investigated by determining tubulin assembly and kinesin-driven motility in cell-free systems. Hg²⁺ inhibits microtubule assembly at concentrations above 1 µM, and inhibition is complete at about 10 µM. In this range, the tubulin assembly is fully (up to 6 µM) or partially (~6–10 µM) reversible. The inhibition of tubulin assembly by mercury is independent of the anion, chloride or nitrate. The no-observed-effect-concentration for inhibition of microtubule assembly in vitro was 1 µM Hg²⁺, the IC₅₀ 5.8 M. Mercury(II) salts at the IC₅₀ concentrations partly inhibiting tubulin assembly did not cause the formation of aberrant microtubule structures. Effects of mercury salts on the functionality of the microtubule motility apparatus were studied with the motor protein kinesin. By using a gliding assay mimicking intracellular movement and transport processes in vitro, HgCl₂ affected the gliding velocity of paclitaxel-stabilised microtubules in a clear dose-dependent manner. An apparent effect is detected at a concentration of 0.1 µM and a complete inhibition is reached at 1 M. Cytotoxicity of mercury chloride was studied in V79 cells using neutral red uptake, showing an influence above 17 µM HgCl₂. Between 15 and 20 µM HgCl₂ there was a steep increase in cell toxicity. Both mercury chloride and mercury nitrate induced micronuclei concentration-dependently, starting at concentrations above 0.01 µM. CREST analyses on micronuclei formation in V79 cells demonstrated both clastogenic (CREST-negative) and aneugenic effects of Hg²⁺, with some preponderance of aneugenicity. A morphological effect of high Hg²⁺ concentrations (100 µM HgCl₂) on the microtubule cytoskeleton was verified in V79 cells by immuno-fluorescence staining. The overall data are consistent with the concept that the chromosomal genotoxicity could be due to interaction of Hg²⁺ with the motor protein kinesin mediating cellular transport processes. Interactions of Hg²⁺ with the tubulin shown by in vitro investigations could also partly influence intracellular microtubule functions leading, together with the effects on the kinesin, to an impaired chromosome distribution as shown by the micronucleus test.