The Nitric Oxide Donor SIN-1 Is Free of Tolerance and Maintains Its Cyclic GMP Stimulatory Potency in Nitrate-Tolerant LLC-PK1 Cells

Purpose. Using an established cell culture model, the present study investigates whether linsidomine (SIN-1), a spontaneous donor of nitric oxide and active metabolite of the antianginal drug molsidomine, induces tolerance to its own cyclic GMP stimulatory action or shows a diminished response after tolerance induction with glyceryl trinitrate. Methods. Incubations with nitric oxide donors were carried out in LLC-PK₁, kidney epithelial cells. Intracellular levels of cyclic GMP, the vasodilatory second messenger of nitric oxide, were determined by radioimmunoassay. Results. A 5-h preincubation with glyceryl trinitrate (0.01–100 M) led to complete inhibition of a subsequent cyclic GMP stimulation by glyceryl trinitrate but left the cyclic GMP response to SIN-1 unaltered. Similarly, cyclic GMP elevations by the spontaneous nitric oxide donors sodium nitroprusside and spermine NONOate were not affected after pretreatment with glyceryl trinitrate. Moreover, pretreatment with SIN-1 (1–1000 M) had no significant effect on SIN-1-dependent cyclic GMP stimulation. Conclusions. Our results show that in LLC-PK₁, cells, SIN-1 is free of tolerance induction and not cross-tolerant to glyceryl trinitrate. This may be due to the spontaneous nitric oxide release from SIN-1, which in contrast to nitric acid esters does not require enzymatic bioactivation and may therefore be unaffected by nitrate tolerance.     

Effects of glyceryl trinitrate on endothelium-dependent and -independent relaxation and cyclic GMP levels in rat aorta and human coronary artery

The effects of glyceryl trinitrate-induced desensitization on relaxations and/or elevated cyclic GMP levels due to the nitrogen oxide-containing vasodilators (glyceryl trinitrate and sodium nitroprusside), the endothelium-dependent vasodilators (acetylcholine and the Ca2+ ionophore A23187), and the atrial peptides (atriopeptin II) were investigated in the rat thoracic aorta and human coronary artery. Prior exposure of rat thoracic aorta to glyceryl trinitrate decreased relaxations to glyceryl trinitrate, sodium nitroprusside, and acetylcholine, whereas relaxations to atriopeptin II and 8-bromo cyclic GMP remained unaltered. In human coronary artery, glyceryl trinitrate pretreatment inhibited relaxations to glyceryl trinitrate, sodium nitroprusside, and the Ca2+ ionophore A23187. Relaxation to glyceryl trinitrate was inhibited more than that to sodium nitroprusside in both tissues. Acetylcholine-induced relaxation in rat thoracic aorta was slightly inhibited, whereas relaxation to the Ca2+ ionophore A23187 in human coronary artery was markedly depressed. Pretreatment with glyceryl trinitrate decreased the elevated cyclic GMP levels due to glyceryl trinitrate and acetylcholine in rat thoracic aorta and to glyceryl trinitrate and the Ca2+ ionophore A23187 in human coronary artery. Removal of the endothelium abolished the increased cyclic GMP levels and relaxation due to the Ca2+ ionophore A23187 and decreased basal cyclic GMP levels in the human coronary artery. In contrast, atriopeptin II-induced increased cyclic GMP levels were unaltered by glyceryl trinitrate pretreatment in rat thoracic aorta. The present results suggest that: glyceryl trinitrate-induced desensitization inhibits relaxation to the nitrogen oxide-containing vasodilators and endothelium-dependent vasodilators in both the rat thoracic aorta and human coronary artery: the inhibition of relaxation is associated with decreased formation of cyclic GMP;(ABSTRACT TRUNCATED AT 250 WORDS)     

Cross-tolerance to L-arginine-dependent guanylate cyclase activators in nitrate-tolerant LLC-PK1 kidney epithelial cells

Recently, it was shown that in LLC-PK1 kidney epithelial cells hormones such as vasopressin or oxytocin increase cyclic GMP in a receptor-mediated and L-arginine-dependent manner. In the present study, the possible existence of cross-tolerance to vasopressin and oxytocin was investigated in nitrate-tolerant LLC-PK1 cells. Pretreatment with 1 mM glyceryl trinitrate for 3 h decreased cyclic GMP stimulation by 1 microM vasopressin and 1 microM oxytocin by 49% and 54%, respectively. Under the same conditions, cyclic GMP stimulation at 1 microM sodium nitroprusside was diminished by 56% whereas the cyclic GMP response to 100 microM glyceryl trinitrate was virtually abolished. Our results demonstrate that a substantial degree of cross-tolerance to L-arginine-dependent guanylate cyclase activators occurs in nitrate-pretreated nonvascular cells which may be due to glyceryl trinitrate-induced desensitization of soluble guanylate cyclase.     

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     

In vitro forecasting of drugs which may interfere with the biotransformation of midazolam

Summary The biotransformation of midazolam is mediated by a cytochrome P-450 isozyme (P-450 IIIA) whose activity is highly variable. The kinetics of the 1- and 4-hydroxylation of midazolam, the major routes of midazolam oxidation, by human liver microsomes have been examined to characterize further the cytochrome isozyme(s) catalysing these reactions, and to screen for drugs that might interfere with them.In hepatic microsomal preparation from two kidney donors (extensive and poor metabolisers of debrisoquine) K_M values for 1-hydroxylation were 4.2 and 6.1 M (extensive and poor metabolisers, respectively), and for the 4-hydroxylation they were 14.7 and 18.1 M, respectively. The corresponding V_max values were 25.8 and 29.8 and 17.0 and 18.1 nmolsdmg P^–1·h^–1. Both reactions appeared to be catalysed by the same or by coregulated isozymes.Midazolam hydroxylations in vitro are inhibited by many drugs, including nifedipine and other dihydropyridine-type calcium channel blockers, ergot alkaloids, cyclosporine, erythromycin and phenothiazine-type neuroleptics.A clinical case report illustrates the consequence of such a drug-drug interference with hepatic biotransformation; midazolam-induced sleep in a patient lasted for 6 days (t_1/2=25 h).Presented in part at the Fifty-seventh Annual Meeting of the Swiss Society for Internal Medicine, Interlaken, May 1989, and at the Fourth World Conference on Clinical Pharmacology and Therapeutics, Mannheim, July 1989.     

Effect of the phosphodiesterase inhibitor 4-(3-cyclopentyloxy-4-methoxyphenyl)-2-pyrrolidone (ZK 62711) on gastric secretion and gastric mucosal cyclic AMP

Summary The effect of the phosphodiesterase inhibitor 4-(3-cyclopentyloxy-4-methoxyphenyl)-2-pyrrolidone (ZK 62711) on gastric secretion and the cyclic AMP system of the gastric mucosa was studied in rats and guinea pigs. In rats, 0.03–0.3 moles/kg ZK 62711 i.v. stimulated acid and pepsin secretion in a dose-dependent manner and 0.03 moles/kg i.v. enhanced the effect of histamine. In guinea pigs no reproducible stimulation was found after intravenous injections of ZK 62711. The stimulation of gastric secretion in the rat by 0.3 moles/kg ZK 62711 i.v. was not affected by vagotomy but was totally inhibited by 10 moles/kg cimetidine i.v. The structurally related phosphodiesterase inhibitor, 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidine (Ro 20-1724), at the dose of 3.3 moles/kg i.v. stimulated gastric secretion in anaesthetised rats to a similar extent as 0.3 moles/kg ZK 62711 i.v. The content of cyclic AMP in the rat gastric mucosa in vivo was slightly increased by 10 moles/kg ZK 62711 i.v, whereas lower doses did not change it. Accumulation of cyclic AMP in the rat gastric mucosa by 50 moles/kg histamine i.v. was enhanced by simultaneous injections of 3.3 moles/kg ZK 62711 i.v. In rat gastric tissue slices in vitro 1–50 M ZK 62711 increased the level of cyclic AMP but 100 M histamine had no effect in the absence or presence of ZK 62711. In gastric mucosal slices of the guinea pig 10 and 50 M ZK 62711 increased the cyclic AMP content which effect was inhibited by 100 M cimetidine and enhanced the stimulatory effect of 100 M histamine on cyclic AMP. The activity of soluble cyclic AMP phosphodiesterase was inhibited by ZK 62711 in the rat (IC₅₀=18 M) and guinea pig gastric mucosa (IC₅₀=1.5 M). Adenylate cyclase was not affected in the homogenate of the guinea pig gastric mucosa. The results indicate that the phosphodiesterase inhibitor ZK 62711 which increases cyclic AMP levels in the gastric mucosa in vivo and in vitro, is a potent stimulator of gastric acid secretion.     

Biphasic relaxant curves to glyceryl trinitrate in rat aortic rings

Summary The concentration-effect curve for the relaxant effects of glyceryl trinitrate (GTN) in rat aortic rings consisted of two phases with IC₅₀ values of 0.1 M for Phase I and 14 M for Phase II. Incubation of tissues with oxyhaemoglobin or the induction of tolerance to GTN abolished responses occurring in Phase I but were without effect on Phase II relaxant responses. Both phases of the relaxant curve appeared to involve cyclic GMP since responses were (i) potentiated by the cyclic GMP phosphodiesterase inhibitor zaprinast (M & B 22948) and (ii) inhibited by methylene blue and LY83583, agents which inhibit soluble guanylate cyclase. The latter agents inhibited Phase I responses in a non-surmountable manner while Phase II responses were shifted to the right without effect on the maximal response. Neither phase of relaxation involved stimulation of the Na⁺/K⁺ ATPase pump since treatment of tissues with ouabain or K⁺-free solutions did not alter the GTN biphasic curve. Phase I relaxant responses to GTN resembled those to the endothelium-dependent relaxant acetylcholine, since oxyhaemoglobin and methylene blue were non-surmountable antagonists; however there was no cross tolerance to acetylcholine in GTN tolerant tissues. Phase II relaxant responses resembled those obtained with sodium nitroprusside (SNP) since neither oxyhaemoglobin nor the induction of tolerance to GTN altered the response to SNP. These results indicate that there are two distinct mechanisms of relaxation for GTN in rat aortic rings; however both mechanisms appear to involve cyclic GMP as the second messenger. Send offprint requests to E. Malta at the above address     

Inhibition of vascular smooth muscle relaxation by LY83583

Summary The ability of LY83583 to antagonize vascular smooth muscle relaxation elicited by a number of vasodilators was examined in rings of rat aorta. LY83583 (0.3–10 M) inhibited relaxant responses to acetylcholine, calimycin (A23187), adenosine triphosphate (ATP) and sodium nitroprusside, whereas responses to atriopeptin III an activator of particulate guanylate cyclase, and papaverine were unaffected. For acetylcholine and calimycin the major effect of LY83583 (0.3–10 M) was to reduce the maximal response without appreciably altering the EC₅₀ values whereas for ATP the EC₅₀ values were markedly increased by low concentrations of LY83583 (0.3–1 M) with depression of maximal responses occurring at higher concentrations (10 M) of the antagonist. In contrast LY83583 produced nonparallel rightward shifts of the curve for sodium nitroprusside without altering the maximal response. In addition, LY83583 (10 M) reduced basal levels of cyclic GMP and prevented acetylcholine and sodium nitroprusside-induced elevations of cyclic GMP, in parallel with reductions in the relaxant responses. In the presence of LY83583 (10 M) higher concentrations of sodium nitroprusside restored both the relaxant response and the elevation of cyclic GMP. The results of this study show that LY83583 antagonises only those vasodilators which are thought to act via stimulation of soluble guanylate cyclase. The nonsurmountable inhibition of relaxation to acetylcholine, calimycin and ATP probably reflects a limited maximal capacity of the endothelium to release EDRF in response to these agents. Send offprint requests to E. Malta     

Nitric oxide synthesis in endothelial cytosol: Evidence for a calcium-dependent and a calcium-independent mechanism

Summary Release of nitric oxide (NO) from endothelial cells critically depends on a sustained increase in intracellular free calcium maintained by a transmembrane calcium influx into the cells. Therefore, we studied whether the free cytosolic calcium concentration directly affects the activity of the NO-forming enzyme(s) present in the cytosol from freshly harvested porcine aortic endothelial cells. NO was quantified by activation of a purified soluble guanylate cyclase coincubated with the cytosol. In the presence of 1 mM L-arginine, 0.1 mM NADPH and 0.1 mM EGTA, endothelial cytosol (0.2 mg of cytosolic protein per ml) stimulated the activity of guanylate cyclase 5.0 + 0.5-fold (from 31 + 9 to 153 + 15 nmol cyclic GMP formed per min per mg guanylate cyclase). Calcium chloride increased this stimulation further in a concentration-dependent fashion by up to 136 + 15% (with 2 M free calcium; EC₅₀ 0.3 M). The calcium-dependent and -independent activation of guanylate cyclase was enhanced by superoxide dismutase (0.3 M) and was inhibited by the stereospecifically acting inhibitor of L-arginine-dependent NO formation N^G-nitro-L-arginine (1 mM) and by LY 83583 (1 M), a generator of superoxide anions. Our findings suggest a calcium-dependent and -independent synthesis of NO from L-arginine by native porcine aortic endothelial cells. Send of fprint requests to A. Mülsch, at the above address     

Stimulatory effects of brain natriuretic peptide on cyclic GMP accumulation and tyrosine hydroxylase activity in cultured bovine adrenal medullary cells

Summary We studied the effect of brain natriuretic peptide (BNP) on the accumulation of cyclic GMP and the phosphorylation and activity of tyrosine hydroxylase, compared with that of atrial natriuretic peptide (ANP), in cultured bovine adrenal medullary cells. 1. BNP as well as ANP increased cellular cyclic GMP accumulation in a concentration-dependent manner (10–1000 nmol/1). BNP (1 mol/1) and ANP (1 mol/1) produced a 60-fold and 30-fold increase in cyclic GMP accumulation, respectively. 2. The stimulatory effects of BNP and ANP on cyclic GMP accumulation were observed even when Ca²⁺ or Na⁺ was removed from the incubation medium. 3. 12-O-Tetradecanoylphorbol 13-acetate (TPA), an activator of protein kinase C, inhibited the stimulatory effect of BNP on cyclic GMP accumulation in a concentration-dependent manner (1–100 nmol/1). Furthermore, the BNP-induced accumulation of cyclic GMP was attenuated by forskolin (1 mol/1), an activator of adenylate cyclase. 4. BNP (1 mol/1) and ANP (1 mol/1) caused a significant increase in phosphorylation and activity of tyrosine hydroxylase in the cells. 5. In digitonin-permeabilized cells, cyclic GMP (1–100 mol/1) activated tyrosine hydroxylase in the presence of ATP and Mg²⁺.These results suggest that BNP stimulates the accumulation of cyclic GMP in a manner similar to that of ANP. The increased accumulation of cyclic GMP by these peptides may be negatively modulated by protein kinase C and cyclic AMP and may cause the phosphorylation and activation of tyrosine hydroxylase-in cultured bovine adrenal medullary cells.     

Effects of dexamethasone on [3H]choline uptake by rat forebrain synaptosomes

Summary Dexamethasone (3–300 mol/l) did not affect uptake of choline (1 mol/l) by rat forebrain isolated nerve terminals (crude synaptosomal fraction). At concentrations which have been shown to increase choline uptake by rat superior cervical ganglion, dexamethasone had no effect on synaptosomal choline uptake at choline concentrations between 0.1 and 30 mol/l, nor on choline uptake which had been partially inhibited either by hemicholinium-3 (0.1 mol/l) or by reducing the NaCl concentration (0-140 mmol/l).     

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     

Dopamine D-2 receptors inhibit D-1 stimulated cyclic AMP accumulation in striatum but not limbic forebrain

Summary The effect of dopamine D-2 receptor activtion on dopamine D-1 stimulated cyclic AMP accumulation was investigated in slices of rat striatum and limbic forebrain (nucleus accumbens and tuberculum olfactorium). In striatal slices the dose-dependent increase in cyclic AMP accumulation due to dopamine (3–100 mol/1) was enhanced by selective D-2 receptor blockade using (–)-sulpiride (30 mol/1). In limbic slices the increase in cyclic AMP due to dopamine (3–50 mol/l) was unaffected by selective D-2 receptor blockade. The enhancement of cyclic AMP accumulation due to the selective D-1 agonist SKF 38393 (2,3,4,5-tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine; 1 gmol/1) in striatal slices was attenuated in the presence of the selective D-2 receptor agonist LY 171555 (quinpirole hydrochloride; 10 mol/l). This attenuation was in turn blocked by (–)-sulpiride (10 mol/1). In limbic slices LY 171555 (10 mol/l) had no effect on SKF 38393 (1 mol/l) stimulated cyclic AMP accumulation. Conversely muscarine receptor activation, using carbachol (10 mol/l), attenuated D-1 stimulated cyclic AMP accumulation in both striatum and limbic forebrain. Dopamine D-2 or muscarine receptor stimulation in either striatal or limbic slices did not attenuate cyclic AMP accumulation due to VIP (vasoactive intestinal polypeptide; 0.5 mol/l), isoprenaline (10 mol/l) or 2-chloroadenosine (100 mol/l). This suggests that in striatal slices, D-2 receptors mediate a selective inhibition of D-1 stimulated cyclic AMP accumulation, but that in the limbic forebrain D-2 receptors are unlikely to be coupled to D-1 receptor-linked adenylate cyclase. These data indicate a fundamental difference in the properties of D-2 receptor-effector coupling in these brain regions. Send offprint requests to S. R. Nahorski 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     

Opposing actions of D-1 and D-2 dopamine receptor-mediated alterations of adenosine-3′,5′-cyclic monophosphate (cyclic AMP) formation during the amphetamine-induced release of endogenous dopamine in vitro

Summary Changes in the formation of cyclic AMP following d-amphetamine (0.1 to 20 pmol/1) were examined in vitro in striatal slices of the rat. d-Amphetamine caused a doserelated increase in cyclic AMP content. This action of d-amphetamine was abolished by tissue pretreatment with reserpine (2.5 mg/kg, i.p.) and 3-iodotyrosine (1 mmol/1). By contrast, both clorgyline (0.1 pmol/l) and nomifensine (30 mol/l) enhanced the d-amphetamine-induced increase in cyclic AMP formation. In superfusion experiments, a strong correlation between endogenous dopamine and cyclic AMP release was observed before, during and after d-amphetamine exposure. Finally, Sch 23390 (10 mol/1) abolished while (–)sulpiride (10 mol/1) enhanced the amphetamine-induced increase in cyclic AMP content. These results suggest that d-amphetamine enhances the formation of cyclic AMP through the release of endogenous dopamine into the synapse where it can interact with both D-1 and D-2 dopamine receptors. These results provide direct evidence that the antagonistic properties of D-1 and D-2 receptors on cyclic AMP formation are apparent at striatal synapses during release of endogenous neuronal dopamine.Abbreviations DA dopamine - 5-HT serotonin - CAMP cyclic AMP adenosine-3,5-cyclic monophosphate Supported in part by the West Virginia University Medical Corporation and a grant from the Fraternal Order of Eagles. Some of the findings were presented at the Annual meeting of the Society for Neurosciences, Washington, DC (Azzaro and Liccione 1986) Send offprint requests to A. J. Azzaro 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     

Coinduction of multiple hepatic cytochrome P-450 proteins and their mRNAs in rats treated with imidazole antimycotic agents

To characterize the molecular basis by which imidazole antimycotic drugs increase cytochrome P-450, we examined the effects of treating female rats with clotrimazole, miconazole, or ketoconazole on expression of the major inducible forms of hepatic cytochromes P-450 (P-450p, P-450b/e, P-450c/d, and P-450j). From measurements of the content of immunoreactive cytochromes P-450 in liver microsomes and of the amounts of liver RNA hybridizing to cloned P-450 cDNAs, we established that the glucocorticoid-responsive P-450p is the form predominantly induced by clotrimazole, miconazole, and ketoconazole, to as much as 382 times above control values. The phenobarbital-responsive cytochromes P-450b/e were also induced strongly by clotrimazole and miconazole, but not by ketoconazole. Aromatic hydrocarbon-inducible cytochromes P-450c/d were modestly elevated by each of these three antifungal drugs whereas ethanol-responsive P-450j was marginally induced by ketoconazole, but not by clotrimazole or miconazole. In some, but not all cases, treatment of rats with antifungal drugs resulted in accumulation of P-450 protein that significantly exceeded the increase in the corresponding P-450 mRNA. In conclusion, imidazole antifungal drugs differentially modulate the expression of at least four distinct gene subfamilies of rat hepatic cytochrome P-450 by separate mechanisms involving accumulation of P-450 mRNA and protein.     

Adenosine modulation of non-adrenergic non-cholinergic neurotransmission in isolated guinea-pig atria

Summary Transmural stimulation of non-adrenergic, non-cholinergic sensory nerves in guinea-pig atria, isolated from reserpine-pretreated animals, in the presence of atropine and the beta-adrenoceptor-blocking drug CGP 20712A, induced a positive inotropic effect. Adenosine (0.1–10 M) concentration-dependently reduced the cardic response to transmural nerve stimulation, without modifying the response to exogenous calcitonin gene-related peptide; the inhibitory effect of adenosine was antagonized by 1 M 8-phenyltheophylline. Moreover, the cardiac response to field stimulation was enhanced by 8-phenyltheophylline (0.1, 1 M) and by adenosine deaminase (1 g/ml), but was reduced by dipyridamole (1 M). These findings indicate the presence of inhibitory adenosine receptors on cardiac sensory nerves and suggest a modulatory effect of endogenous adenosine on cardiac non-adrenergic, non-cholinergic neurotransmission.Send offprint requests to A. Rubino at the above address     

The dihydropyridine derivative,Bay K 8644, enhances insulin secretion by isolated pancreatic islets

Summary The effects of the dihydropyridine derivative Bay K 8644 upon insulin secretion by perifused isolated mouse pancreatic islets were examined. At a non-stimulatory glucose concentration (5 mmol/l) Bay K 8644 (1 mol/l) did not stimulate insulin release. However, the same drug concentration enhanced the insulin secretory responses to an intermediate (15 mmol/l) or high (30 mmol/l) glucose concentration by 80 or 90%, respectively. Bay K 8644 was half maximally effective at 0.1 mol/l and maximally effective at 1 mol/l. The results are compatible with the view that voltage-dependent calcium channels are essential for stimulus-secretion coupling in pancreatic B-cells.Part of the medical thesis of M.-T. Schrader     

α2-Adrenoceptor-mediated autoinhibition of sympathetic transmitter release in guinea-pig vas deferens studied by intracellular and focal extracellular recording of junction potentials and currents

Summary Excitatory junction potentials (e j.ps; intracellular electrodes) and excitatory junction currents (e.j.cs; extracellular electrodes) elicited by stimulation (20 pulses at 1 Hz every minute) of the hypogastric nerve trunk were recorded from guinea-pig isolated vas deferens. Intracellular recording. At a variety of stimulation intensities, bath-applied yohimbine (0.1–1 mol/l) did not change the first one to three e j.ps in a train but increased the amplitude of subsequent e j.ps. The effect of yohimbine was abolished in tissues from reserpinepretreated guinea pigs. Bath-applied desipramine (0.1 mol/l) diminished the amplitude of all but the first one to three e j.ps in a train. - Extracellular recording. Yohimbine (0.1–1 mol/l), when applied locally through the recording suction electrode, increased the number of e.j.cs per given number of stimuli, i. e., enhanced the probability of occurrence of e.j.cs. When desipramine (0.1 mol/l) was present both in the bath and in the recording electrode, the probability of the occurrence of e.j.cs was decreased. In the presence of desipramine, yohimbine (0.1–1 mol/l) increased the number of e j.cs even more markedly. Neither the nerve terminal impulse nor the number of spontaneous e j.cs was changed by yohimbine. A mixture of tetraethylammonium (2 mmol/l) and 4-aminopyridine (0.2 mmol/l), when applied locally, both increased the number of e.j.cs and changed markedly the shape of the nerve terminal impulse.These experiments demonstrate presynaptic ₂-autoinhibition at a high degree of resolution, i. e., when the intermittent release of transmitter from only a few varicosities along a single terminal axon is monitored by the e j.c. ₂-Autoinhibition is not due to a depression of impulse conduction but to a depression of stimulus-secretion coupling in varicosities reached by the impulse. Taken together with the low probability of transmitter release at the level of individual varicosities, the results support the idea of lateral inhibition by noradrenaline released from distant varicosities rather than inhibition due to noradrenaline released from the same varicosity. The mode of action of yohimbine differs from that of K⁺ channel blocking agents. Send offprint requests to K. Starke at the above address