Mechanisms of vasorelaxation induced by N-allylsecoboldine in rat thoracic aorta

N-Allylsecoboldine was shown to be the most effective of several boldine derivatives that were tested for their vasorelaxing effect on the rat aorta. In KCl (60 mmol/l) medium, Ca²⁺ (0.03–3 mmol/l)-induced vasoconstriction was inhibited, concentration-dependently, by N-allylsecoboldine. The IC₅₀ for N-allylsecoboldine was calculated to be about 4 mol/l (for a Ca²⁺ concentration of 1 mmol/1). The vasorelaxant effect on KCl-induced responses was more pronounced at 60 mmol/l KCl than at 15 mmol/1 KCI. Contraction of rat aorta in response to phenylephrine (0.01-100 mol/l) was concentration-dependently inhibited by N-allylsecoboldine and by verapamil (3–30 mol/l), while contraction in response to B-HT 920, serotonin or PGF₂ was not affected. This relaxing effect of N-allylsecoboldine persisted in endothelium-denuded aorta. In cultured A 10 vascular smooth muscle cells, N-allylsecoboldine and verapamil displaced the binding of [³H]-prazosin (K _i values = 0.4±0.2 and 0.6±0.2 mol/l, respectively). The increase of inositol monophosphate caused by phenylephrine in rat aorta was completely suppressed by chloroethylclonidine, but only slightly inhibited by N-allylsecoboldine and by verapamil. Glibenclamide or charybdotoxin did not affect the relaxation induced by N-allylsecoboldine of aortic rings precontracted with phenylephrine. Neither the cGMP nor the cAMP content was changed by N-allylsecoboldine. We conclude that N-allylsecoboldine relaxes the rat aorta by blocking Ca²⁺ channels and that it also has an antagonistic effect at ₁-adrenoceptors. Correspondence to: C.M. Teng at the above address     

Direct inhibition of chicken gizzard smooth muscle contractile apparatus by caffeine

Summary The mechanism of the inhibitory effect of caffeine on smooth muscle contraction was examined using chicken gizzard. Caffeine (0.1–5 mmol/l) inhibited the KCl-induced contraction of the muscle with an IC₅₀ of 1.1 mmol/l. Forskolin (0.01–10 mol/l) also inhibited KCl-induced contraction. The inhibitory effect of caffeine was potentiated by a low concentration of forskolin (0.3 mol/l) and the inhibitory effect of forskolin was potentiated by a low concentration of caffeine (0.1 mmol/l). Although caffeine and forskolin increased tissue cyclic AMP levels, caffeine inhibited the KCl-induced contraction more strongly than forskolin at a given cyclic AMP level. Caffeine (1–40 mmol/l) inhibited the contractions induced by 3 mol/l Ca²⁺ in Triton X-100-permeabilized muscle. Caffeine (1–40 mmol/l) inhibited the phosphorylation of 20 kDa myosin light chain (MLC) in native actomyosin preparation and the inhibition was enhanced by decreasing the ATP concentration in the reaction medium. Calmodulin (CaM) activity, as monitored by Ca²⁺/CaM-dependent erythrocyte membrane (Ca²⁺+Mg²⁺)-ATPase, was not affected by 20 mmol/l caffeine. Time-dependent dephosphorylation of MLC upon removal of Ca²⁺, an indicator of phosphate activity, was not affected by caffeine. Caffeine also inhibited the Ca²⁺-independent contraction in thiophosphorylated permeabilized muscle. These results indicate that caffeine inhibits smooth muscle contraction by a direct inhibition of MLC kinase and actinmyosin interaction. A part of the inhibitory effect may be mediated by cyclic AMP-dependent mechanism(s). Send offprint requests to H. Karaki at the above address     

The relaxant effects of cromakalim (BRL 34915) on human isolated airway smooth muscle

Summary Cromakalim (BRL 34915) is a potassium channel opener with therapeutic potential as a bronchodilator in asthma. Cromakalim (0.1–30 mol/l) inhibited the spontaneous tone of human isolated bronchi n a concentration-related manner being nearly as effective as isoprenaline or theophylline. The order of relaxant potencies (expressed as -log₁₀ IC₅₀ mol/l; mean ±SEM) was isoprenaline (7.29 ± 0.27; n = 8) > cromakalim (5.89 ± 0.12; n = 7) > theophylline (4.07 ±0.13; n = 10). In human bronchi where tone had been raised by addition of histamine (0.1 mmol/l), acetylcholine (0.1 mmol/l) or leukotriene D₄ (LTD₄, 0.1 mol/l), the relaxant effect of cromakalim was substantially reduced. Cromakalim suppressed the contraction produced by KCI (25 mmol/l) but not that produced by KCl (120 mmol/l). Tetraethylammonium (8 mmol/l) was without effect against the relaxant action of cromakalimbut procaine (0.5 – 5 mmol/l) and glibenclamide (0.3 mol/l) antagonised it. Cromakalim (10 mol/l) produced an upward displacement of concentration-effect curves forKCI (1–100 mmol/l), acetylcholine (1 nmol/l-1 mmol/) and histamine (1 nmol/l-1 mmol/l) but it did not alter the concentration-effect curve for LTD₄ (0.1 nmol/l-0.1 mol/l). When tissues were challenged in the presence of cromakalim (10 mol/l) with KCI (100 mmol/l), acetylcholine (1 mmol/l) or histamine (1 mmol/l), an enhanced contraction was observed compared to control tissues. This enhancement by cromakalim was absent when tissues were challenged with acetylcholine or histamine in either a Ca²⁺-free medium (plus EGTA 0.1 mmol/l) or in the presence of verapamil (10 mol/l). It is concluded that cromakalim is an effective relaxant of human airway smooth muscle in vitro and this activity may depend on the opening of K⁺ channels in the plasma membrane of smooth muscle cells but other actions cannot be ruled out. Correspondence to: J. Cortijo at the above address     

Vasorelaxing effect in rat thoracic aorta caused by fraxinellone and dictamine isolated from the Chinese herb Dictamnus dasycarpus Turcz: comparison with cromakalim and Ca2+ channel blockers

Summary The components of Dictamnus dasycarpus Turcz were tested for their vasorelaxing effect on the rat aorta, and fraxinellone and dictamine were shown to be effective vasorelaxants. In high K⁺ (60 mmol/l) medium, Ca²⁺ (0.03 to 3 mmol/l)-induced vasoconstriction was inhibited concentration-dependently by both agents. The IC₅₀ for fraxinellone and dictamine were calculated to be about 25 mol/l and 15 mol/l (for Ca²⁺) concentration of (1 mmol/l), respectively. Cromakalim (0.2–10) mol/l relaxed aortic rings precontracted with 15 but not 60 mmol/l of K⁺. Fraxinellone and verapamil were more potent and effective in producing relaxation in 60 mmol/l than in 15 mmol/l K⁺-induced contraction. However, dictamine was more potent in producing relaxation in 5 mmol/l K⁺-induced contraction. Nifedipine (1 mol/l), dictamine (100 mol/l) and fraxinellone (100 mol/l) relaxed the aortic contraction caused by KCl or Bay K 8644. The tonic contraction elicited by nor adrenaline (NA, 3 mol/l) was also relaxed by dictamine (500 mol/l), but not by fraxinellone (500 mol/l) in the nifedipine (1 mol/l)-treated aorta. This relaxing effect of dictamine persisted in endothelium-denuded aorta. Glibenclamide (10 mol/l) shifted the concentration-relaxation curve of cromakalim, but not that of dictamine, to the right in rat aortic rings precontracted with NA. Dictamine (500 mol/l) did not affect tonic contraction of NA which are reduced by H-7 (1 mol/l) in Ca²⁺ depleted medium. In conclusion, fraxinellone is a selective blocker of voltage-dependent Ca²⁺ channel, while dictamine relaxed the rat aorta by suppressing the Ca²⁺ influx through both voltage-dependent and receptor-operated Ca²⁺ channels.This work was supported by a research grant from the Nationat Science Council of the Republic of China (NSC80-0420-B002-18) Send offprint requests to C. M. Teng, Pharmacological Institute, College of Medicine, National Taiwan University, No. 1, Jen-Ai Road, Sect. 1, Taipei, 10018, Taiwan     

Interaction of two sulfhydryl reagents with a cation recognition site on the neuronal dopamine carrier evidences small differences between [3H]GBR 12783 and [3H]cocaine binding sites

We have compared the effect of treating rat striatal cell membranes with ionic hydrophilic sulfhydryl reagents on the specific bindings of [³H]cocaine and of [³H]GBR 12783 (1-[2-(diphenylmethoxy)ethyl]4-(3-phenyl-2-[1-³H]propenyl)-piperazine) to the neuronal transporter of dopamine. Treatment with 1 mmol/1 5,5-dithiobis(2-nitrobenzoic acid) (DTNB) resulted in similar time-and concentration-dependent reductions of the specific binding of both radioligands. None of the uptake blockers tested afforded any protection against 1 mmol/1 DTNB. Addition of (sub)millimolar concentrations of CaCl₂ or MgCl₂, or 250 mmol/1 KCl to a treatment medium containing 10 mmol/l Na + significantly increased the DTNB-induced reduction of the specific binding of both radioligands. Cations were likely to be responsible for this effect since ions in combination with DTNB induced similar reductions in binding when either 1 mmol/l CaCl₂ or 50–250 mmol/l NaCl were added. Effects of cations on the DTNB-induced inhibition of binding were generally more marked on [³H]GBR 12783 than on [³H]cocaine binding. When added to a medium containing 10 mmol/1 Na⁺ 1 mmol/1 DTNB induced a reduction in the B_max of the specific binding of both radioligands. Addition of 1 mmol/l Ca²⁺ maintained or increased this B_max reduction and elicited a decrease in affinity which was significant for [³H]GBR 12783 binding.Treatment of membranes with the sodium salt of p-hydroxymercurybenzenesulfonate (pHMBS) induced time-and concentration-dependent decreases in [³H]GBR 12783 binding which were significantly greater than decreases in [³H]cocaine binding. However, 50mol/lpHMBS produced a similar decrease in the B_max of the specific binding of both radioligands. The pHMBS-induced reduction of [³H]GBR 12783 binding was not reversed by drugs whose action is purely that of uptake inhibition or by substrates of the dopamine carrier. Some of these drugs (100 mol/l dopamine, 1 mol/l mazindol or 100 mol/l cocaine) protected the specific binding of [³H]cocaine against the effects of pHMBS, whereas 1 mmol/1 p-tyramine, 10 mol/l nomifensine and 10 nmol/l GBR 12783 were ineffective. Addition of 120 mmol/l Na⁺, 1 mmol/l Ca²⁺ or 10 mmol/l Mg²⁺ to a treatment medium containing 10 mmol/l Na⁺ significantly reduced the effects of pHMBS on the specific binding of both radioligands. When striatal cell membranes were treated in a medium containing 130 mmol/1 Na⁺, there was a general decrease in the effects of ions on the reductions of specific binding produced by DTNB or pHMBS. Cation concentrations which interfered with the actions of DTNB and pHMBS were approximately those which blocked the specific binding of [³H]GBR 12783 when they were present during association of the radioligand (K⁺, Ca ²⁺, Mg²⁺), or, in the case of Na⁺, which are effective in reducing this blockade (Bonnet et al. 1988).The present data are consistent with the existence of mutually exclusive binding sites for [³H]GBR and [³H]cocaine on the neuronal dopamine transporter. The hypothesis of a cation recognition site which could gate admission of uptake inhibitors or carrier substrates to their binding domain on the transporter is discussed.     

Cyclic GMP stimulation by vasopressin in LLC-PK1 kidney epithelial cells is l-arginine-dependent

Summary The l-arginine antagonist N^G-monomethyl-l-arginine has been shown to inhibit nitric oxide formation from l-arginine in endothelial cells. In the present study N^G-monomethyl-l-arginine was used to assess the role of l-arginine for cyclic GMP stimulation by vasopressin in a kidney epithelial cell line (LLC-PK₁). Preincubation of cells with 1 mol/l, 10 mol/l and 100 mol/l N^G-monomethyl-l-arginine decreased cyclic GMP stimulation at 1 mol/l vasopressin by 25%, 71% and 90%, respectively. This inhibition by N^G-monomethyl-l-arginine was markedly reduced by l-arginine (2 mmol/1) but not d-arginine (2 mmol/1). Cyclic GMP stimulation by the calcium ionophore A23187 was also inhibited by N^G-monomethyl-l-arginine and enantioselectively restored by l-arginine. However, N^G-monomethyl-l-arginine did not affect cyclic GMP stimulation by sodium nitroprusside that spontaneously releases nitric oxide. These results suggest that, in kidney epithelial cells, vasopressin induces nitric oxide formation from l-arginine leading to activation of soluble guanylate cyclase. It is concluded that nitric oxide formation from l-arginine is not only responsible for endothelium-dependent relaxation but may be a more general pathway with regulatory function for intracellular guanylate cyclase activity.Send offprint requests to K. Schror at the above address     

Effects of the calcium entry blocker bepridil on repolarizing and pacemaker currents in sheep cardiac Purkinje fibres

Summary (1) Effects of bepridil (0.3–100 mol/l) on transmembrane currents which are active during the repolarization of the cardiac action potential were studied in sheep cardiac Purkinje fibres with the two-microelectrode voltage-clamp technique. Transmembrane currents were activated at a frequency of 0.03 Hz. (2) The initial inwardly rectifying current (i _K1) was reduced by 1.8 mol/l bepridil to 70% of the reference i _K1-current in the absence of the drug. (3) An initial outward current, which is activated at positive membrane potentials (i _inst) was depressed to 70% of reference by 14 mol/l bepridil. (4) The time-dependent outward current (i _K) was decreased by 1.8 mol/l bepridil to 70% of its amplitude in the absence of bepridil. The biexponential time course of i _K-current activation changed to be monoexponential with 100 mol/l bepridil. The effect of bepridil on i _K-current resulted in a shift of the activation curve of i _K-current to more positive membrane potentials (10 mol/l bepridil) and an additional decrease of driving force and/or conductance of the i _K-channels with higher bepridil concentrations (100 mol/l). (5) The transient outward current (i _to) was completely blocked by 30 mol/l bepridil. Inhibition to 70% of reference occurred with 1 mol/l bepridil. The voltage-dependent inactivation of i _to-current was affected by bepridil: the amplitude of the steady-state inactivation curve was reduced and i _to-current was inactivated faster after application of bepridil. Bepridil caused no pronounced shift of the steady-state inactivation curve along the voltage axis. (6) The pacemaker current (i _f) was slightly increased under the influence of low bepridil concentrations (0.3, 1 mol/l) while it was reduced to 70% of reference by 100 mol/l bepridil. (7) The blocking action of bepridil on outward currents in sheep cardiac Purkinje fibres will explain the action potential prolongation, which is observed in different mammalian cardiac tissues under the influence of bepridil.Supported by the Deutsche Forschungsgemeinschaft SFB 242, C 1 Send offprint requests to U. Borchard at the above address     

cAMP- and diacylglycerol-mediated pathways elevate cytosolic free calcium concentration via dihydropyridine-sensitive, ω-conotoxin-insensitive calcium channels in normal rat anterior pituitary cells

Summary The effect of 8-bromocyclic AMP (8-Br-cAMP) and phorbol 12-myristate 13-acetate (PMA), a protein kinase C activator, on cytosolic free calcium concentration ([Ca²⁺]_i) in normal rat anterior pituitary cells was examined. [Ca²⁺]_i was monitored directly by the fluorescent indicator fura-2. 8-Br-cAMP as well as PMA elevated [Ca²⁺]_i in a concentration-dependent manner. Forskolin (10 mol/l), which activates adenylate cyclase, and 1-oleoyl-2-acetyl-glycerol (10 mol/l), another activator of protein kinase C, also increased [Ca²⁺]_i. Both the 8-Br-cAMP (2 mmol/l)- and the PMA (100 nmol/l)-induced increase in [Ca²⁺]_i was dependent on the presence of extracellular calcium and could be inhibited by the calcium channel blockers Mg²⁺ and nifedipine, but not by onotoxin (100 nmol/l). The half-maximally inhibitory concentrations of Mg²⁺ and nifedipine were about 12 mmol/l and 160 nmol/l, respectively, for the [Ca²⁺]_i response to 8-Br-cAMP (2 mmol/l), and were about 6 mmol/l and 400 nmol/1, respectively, for the PMA (100 nmol/1)-induced increase in [Ca²⁺]_i. The sodium channel blocker tetrodotoxin (5 mol/l) had no influence on the effect of 8-Br-cAMP (2 mmol/l) or PMA (100 nmol/l) on [Ca²⁺]_i. After pretreatment for 3 min with PMA (100 nmol/l), the subsequent K⁺ (100 mmol/l)- or arachidonic acid (3 mol/l)-induced increase in [Ca²⁺]_i was decreased by about 50%. By contrast, pretreatment (3 min) with 8-Br-cAMP (2–10 mmol/1) markedly enhanced the subsequent [Ca²⁺]_i response to K⁺ (100 mmol/l), and left the effect of arachidonic acid (3 mol/l) on [Ca²⁺]_i unimpaired. These data indicate that both cAMP- and diacylglycerol-mediated pathways increase [Ca²⁺ _i in normal rat anterior pituitary cells via an influx of extracellular Ca²⁺ through dihydropyridine-sensitive, -conotoxin-insensitive voltage-dependent calcium channels. These second messengers may thus be involved in Ca²⁺ channel activation by hypothalamic releasing hormones. Effects of cAMP- or diacylglycerol-induced pathways on anterior pituitary function may not be independent of but be mediated also by changes in [Ca²⁺]_i. However, substantial differences appear to exist in how cAMP and diacylglycerol influence voltage-dependent calcium channels.     

The relaxant action of osthole isolated from Angelica pubescens in guinea-pig trachea

The effect of osthole, isolated from Angelica pubescens, on the contraction of guinea-pig trachea was studied. Osthole (25–100 mol/l), theophylline (10–1000 mol/l) and higher concentrations of nifedipine (0.1–100 mol/l) suppressed the contraction response curves of tracheal smooth muscle caused by carbachol, prostaglandin F₂ (PGF₂), U46619 (thromboxane A₂ analogue) and leukotriene C₄ (LTC₄) in a concentration-dependent manner. The contraction caused by high K⁺ (120 mmol/1) and cumulative concentrations of CaCl₂ (0.03–3 mmol/1) was also inhibited concentration-dependently by osthole (25–100 mol/l), theophyl line(10–1000 mol/l) and lower concentrations of nifedipine (0.01–0.1 mol/l). The relaxant actions of osthole were not affected by propranolol (1 mol/l), glibenclamide (10 mol/l) or removal of tracheal epithelium. Osthole (100 mol/l) was still effective in causing tracheal relaxation in the presence of nifedipine (1 mol/l). In Ca²⁺-free- and EGTA (0.2 mmol/1)-containing medium, the relaxing effect of osthole was more potent than in normal Krebs solution. Osthole (25 and 50 mol/l) caused 2.9 and 6.5, or 3.0 and 5.6 fold, respectively, increase in potency of forskolin or sodium nitroprusside in causing tracheal relaxation but did not affect that by cromakalim. Osthole (50 mol/l) enhanced the increase in tissue cAMP and cGMP levels induced by forskolin and sodium nitroprusside, respectively, and in higher concentrations (100 and 250 mol/l), itself increased markedly tissue cAMP and cGMP contents. Osthole (10–250 mol/l) inhibited the activity of cAMP and cGMP phosphodiesterases in a concentration-dependent manner. It is concluded that osthole exerts a nonspecific relaxant effect on the trachealis by inhibiting the cAMP and cGMP phosphodiesterases. Correspondence to: C. M. Teng at the above address     

Sodium dependent 3H-noradrenaline release from rat neocortical slices in the absence of extracellular calcium presynaptic modulation by μ-opioid receptor and adenylate cyclase activation

Summary In Ca²⁺-free EGTA (1 mmol/l)-containing medium veratrine (3 mol/l) and ouabain (100 mol/l) strongly enhanced the efflux of ³H-noradrenaline from superfused rat brain neocortical slices prelabelled with the radioactive amine. In both cases ³H-noradrenaline release was prevented by tetrodotoxin (1 mol/l). These effects of veratrine and ouabain were virtually additive and independent of whether the noradrenaline uptake carrier was blocked with 1 mol/l desipramine or not. The adenylate cyclase activator forskolin (10 nmol/l–10 mol/l) strongly enhanced veratrine- and ouabain-induced ³H-noradrenaline release, without affecting spontaneous tritium efflux. The release induced by both stimuli was profoundly inhibited by the selective -opioid receptor agonist [d-Ala, MePhe⁴, Gly-ol⁵]enkaphalin (DAGO, 3 nmol/l–1 mol/l) in a concentration-dependent manner. The inhibitory effects of 1 mol/l DAGO were abolished by 1 mol/l naloxone. On the other hand, preincubation of the slices for 1 h with the -opioid receptor-selective irreversible ligand fentanyl isothiocyanate (1 pmol/l) did not change the inhibitory effects of DAGO.These data show that veratrine- and ouabain-induced ³H-noradrenaline release from central noradrenergic nerve terminals is facilitated by increasing intracellular cyclic AMP levels and reduced by activation of presynaptic -opioid receptors, indicating the involvement of exocytotic neurotransmitter release. The results provide further evidence for the hypothesis that under these conditions neurotransmitter release from central noradrenergic neurons is triggerred by a Na⁺-induced efflux of Ca²⁺ ions from intracellular stores.Abbreviations DAGO [d-Ala², McPhe⁴, Gly-ol⁵]enkephalin Send offprint requests to A. N. M. Schoffelmeer at the above address     

Effects of para-substituted beta-adrenoceptor blocking agents and methyl-substituted phenoxypropanolamine derivatives on maximum upstroke velocity of action potential in guinea-pig papillary muscles

Summary The effects of atenolol (2–5 mmol/l), sotalol (1–2 mmol/l) and pamatolol (0.1–1 mmol/l), together with N-tertiary butyl phenoxypropanolamines with o-methyl (D-2T: 50–100 mol/l) m-methyl (D-3T: 50–100 mol/l) and p-methyl (D-4T:100–200 mol/l) group as well as with o,p-methyl groups (D-24T) (50–100 mol/l) on action potentials (APs) were investigated in isolated guinea-pig papillary muscles. All the drugs in these concentrations produced a concentration-dependent reduction of the maximum upstroke velocity (V _max). The reduction ofV _max in premature APs induced by stimuli interpolated between the basic driving rate of 0.25, 0.1 or 0.027 Hz decayed exponentially during diastolic intervals. The time constants of these decay processes for atenolol, pamatolol and sotalol ranged between 260–541 ms, those for D-3T and D-4T between 655–1,166 ms, and D-2T and D-24T between 1,565–1,931 ms. A drug which provided larger values caused the reduction ofV _max in a wider range of the frequency. With respect to the aryloxypropanolamine derivatives so far studied (Sada and Ban 1980, 1981 a, b; Sada et al. 1983) fairly good correlations were found as follows: between logn-octanol/water partition coefficient (logP) and ED₂₀ at 0.25 Hz, ED₃₀ at 1 and 4 Hz for 11–14 compounds; between logP and resting block, between molecular weight and A _o ^c i.e. the value extrapolated to the time of APD₉₀ of the conditioning response relative to the predrugV _max value which may represent a fraction of channels blocked per AP for 100 mol/l of 20–22 compounds. With respect to 8 compounds with methyl substituents in the benzene ring or amine part the ortho methyl group makes a major contribution to increase the resting block and to increase log values.     

Blockade of γ-aminobutyric acid-receptors of the B-subtype inhibits the dopamine-induced enhancement of the release of cholecystokinin-like immunoreactivity from slices of rat dorsal caudatoputamen

Summary When slices of rat dorsal caudatoputamen (= neostriatum) are incubated in vitro, Choecystokinin-like immunoreactivity (CCK-LI) is released upon addition of veratridine (3.75 mol/l). This release is affected by dopamine and by -aminobutyric acid (GABA)-receptor agonists. Dopamine enhances the release by stimulating dopamine D₂-receptors and decreases it via D₁-receptors. GABA_A-receptor agonists enhance the veratridine-induced release of CCK-LI, while GABA_B-receptor agonists decrease it. In the present investigation, it was examined whether GABA-receptors are involved in the effect which dopamine exerts via D₂-receptors. The GABA_A-receptor antagonist bicuculline (10 mol/l)and the blocker of the GABA_A-receptor ionophore picrotoxin (1 mol/l) did not affect the dopamine (0.1 mol/1)-induced increase in the release of CCK-LI. However, the GABA_A-receptor agonist muscimol (1 mol/l) not only enhanced the release of CCK-LI, but also prevented a further enhancement by dopamine (0.1 mol/l). This effect of muscimol was blocked by bicuculline (10 mol/l). In the presence of -amino-n-valeric acid (0.1 mmol/l), which has been described to block GABA_B-receptors, dopamine no longer enhanced the veratridine-induced release of CCK-LI. -Amino-n-valeric acid also inhibited the pronounced enhancement of the release of CCK-LI caused by dopamine (0.1 mol/l) and 1 mol/l in the presence of the preferential D₁-receptor antagonist SCH 23390. The effect of -amino-n-valeric acid persisted in the presence of bicuculline (10 mol/l and 100 mol/l). (+)-Baclofen, a partial agonist at GABA_B-receptors, and the stereoisomer (–)-baclofen, a full agonist, also prevented the effect of dopamine on the veratridine-induced release of CCK-LI. The effects of both drugs may be due to desensitization of GABA_B-receptors, which has been described to develop quite rapidly. It is concluded that -amino-n-valeric acid blocks GABA_B-receptors and in this way prevents the enhancement of the veratridine-induced release of CCK-LI caused by dopamine via D₂-receptors. These data are interpreted as evidence that dopamine and GABA-neurons can directly or indirectly interact in the rat neostriatum. Send offprint requests to D. K. Meyer at the above address     

Acetylcholine release in rat nucleus accumbens is regulated through dopamine D2-receptors

Summary Experiments in slices of rat nucleus accumbens were carried out in order to investigate whether the release of acetylcholine in this tissue is modulated through dopamine receptors. The slices were preincubated with ³H-choline and then superfused and stimulated electrically twice for 2 min each at a frequency of 3 Hz.The electrically evoked overflow of tritium averaged 2.9–3.9% of the tritium content of the tissue in the various groups. The D₂-selective agonist quinpirole (0.01–1 mol/l) reduced the evoked overflow of tritium by maximally 56%, an effect antagonized by the D₂-selective antagonist (–)-sulpiride (1 mol/l). The D₁-selective agonist 2,3,4,5-tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine (SKF 38393) caused a slight decrease only at the high concentration of 10 mol/l. (–)-Sulpiride (0.1–10 mol/l) moderately increased the evoked overflow of tritium when given alone. The dopamine uptake inhibitor nomifensine (10 mol/l) caused a decrease, and in its presence the increase produced by (–)-sulpiride became much more marked, amounting to maximally 149%. (+)-Sulpiride (0.1–1 mol/l) failed to change the evoked overflow of tritium in the presence of nomifensine. The dopamine-releasing agent (±)-amphetamine (1 mol/l) also reduced the evoked overflow, an effect abolished by (–)-sulpiride. Finally, bretylium (1 mmol/l), which blocks the release of dopamine, increased the evoked overflow. (–)-Sulpiride (1 mol/l) lost its facilitatory effect in slices treated with bretylium.We conclude that the release of acetylcholine in rat nucleus accumbens, like its release in the nucleus caudatusputamen, is modulated through dopamine D₂-receptors. The receptors are activated by endogenous dopamine under the conditions of these experiments. Send offprint requests to K. Starke at the above address     

Studies on the effects ofl(αS,5S)-α-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (AT-125) on 4-aminophenol-induced nephrotoxicity in the Fischer 344 rat

4-Aminophenol (para-aminophenol; PAP) causes selective necrosis to the S₃ segment of the proximal tubule in experimental animals. The mechanism of PAP nephrotoxicity has not been fully elucidated, although it has been suggested to involve glutathione (GSH)-dependentS-conjugation followed by processing by the enzyme -glutamyl transpeptidase (GT) to the corresponding cysteineS-conjugate. This proposed toxicity mechanism was probed further by administering L-(S,5S)--amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (AT-125), a potent GT inhibitor, to Fischer 344 (F344) rats before treatment with PAP (100 mg/kg). AT-125 pretreatment did not appear to protect against PAP-induced nephrotoxicity as assessed by renal histopathology, clinical chemistry and proton nuclear magnetic resonance (¹H NMR) spectroscopy of urine. These data suggest that renal GT activity is not a prerequisite for PAP nephrotoxicity and that the generation of a cysteineS-conjugate is not a unique requirement for the induction of PAP nephrotoxicity.     

Evidence against a functional link between noradrenaline uptake mechanisms and presynaptic alpha-2 adrenoceptors

Summary Slices prepared from rat cerebral cortex were labelled with ³H-noradrenaline and superfused. Electrical field stimulation was carried out 15 min (S₁) and 45 min (S₂) after the start of collection of 5-min samples using 4 pulses delivered at 100 Hz. Drugs acting at ₂-adrenoceptors were added 20 min before S₂, and their effects were evaluated using the S₂/S₁-ratio. The ₂-adrenoceptor antagonists idazoxan (1 mol/l) and rauwolscine (1 mol/l) failed to increase stimulation-evoked overflow of radioactivity in the absence or presence of the noradrenaline reuptake inhibitor desipramine (1 gmol/l). This indicates that the duration of electrical stimulation was too short to allow development of ₂-adrenoceptor-mediated auto-inhibition by released noradrenaline. The effect of clonidine (3–1000 nmol/l) on stimulation-evoked overflow of radioactivity was tested in the absence and presence of three different reuptake inhibitors (desipramine, 1 ol/l; maprotiline, 1 ol/l; cocaine, 10 mol/l). The analysis yielded identical concentration-response curves under all conditions. These results argue against an action of inhibitors of neuronal reuptake of noradrenaline at the presynaptic ₂-adrenoceptor and against the concept of a functional link between uptake site and receptor. Send offprint requests to E. A. Singer     

Phorbol 12,13-dibutyrate,an activator of protein kinase C,stimulates both contraction and Ca2+ fluxes in dog saphenous vein

Summary The vascular effects of phorbol 12,13-dibutyrate (PDBu) were studied in the dog saphenous vein. PDBu (1 M) caused contraction (0.58 ± 0.22 g/mg wet wt.) and Ca uptake (74.2 ± 41.2 mol/kg wet wt.) which were unaffected by 10 M phentolamine (N = 6). The PDBu-induced contraction was greatly (60–80%) inhibited in Ca²⁺-free solution. 15 Ca efflux measurements performed in Ca²⁺-free solution showed that PDBu did not cause Ca release from intracellular storage sites. The contractile response to PDBu (1 nM-1 M) was significantly correlated with the magnitude of Ca uptake; contraction and the rise in tissue Ca²⁺ also had a similar time course. Correlation between the two measures persisted when the responses to PDBu were augmented by co-administration with 20 mM KCl. However, no synergism occurred between the two agonists. Both the contraction and Ca uptake responses to PDBu were reduced by nifedipine and verapamil, each at 1 M. In the Triton X-100 skinned saphenous vein, where the voltage-dependent Ca channel is not functional, 10 M PDBu did not cause contractions in the presence of 0.1 M Ca²⁺. Thus, contraction of the intact saphenous vein by PDBu characteristically exhibits great Ca dependence and PDBu seems to activate the voltage-dependent Ca channel, presumably through stimulation of protein kinase C; the ensuing Ca entry is primarily responsible for contraction. However, the mechanism responsible for the PDBu-induced contractions that are resistant to Ca²⁺-free PSS or Ca entry blockers remains to be defined. It appears that the dog saphenous vein differs from dog femoral artery, rabbit aorta and pig carotid artery where PDBu contractions do not display dependence on external Ca²⁺. Send offprint requests to P. J. S. Chin at the above address     

Opposite modulation of ouabain cardiotoxicity by hexamethyleneamiloride and phenylephrine

Summary To see whether the Na/H antiporter plays a role in digitalis cardiotoxicity, we investigated the influence of modulators of Na/H exchange on the toxic effects of ouabain in isolated, paced (0.4 Hz) rat left atria. Ouabain (1 mmol/l) caused a transient positive inotropic effect followed by toxic events, including a complete loss of developed force and a gradual increase in resting force. In the presence of hexamethyleneamiloride (3 and 10 mo1/l), an inhibitor of Na/H exchange, ouabain (1 mmol/l) caused a sustained positive inotropic effect without toxicity. By contrast, phenylephrine (100 mol/ 1) an -adrenoceptor agonist reported to stimulate the antiporter, hastened the development of ouabain's toxicity. Neither ouabain, at a subtoxic concentration (650 ol/l), nor phenylephrine (100 mol/l) affected diastolic force, but in their combined presence, a substantial contracture developed and twitch contractions disappeared. Phenylephrine (30 or 100 mol/l) or adrenaline (30 mol/l), in the presence of a -adrenoceptor antagonist, increased the intracellular pH by up to 0.15 pH unit, as measured using ion-selective microelectrodes in quiescent preparations. This effect on pH₁ was prevented by hexamethyleneamiloride (10 mol/l). Consistent with phenylephrine's ability to stimulate Na⁺ influx via the Na/H antiporter, phenylephrine (100 mol/l) increased intracellular Na⁺ activity by about 3 mmol/l in ouabain (650 mol/l)-treated atria. These findings indicate that modulators of Na/H exchange affect the cardiotoxicity of digitalis glycosides and imply that the stimulation of myocardial -adrenoceptors may aggravate digitalis toxicity.This work was conducted in part under the auspices of the Association for US/French Biomedical Cooperation Send offprint requests to S. M. Vogel at the above address     

Effect of histamine on human bronchial arteries in vitro

Summary Histamine caused a concentration-dependent relaxation at lower concentrations (1 pmol/1–1 mol/l) and contraction at higher concentrations (0.01–1 mmol/l) of isolated precontracted human bronchial arteries. In the vessels at resting tension only concentration-dependent contraction was evoked by histamine (0.01–1 mmol/l). Both the contractile and relaxant responses were significantly antagonised by mepyramine (1 gmol/l), with an estimated pK_B value of 8.4, but not by cimetidine (100 mol/l). Our results indicate that histamine induces biphasic effects on human bronchial arteries via H₁-re-ceptors. Send offprint requests to P. J. Barnes at the above address     

Evidence for a non-precursor dopamine pool in noradrenergic neurones of the dog mesenteric artery

Summary Isolated tracheal strip-chain preparation of the guinea-pig was used to study the effect of temperature on carbachol-induced contraction. The preparation was suspended in the organ bath containing Krebs bicarbonate solution for isometric tension recording. A decrease of bath temperature from 37°C to 20°C (cooling) caused a transient increase in tension and thereafter inhibited the contractile response of the trachea caused by carbachol (30 nmol/l–3 mol/l). Isosmotic potassium chloride (KCl, 64.7 mmol/l)-induced contraction or calcium chloride (CaCl₂, 0.1–3 mmol/l)-induced contraction in K⁺-depolarized muscle was markedly inhibited by cooling. Verapamil in concentrations of 1 mol/l or greater, which markedly depressed the CaCl₂-induced contraction, caused partial depression of the contractile response to carbachol.On the other hand, carbachol-induced contraction of the trachea which was incubated with K⁺-rich, verapamil (3 mol/l) containing Krebs solution and with Ca²⁺-free, EGTA (0.4 mmol/l) containing Krebs solution were both augmented at 20°C. From these observations, it is concluded that decreased responsiveness of the guinea-pig airway smooth muscle to carbachol with lowered temperature may be due to an inhibition of Ca²⁺ influx through voltage-dependent Ca²⁺ channels which involves part of the contraction.     

Raising the ambient potassium ion concentration enhances carbachol stimulated phosphoinositide hydrolysis in rat brain hippocampal and cerebral cortical miniprisms

Summary The influence of the ambient potassium ion concentration ([K⁺]_ upon agonist stimulated hydrolysis of phosphoinositides (PI) has been studied in isolated miniprisms of rat hippocampus and cerebral cortex. When the external [K⁺] was raised from 6 to 18 mmol/l, there was little or no increase in the hydrolysis of PI in the absence of agonist, however, carbachol (100 mol/l) stimulated hydrolysis was greatly enhanced in both brain regions studied. Thus, carbachol stimulated the hydrolysis of PI to 146% and 386% of control levels at potassium concentrations of 5.8 and 18.2 mmol/l, respectively, in the rat hippocampus. A similar enhancement of muscarine (100 mol/l) stimulation was observed in cortical miniprisms with 18 mmol/l [K⁺]. A further enhancement was seen at higher ambient [K⁺], although basal hydrolysis of PI was then also increased. The carbachol-stimulated hydrolysis of PI found at both 6 and raised [K⁺] was prevented by atropine (1 and 10 mol/l) and tetraethylammonium (20 mmol/l), but not by 10 mmol/l Mg²⁺. Pirenzepine (50 nmol/l) also reduced this response. The ions Cs⁺ and Rb⁺ (but not Li⁺ or Tris⁺) produced a similar enhancement of the carbachol stimulation to that found with K⁺. At a buffer [K⁺] of 6 mmol/l, noradrenaline (100 mol/l) produced a 2-fold increase in the hydrolysis of PI whereas 5-hydroxytryptamine (100 mol/l) and histamine (500 mol/l) had little or no effect. However, histamine and 5-hydroxytryptamine did stimulate the hydrolysis of PI when [K⁺] was increased. Miniprism ATP content was not changed by a rise in [K⁺] to 18 mmol/l. The significance of these results is discussed in terms of the postsynaptic cellular events following cholinergic stimulation.