Effects of avitriptan, a new 5-HT1B/1D receptor agonist, in experimental models predictive of antimigraine activity and coronary side-effect potential

Several acutely acting antimigraine drugs, including ergotamine and sumatriptan, have the ability to constrict porcine arteriovenous anastomoses as well as the human isolated coronary artery. These two experimental models seem to serve as indicators, respectively, for the therapeutic and coronary side-effect potential of the compounds. Using these two models, we have now investigated the effects of avitriptan (BMS-180048; 3-[3-[4-(5-methoxy-4-pyrimidinyl)-1-piperazinyl]propyl]-N-methyl-1H-indole-5-methanesulfonamide monofumarate), a new 5-HT_1B/1D receptor agonist. In anaesthetized pigs, avitriptan (10, 30, 100 and 300 μg·kg^–1) decreased the total carotid blood flow by exclusively decreasing arteriovenous anastomotic blood flow; capillary blood flow was increased. The mean ± SEM i.v. dose of avitriptan eliciting a 50% decrease (ED₅₀) in the porcine carotid arteriovenous anastomotic blood flow was calculated to be 76 ± 23 μg·kg^–1 (132 ± 40 nmol·kg^–1) and the highest dose (300 μg·kg^–1) produced a 72 ± 4% reduction. In recent comparative experiments (DeVries et al. 1996), the mean ± SEM ED₅₀ (i.v.) of sumatriptan in decreasing carotid arteriovenous anastomotic blood flow was 63 ± 17 μg·kg^–1 (158 ± 43 nmol·kg^–1), with a reduction of 76 ± 4% by 300 μg·kg^–1, i.v. Both avitriptan (pD₂: 7.39 ± 0.09; E_max: 13.0 ± 4.5% of the contraction to 100 mM K⁺) and sumatriptan (pD₂: 6.33 ± 0.09; E_max: 15.5 ± 2.3% of the contraction to 100 mM K⁺) contracted the human isolated coronary artery. The above results suggest that avitriptan should be able to abort migraine headaches in patients, but may exhibit sumatriptan-like effects on coronary arteries. Initial clinical studies have demonstrated the therapeutic action of the drug in acute migraine. Received: 23 August 1996 / Accepted: 19 October 1996     

Potassium channels are involved in testosterone-induced vasorelaxation of human umbilical artery

Recent studies have shown that testosterone induces relaxation of different arteries, although the mechanism of this action is still under debate. We investigated the involvement of potassium channels in this mechanism. Using standard organ bath techniques, rings of human umbilical arteries (HUA) without endothelium were contracted by serotonin (5-HT, 1 μM), histamine (10 μM) and potassium chloride (KCl, 30 and 60 mM), and the vasorelaxant effect of testosterone was analysed. Testosterone (100 μM) relaxed human umbilical arteries contracted with 5-HT (30.1 ± 3.2%), histamine (55.1 ± 2.6%), KCl 30 mM (52.9 ± 8.3%) and KCl 60 mM (54.8 ± 6.3%). Flutamide (10 μM), an inhibitor of classical intracellular testosterone receptor, and glibenclamide, an ATP-sensitive potassium-channels (K_ATP) inhibitor, did not influence the testosterone relaxant effect. 4-aminopyridine, a voltage-sensitive potassium-channels (Kv) inhibitor, decreased the effect of testosterone on histamine- and 5-HT-contracted arteries. Tetraethylammonium (TEA), which inhibits Kv channels and large-conductance Ca²⁺-activated potassium channels (BK_Ca), decreased the effect of testosterone on KCl (60 mM)-contracted and 5-HT-contracted HUA. In conclusion, testosterone induces relaxation of HUA, and this effect does not appear to be mediated via a classic intracellular testosterone receptor-dependent mechanism. Our results suggest that this relaxation is partially mediated by activation of BK_Ca and K_V channels. The involvement of these two channels in testosterone-relaxant mechanism is dependent on the pathways activated by the contractile agent used.     

Mechanisms of changes in coronary arterial tone induced by bee venom toxins

Our study elucidates some mechanisms of contractions or relaxations of isolated porcine left anterior descending coronary artery (LAD) induced by two peptides from the honeybee venom, melittin and apamin. Contractions or relaxations were measured on relaxed or precontracted arteries, respectively. Melittin at lower concentrations (0.1-10 μg/ml) induced transient relaxation, and contraction at higher concentrations (≥7 μg/ml). The removing of the endothelium diminished the melittin-induced relaxation but did not affect the maximal contraction. The inhibition of prostaglandin and nitric oxide (NO) synthesis (by indomethacin and by N-omega-Nitro-l-arginine, respectively) and the use of K⁺ channel inhibitors (apamin and charybdotoxin) showed that melittin evoked relaxation via an endothelium-dependent mechanism (NO production), and by activation of charybdotoxin-sensitive K⁺ channels of smooth muscle. Apamin alone did not affect contraction or relaxation, but the inhibition of NO and prostanoid production revealed the involvement of apamin-sensitive K⁺ channels of smooth muscle in melittin-induced relaxation. Our data show that melittin and apamin could affect contractility of porcine LAD at concentrations similar to those encountered in multiple honeybee stings in humans. Melittin could directly affect contractility of porcine LAD, whereas apamin acts as a modulator of the relaxant response to melittin.     

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.     

Cytotoxicity and genotoxicity of capsaicin in human neuroblastoma cells SHSY-5Y

Capsaicin, a natural product of Capsicum species, induces excitation of pain perception at nociceptive terminals. Our previous studies have shown that capsaicin inhibits protein synthesis in cultured monkey kidneys cells (Vero cells) and in primoculture of rat astrocytes. We have now investigated the effect of capsaicin on human neuroblastoma cells SHSY-5Y. The cytotoxicity has been assessed by incorporation of [³H]L-leucine into cellular protein in the presence of capsaicin and the genotoxicity has been evaluated using the comet assay and the fragmentation assay after incubation of neuroblastoma cells with 25–100 μM capsaicin. The concentration required to inhibit 50% of the protein synthesis (IC₅₀) was found to be 60 μM after incubation with the toxin during one cellular cycle (5 days) of SHSY-5Y. The results of the comet test and DNA fragmentation assay clearly suggest that capsaicin is able to induce DNA strand breaks already with concentrations in the range of 50 μM, corresponding to 29.3 μM of capsaicin not bound to alpha-1 acid glycoprotein. Several daily topical applications of preparations containing 0.075% of capsaicin could lead to blood capsaicin concentration of this order of magnitude following transdermal passage (5% of the total quantity applied). Because DNA strand breaks or DNA lesions may affect cellular functions, lead to cell death and/or mutagenesis, our data in case of inappropriate DNA repair may have important implications for the possible health threats of capsaicin, specially in the case of misuse of capsaicin preparations in pathological situations. Received: 30 November 1998 / Accepted: 16 June 1999     

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.     

The inhibitory modulation of guinea-pig intestinal peristalsis caused by capsaicin involves calcitonin gene-related peptide and nitric oxide

The effect of capsaicin-induced stimulation of afferent neurons on peristalsis and the possible neural mediators involved in this action were examined in the guinea-pig isolated ileum. The intraluminal pressure threshold for eliciting peristaltic waves was used to quantify facilitation (decrease in threshold) or inhibition (increase in threshold) of peristalsis. Capsaicin (0.1–1 M) caused an initial short-lasting stimulation of peristalsis followed by a prolonged inhibition of peristaltic activity. Capsaicin (1 M) was ineffective when the gut segments had been pretreated with 3.3 M capsaicin, which is indicative of an afferent neuron-dependent action of the drug. In contrast, the abolition of peristalsis caused by a high concentration of capsaicin (33 M) was fully reversible on removal and reproducible on readministration of capsaicin, a feature characteristic of a nonspecific depression of smooth muscle excitability. Baseline peristalsis and the excitatory/inhibitory effect of capsaicin (1 M) on peristalsis remained unaltered by a combination of the tachykinin NK₁ receptor antagonist ( + )-(2S, 3S)-3-(2-methoxybenzylamino)-2-phenyl piperidine (CP-99,994; 0.3 M) and the tachykinin NK₂ receptor antagonist L(-)-N-methyl-N[4-acetylamino-4-phenyl-piperidino-2-(3,4-dichlorophenyl)butyl]-benzamide (SR-48,968; 0.1 M). Further experiments, performed in the presence of a low concentration of atropine (10 nM) showed that the catcitonin gene-related peptide (CGRP) antagonist human -catcitonin gene-related peptide (8–37) [hCGRP (8–37); 10 M] attenuated the delayed inhibitory effect of capsaicin on peristalsis, but did not influence baseline peristaltic activity and the capsaicin-induced facilitation of peristalsis. Blockade of nitric oxide (NO) synthesis by N ^G-nitro-l-arginine methylester (l-NAME, 300 M) facilitated baseline peristaltic activity and reduced the delayed inhibition of peristalsis caused by capsaicin (1 M) without affecting the initial peristalsis-stimulating action of capsaicin. The effects of l-NAME were prevented by l-arginine (1 mM). The data of the current study indicate that capsaicin-sensitive afferent neurons do not participate in the neural pathways subserving peristalsis in the guinea-pig small intestine, but modulate peristaltic activity upon stimulation with capsaicin. The initial stimulant action of capsaicin on peristalsis is independent of tachykinins acting via NK₁ or NK₂ receptors, while the delayed capsaicin-induced depression of peristalsis involves CGRP and NO.     

Cytotoxicity of capsaicin in monkey kidney cells: lack of antagonistic effects of capsazepine and Ruthenium red

Capsaicin is a natural product of Capsicum peppers, excitatory effects of which have been shown to be mediated by the recently cloned vanilloid receptor 1 (VR1). Since previous studies have shown that capsaicin inhibits protein synthesis, experiments were performed to investigate whether this effect is mediated by VR1 receptor on cultured monkey kidney cells (Vero cells). The capsaicin uptake was assessed in cellular homogenate and in medium by high-performance liquid chromatography (HPLC) separation and quantification on C18 reverse-phase column and fluorescence detection. Toxic effects were assessed by incorporation of [³H]L-leucine into cellular proteins in the presence of capsazepine, the VR1 vanilloid receptor antagonist and Ruthenium red or tyrosine or calcium. Capsazepine (1 to 256 μM) did not modify the uptake rate of capsaicin for incubation times up to 24 h and did not antagonize capsaicin-induced protein synthesis inhibition. It rather inhibited protein synthesis per se from 100 to 256 μM. Ruthenium red which blocks mitochondrial calcium uptake, inhibited protein synthesis and did not antagonise or increase synergistically the effects of capsaicin. Interestingly in a medium deprived of calcium and supplemented by calcium chloride (10–50 μM) the protein synthesis inhibition induced by capsaicin is antagonised somehow. There was no prevention of capsaicin diffusion into the cells. Tyrosine, which seems to be the best preventive agent of capsaicin inhibitory effects, prevents its metabolism but not its diffusion. Capsaicin might enter cells by diffusion and interfere with protein synthesis machinery by competition with tyrosine which in turn prevents the metabolism of capsaicin. The results of the present study suggest that cell responses to capsaicin may be transduced through at least two molecular pathways, one involving VR1, since the receptor antagonist capsazepine fails to prevent the inhibitory effect of capsaicin in Vero cells of renal origin. Received: 27 August 1999 / Accepted: 3 November 1999     

Overnight storage of the porcine isolated splenic artery enhances endothelium-dependent contractions to NG-nitro-l-arginine methyl ester without impairing endothelium-dependent dilator function

This study examined the influence of overnight storage on endothelium-independent contractions to 5-hydroxytryptamine (5-HT), endothelium-dependent contractions to N^G-nitro-l-arginine methyl ester (L-NAME), and endothelium-dependent relaxations to substance P (SP) and l-arginine, using the porcine isolated splenic artery. In endothelium-intact (E+) segments from fresh porcine isolated splenic arteries or segments from the same vessels stored overnight at 4°C, either in Krebs-Henseleit saline or in Krebs-Henseleit saline containing 1 MM l-arginine, 5-HT caused concentration-related contractions that were similar under all three conditions. Overnight storage enhanced contractions of the splenic artery to L-NAME, an effect not observed if the vessels were co0-stored with 1 MM l-arginine. L-NAME failed to contract endothelium-denuded (E–) segments from fresh tissues or tissues stored overnight, indicating that its constrictor effects were endothelium-dependent. SP caused concentration-related, endothelium-dependent relaxations of the splenic artery that were inhibited by 100 gM L-NAME, indicating that the relaxations could be attributed to the stimulated release of NO from endothelial cells. Established contractions to 100 M L,NAME in E+ segments from fresh tissues, or segments from the same tissues stored overnight at 4°C, either in KrebsHenseleit saline or in Krebs-Henseleit saline containing 1 MM l-arginine, were all reversed by 1 MM l-arginine to similar extents, indicating that overnight storage did not affect endothelium-dependent dilator responses to l-arginine. Our findings indicate that overnight storage enhances endothelium-dependent contractions to L-NAME without affecting endothelium-independent contractions to 5-HT, or endothelium-dependent dilator responses to SP or l-arginine. Correspondence to: V.G. Wilson at the above address     

Relaxation by adenosine and its analogs of potassium-contracted human coronary arteries

Summary The present study was an attempt to characterize the adenosine receptor in human coronary arteries, and to establish the dependence of the relaxations mediated by this receptor on a functional endothelium. Human coronary arteries were obtained from organ donors. Adenosine and its analogs (5-N-ethyl-carboxamido-adenosine, NECA; N⁶-l-phenylisopropyladenosine, LPIA; 2-chloroadenosine, CAD), all inhibited the contraction induced by 25 mmol/l KCl in a concentration-dependent manner and the order of potency was found to be NECA > CAD > L-PIA > adenosine. These relaxations were antagonized by 8-phenyltheophylline (8PT). At higher concentrations of KCl, the relaxations were attenuated. In rings which relaxed in response to endothelium-dependent relaxing agents (bradykinin and A23187), NECA and CAD produced relaxations similar to those produced in rings which did not show endothelium-dependent responses. The results suggest that the coronary adenosine receptor (probably A₂) mediates relaxations which may not be dependent on the relaxing function of the endothelium. Send offprint requests to S. J. Mustafa at the above address     

Pharmacological differentiation of synergistic contribution of L-type Ca2+ channels and Na+/H+ exchange to the positive inotropic effect of phenylephrine,endothelin-3 and angiotensin II in rabbit ventricular myocardium

The present study was designed to delineate pharmacologically the role of sarcolemmal L-type Ca²⁺ channels and Na⁺/H⁺ exchange in the positive inotropic effect (PIE) of phenylephrine mediated by alpha-1 adrenoceptors, endothelin (ET) and angiotensin II (Ang II) that stimulate phosphoinositide (PI) hydrolysis in the rabbit ventricular muscle. The PIE of these receptor agonists was compared with the PIE of isoprenaline that accumulates cyclic AMP. For this purpose, we investigated the influence of a Ca²⁺ antagonist, verapamil, and of an inhibitor of Na⁺/H⁺ exchange, 5-(N-ethyl-N-isopropyl) amiloride (EIPA), alone or in combination, on the cumulative concentration-response curve (CRC) for phenylephrine (with 0.3 μM bupranolol), ET-3 and Ang II in isolated right ventricular papillary muscles of the rabbit, which were electrically stimulated at 1 Hz in Krebs-Henseleit solution at 37°C. Verapamil at 0.3 and 1 μM decreased the basal force of contraction to 37.0 ± 4.0% and 13.2 ± 1.1% of the control, respectively, while EIPA even at 10 μM affected the basal force to much less extent and decreased it to 87.0 ± 1.4%. Verapamil (0.3 and 1 μM) and EIPA (1 and 10 μM), when used alone, each significantly attenuated but did not abolish the PIEs induced by phenylephrine, ET-3 and Ang II, while the simultaneous administration of verapamil (1 μM) and EIPA (10 μM) consistently and almost completely inhibited the PIE induced by these receptor agonists. By contrast, the PIE of isoprenaline was retained even in the presence of verapamil and EIPA. These results indicate that both the influx of Ca²⁺ ions through L-type Ca²⁺ channels and activation of Na⁺/H⁺ exchange contribute synergistically to the PIE that is mediated by alpha-1 adrenergic, ET and Ang II receptor agonists, while these mechanisms are not essential for the beta-adrenoceptor-mediated PIE. Received: 20 February 1996 / Accepted: 20 August 1996     

Sensory nerves,vascular endothelium and neurogenic relaxation of the guinea-pig isolated pulmonary artery

Summary 1. In the presence of atropine and guanethidine (3 mol/l each), electrical field stimulation (1–20 Hz) produced frequency-dependent relaxations of the histamine-(3 mol/l) induced vascular tone in isolated rings from the guinea-pig pulmonary artery. The electrically-evoked relaxations were abolished by tetrodotoxin (1 mol/l). The amplitude of these nerve-mediated, nonadrenergic non-cholinergic (NANC) relaxations was unaffected by removal of the vascular endothelium produced through rubbing of the internal surface. 2. Capsaicin (1 mol/l) produced a prompt and sustained relaxation of the histamine-induced tone which was unaffected by removal of the endothelium. A second application of capsaicin 60–120 min later had no further relaxant effect, indicating desensitization. After in vitro capsaicin desensitization, the electrically-evoked NANC relaxations were abolished, both in the presence or absence of the vascular endothelium. 3. Substance P evoked a prompt and transient relaxation in precontracted arterial rings with intact endothelium and a transient small contraction in rings in which the endothelium had been mechanically removed. The selective NK-1 receptor agonist, [Pro⁹]-substance P sulfone closely mimicked the relaxation produced by substance P while the selective NK-2 or NK-3 receptor agonists had no relaxant effect. Tachyphylaxis to substance P did not modify the amplitude of the capsaicin-induced relaxation. 4. Human alpha calcitonin gene-related peptide (CGRP) produced a prompt and sustained relaxation both in the presence and absence of the vascular endothelium. 5. Ruthenium red (10 mol/l) blocked the relaxation to capsaicin while leaving unaffected the relaxation to electrical field stimulation or CGRP (0.1 mol/l). 6. Both substance P (SP)-and CGRP-like immunoreactivities (LI) were detected in extracts of the guinea-pig pulmonary artery. Capsaicin (1 mol/l) evoked a prompt and simultaneous outflow of both SP- and CGRP-LI. A second application of capsaicin 60 min later failed to increase SP- or CGRP-LI outflow, indicating complete desensitization. A small but clearly detectable release of both SP-LI and CGRP-LI was also evoked by electrical field stimulation. 7. These findings provide evidence that the neurogenic NANC vasodilation in the guinea-pig pulmonary artery is due to antidromic activation of peripheral endings of capsaicin-sensitive primary afferents. Endogenous CGRP is a likely mediator for this vasodilation. No evidence was found that endogenous SP might contribute to vasodilation by activating NK-1 receptors on endothelial cells. Send offprint requests to C. A. Maggi at the above address     

Characterization of calcitonin gene-related peptide(CGRP) receptors in intramural coronary arteries from male and female Sprague Dawley rats

1. In this study we characterized the CGRP-receptor subtype by Schild-plot analysis using the C-terminal fragment, human-αCGRP(8–37), a putative competitive CGRP₁-receptor selective antagonist. In addition, the effect of rat-αCGRP was compared with that of homologous peptides rat-βCGRP, rat-amylin, rat-adrenomedullin and [Cys(Acm)^2,7]-human-αCGRP, a putative selective CGRP₂-receptor agonist, in the left coronary arteries of 3 months old male and female Sprague Dawley rats.
2. Isolated rings from the distal, intramural part of the left anterior descending (LAD) coronary artery in both groups of rats were mounted on a double wire-myograph. The arteries were then stretched to their optimal lumen diameter for active tension development and precontracted with 10⁻⁵ M prostaglandin F_2α (PGF_2α), after which agonists were added to the organ bath in a cumulative manner.
3. Rat-αCGRP induced endothelium-independent relaxations in male and female Sprague-Dawley rats. Rat-βCGRP concentration-response relations (10⁻¹¹–10⁻⁷ M) were similar to those of rat-αCGRP in either sex. The maximal relaxations induced by rat-amylin and rat-adrenomedullin, both at 10⁻⁶ M, were significantly (P<0.05) lower than those induced by rat-α- and rat-βCGRP. In contrast, the selective CGRP₂-receptor agonist [Cys(Acm)^2,7]-human-αCGRP failed to induce significant relaxations at the highest concentration used (10⁻⁷ M) in the coronary arteries of male and female rats.
4. The C-terminal fragment, human-αCGRP(8–37) blocked concentration-dependently (10⁻⁷–10⁻⁶ M) the rat-αCGRP-induced relaxation in 10⁻⁵ M PGF_2α-precontracted coronary arteries. The slopes of the regression lines of the Schild-plots for both male and female rats were not significantly (P>0.05) different from unity and the pA₂ values for human-αCGRP(8–37) were 6.93 and 6.98 in arteries from male and female rats, respectively. There was no significant (P>0.05) difference in estimated pK_B values for human-αCGRP(8–37) between male (6.99±0.10, n=13) and female (6.95±0.08, n=13) rats.
5. The concentration-response relationships for rat-α- and rat-βCGRP were similar in male and female Sprague Dawley rats. The predominant CGRP receptor subtype in small intramural coronary arteries appeared to belong to the CGRP₁-receptor subtype in both sexes.

     

β-Adrenoceptor mediated responses in rat pulmonary artery: putative role of TASK-1 related K channels

The effect of isoprenaline on tone, cyclic adenosine 3′:5′ monophosphate (cAMP), and smooth muscle membrane potential (E _m ) were assessed in rat isolated pulmonary arteries. N^ω-nitro-L-arginine methyl ester (10.0 μM) or removal of endothelium partially inhibited relaxant responses to isoprenaline, but glibenclamide (10.0 μM) and indomethacin (10.0 μM) did not. While Rp-8-Br-cAMP (30.0 μM), tetraethylammonium (0.3 & 1.0 mM), 4-aminopyridine (100 μM), anandamide (10.0 μM), charybdotoxin (0.1 μM), ouabain (100 μM), and barium chloride (100 μM), incompletely blocked relaxation to isoprenaline, cyclopiazonic acid (1.0 μM), apamin (3.0 μM) and zinc acetate (300 μM) were without effect. Increasing extracellular K⁺ ([K⁺]_e) inhibited relaxant responses to isoprenaline, completely abolishing the response at 30 mM [K+]_e. Vasorelaxant effects of isoprenaline were significantly attenuated in buffer pH 6.4, and concomitant presence of Rp-8-Br-cAMP (30.0 μM) in pH 6.4 produced significant additive inhibition when compared to pH 6.4 without Rp-8-Br-cAMP. Isoprenaline increased cAMP turnover (1.55±0.24 fold; mean ± SEM), which was inhibited by propranolol (1.0 μM). Resting E _m of smooth muscle cells was -63.0±0.50 mV, and isoprenaline (1.0 μM) produced hyperpolarisation (−73.3±0.80 mV). While glibenclamide failed to affect isoprenaline-induced hyperpolarisation, ICI 118,551 (1.0 μM), anandamide or buffer pH 6.4 prevented it, and barium chloride and oubain combined caused partial inhibition. Isoprenaline-mediated relaxation seems to arise from several processes, including the generation of nitric oxide, the cAMP-cascade and, more importantly, a hyperpolarisation that is not due to activation of ATP-sensitive K channels but possibly of two-pore domain K channels of the TASK family.     

Structure-activity relationship of a novel K+ channel opener,KRN4884, and related compounds in porcine coronary artery

• 1.1. KRN4884 (5-amino-N-[2-(2-chlorophenyl)ethyl]-N′-cyano-3-pyridinecarboxamidine), Ki3005 (5-deamino KRN4884), Ki5624 (2-dechloro KRN4884) and Ki1769 (5-deamino-2-dechloro KRN4884) produced concentration-dependent relaxations in isolated porcine coronary arteries contracted by 25 mM KCI. The order of relaxant potency was KRN4884>Ki3005>Ki5624>Ki1769.
• 2.2. The relaxation induced by these compounds was antagonized by glibenclamide; they had almost no effect on coronary arteries contracted by 60 mM KCl.
• 3.3. The present results suggest that these pyridinecarboxamidine derivatives have vasodilating ability based on a K⁺ channel opening action, and that both the amino groups in the pyridine nucleus and the chlorine atom in the benzene nucleus in pyridinecarboxamidine are important for their potency as a K⁺ channel opener.

     

Relationship between acute toxicity of (bis)aziridinylbenzoquinones and cellular pyridine nucleotides

The toxicity of aziridinylbenzoquinones may occur by a number of mechanisms, including oxidative stress caused by redox cycling and the activation of the aziridine groups. Isolated hepatocytes were used to assess the relationship between the redox status of NADP(H) associated with oxidative stress, the level of NAD(H) closely linked with DNA repair and the cytotoxicity of three 2,5-bis(aziridinyl)-1,4-benzoquinones (BABQ). Exposure of hepatocytes to the BABQ TW13 (200 μM) and TW25 (100 μM), which are able to arylate and to redox cycle, resulted in increased intracellular NADP⁺ from <0.3 nmol/mg protein to 1.5 nmol/mg protein within 60 min. The increase in intracellular NADP⁺ was followed by the onset of cell death by 180 min. In contrast, exposure to lower concentrations of TW13 (100 μM), TW25 (50 μM) and carboquone (100–200 μM) (which neither arylates nor redox cycles via a one-electron reduction) resulted in a less pronounced (<1.0 nmol/mg) increase in NADP⁺ and there was no evidence of cell death within the 180 min incubation. BABQ had a concentration dependent effect on intracellular NAD⁺. Exposure of hepatocytes to TW13 (200 μM) and TW25 (100 μM) resulted in a decrease in intracellular NAD⁺ from >2.7 to <1.0 nmol/mg protein within 60 min. At concentrations of the BABQ where the level of NAD⁺ remained  >1.0 nmol/mg protein after 30 min, the hepatocytes remained viable at 180 min. These changes in intracellular pyridine nucleotides suggests two mechanisms may be involved in BABQ cytotoxicity. At high concentrations, aziridinylbenzoquinones may cause cytotoxicity via oxidative stress following redox cycling. At lower concentrations, however, the predominant pyridine nucleotide change is a prolonged depletion of NAD⁺, suggesting extensive DNA damage which may lead to delayed cell death. Received: 30 October 1996 / Accepted: 13 January 1997     

RELAXANT EFFECTS OF VASODILATOR PEPTIDES ON ISOLATED BASILAR ARTERIES FROM STROKE-PRONE SPONTANEOUSLY HYPERTENSIVE RATS

1. The relaxant effects of vasodilator peptides were examined in ring preparations of basilar arteries from stroke-prone spontaneously hypertensive rats (SHRSP) and Wistar-Kyoto (WKY) rats. 2. Vasoactive intestinal peptide and peptide histidine iso-leucine produced similar endothelium-independent relaxations in basilar arteries from WKY rats and SHRSP. Calcitonin gene-related peptide (CGRP) elicited endothelium-independent relaxations in both groups and the CGRP-induced relaxation was greater in SHRSP than in WKY rats. Substance P and neurokinin A did not relax basilar arteries from either group. 3. Both WKY rat and SHRSP basilar arteries were relatively insensitive to atrial natriuretic peptide, brain natriuretic peptide and C-type natriuretic peptide. 4. Bradykinin (BK) potently relaxed basilar arteries with endothelium, but BK produced contractions in endotheliumrubbed arteries in both WKY rats and in SHRSP. There was no significant difference in the relaxant response to BK between WKY rat and SHRSP arteries. 5. Adrenomedullin (AM) produced endothelium-independent relaxations in both groups and the relaxant response to AM was significantly greater in SHRSP than in WKY rats. 6. Human CGRP(8–37; 1 μmol/L), a CGRP receptor antagonist, significantly inhibited both relaxant responses induced by CGRP and AM in WKY rats and in SHRSP arteries. 7. Among various peptides tested, the responses to CGRP and AM were higher in basilar arteries from SHRSP than in those from WKY rats. The relaxation produced by AM may be via CGRP receptors in WKY rat and SHRSP basilar arteries.     

Involvement of Recognition and Interaction of Carnitine Transporter in the Decrease of l-Carnitine Concentration Induced by Pivalic Acid and Valproic Acid

Purpose Prodrugs with pivalic acid and valproic acid decrease l-carnitine concentration in plasma and tissues by urinary excretion of acylcarnitine as pivaloylcarnitine (PC) and valproylcarnitine (VC), respectively. We investigated the role of the Na⁺/l-carnitine cotransporter in the porcine kidney epithelial cell line, LLC-PK₁ for the decrease of l-carnitine concentration.Methods The uptake of l-[³H]carnitine, acetyl-l-[³H]carnitine (AC), l-[³H]PC and l-[³H]VC were investigated in LLC-PK₁ cells seeded in a 6-well culture plate.Results l-Carnitine and AC uptake in LLC-PK₁ cells exhibited Na⁺ dependency. The K _m values for l-carnitine and AC uptake were 11.0 and 8.18 μM, respectively. These results indicated expression of Na⁺/l-carnitine cotransporter in LLC-PK₁ cells. PC and VC inhibited Na⁺/l-carnitine cotransporter in the competitive (K _i = 90.4 μM) and noncompetitive (K _i = 41.6 μM) manners, respectively. PC and VC uptake by Na⁺/l-carnitine cotransporter were not observed in LLC-PK₁ cells.Conclusions These data suggested that PC and VC formed in the body could not be reabsorbed in the kidney, resulting in the decrease of l-carnitine concentration. In addition, inhibition of l-carnitine reabsorption by VC with lower K _i value could induce the decrease of l-carnitine concentration. Collectively, the recognition and interaction of Na⁺/l-carnitine cotransporter are important factors for carnitine homeostasis.     

Inhibitory effects of large Ca2+-activated K+-channel blockers on β-adrenergic- and NO-donor-mediated relaxations of human and guinea-pig airway smooth muscles

In human bronchi, relaxations to salbutamol and sodium nitroprusside were performed in the presence or absence of blockers of the large Ca²⁺-activated K⁺-channels (BK_Ca): charybdotoxin (Chtx), iberiotoxin (Ibtx) or tetraethylammonium (TEA). In bronchi under basal tone in presence of indomethacin (1 μM) or precontracted with acetylcholine (in presence or absence of indomethacin), the relaxations to salbutamol or sodium nitroprusside were unaffected or weakly inhibited by pretreatment with the BK_Ca blockers (Chtx (100 nM), Ibtx (100 and 300 nM) and TEA (1 mM)). Significant inhibitions were mainly observed with TEA (1 mM) and iberiotoxin at high concentration (300 nM). These results contrasts with the potent inhibitory effects exerted by Chtx (100 nM) or Ibtx (100 nM) in guinea-pig trachea precontracted with acetylcholine in absence or presence of indomethacin indicating that human airways are less susceptible to BK_Ca blockade than guinea-pig airways. In addition, the BK_Ca blockers induced slowly developing contractions of human bronchi at basal tone. The contraction induced by TEA (1 mM) was abolished by verapamil (10 μM) suggesting that BK_Ca blockade promotes an increase in membrane Ca²⁺-conductance through activation of voltage-gated Ca²⁺-channels. Verapamil also reversed the effects of TEA on salbutamol-induced relaxations in human bronchi as well as the effects of Ibtx on salbutamol- or sodium nitroprusside-induced relaxations in guinea-pig trachea. These data suggest that BK_Ca blockers induce activation of voltage-gated Ca²⁺-channels and therefore influx of Ca²⁺ which in turn cause a functional antagonism of β₂-adrenoceptor-agonist- and NO-donor-induced relaxations. Moreover, the BK_Ca opener, NS-1619, induced weak relaxations in human bronchi and guinea-pig trachea which were not blocked by TEA or Ibtx suggesting that BK_Ca opening is of minor significance for the relaxation of human airway smooth muscles. In conclusion, although a wealth of studies have demonstrated that β-adrenoceptor agonists or NO-donors activate BK_Ca, the present study provides evidence that in human bronchi, as recently suggested in guinea-pig trachea, opening of BK_Ca does not appear to functionally participate in the relaxation to these relaxant agents. Received: 25 April 1997 / Accepted: 15 September 1997     

Mitochondrial K<Subscript>ATP</Subscript> channels participate in the limitation of infarct size by cariporide

The objective of this study is to assess the participation of mitochondrial ATP-sensitive potassium (mitoK_ATP) channels in the cardioprotective effects of the Na⁺/H⁺ exchanger (NHE-1) blocker cariporide in isolated rat hearts. Regional ischemia was induced by occlusion of left anterior descending coronary artery during 40 min followed by 2-h reperfusion (IC). Cariporide (C, 10 μΜ), or C plus 5-hydroxydecanoate (5-HD, 100 μM, a selective mitoK_ATP channel inhibitor), or C plus chelerythrine (Chele, 1 μM, a PKC inhibitor), or an opener of mitoK_ATP channels, diazoxide (Dz, 100 μM) was applied at the onset of reperfusion. Infarct size (IS) and myocardial function were evaluated. The calcium-induced permeability transition pore (mPTP) opening was determined by measuring the light scattering decrease (LSD, a.u.) in isolated mitochondria in the absence and presence of C, C + 5-HD and Dz. IS was 33 ± 2% of the risk area in IC and was significantly diminished by C (15 ± 2%, p < 0.05), which also improved myocardial function [LVDP = 58 ± 5% (IC) vs 80 ± 5% (C)] and blunted LSD [0.80 ± 0.04 (IC) vs 0.51 ± 0.04 (C) a.u.]. 5-HD and Chele were both able to abolish the cardioprotective effects of C on IS. Dz treatment decreased IS and LSD to a similar extent to that produced by C (15 ± 4% and 0.52 ± 0.04 a.u., respectively). The present data suggest that attenuation of mPTP opening after PKC-mediated mitoK_ATP channel activation is a crucial step for the cardioprotective effects of cariporide.