Effects of inhibitors of small- and intermediate-conductance calcium-activated potassium channels, inwardly-rectifying potassium channels and Na(+)/K(+) ATPase on EDHF relaxations in the rat hepatic artery.

1. In the rat hepatic artery, the SK(Ca) inhibitors UCL 1684 (300 nM) completely blocked, and scyllatoxin (1 microM) and d-tubocurarine (100 microM) partially inhibited EDHF relaxations when each of them was combined with charybdotoxin (300 nM). 2. The IK(Ca) inhibitors clotrimazole (3 microM) and 2-chlorophenyl-bisphenyl-methanol (3 microM) strongly depressed EDHF relaxations when each of them was combined with apamin (300 nM). The cytochrome P450 mono-oxygenase inhibitor ketoconazole (10 microM) had no effect in the presence of apamin. 3. Ciclazindol (10 microM), which abolishes EDHF relaxations in the presence of apamin, almost completely prevented the calcium ionophore (A23187) stimulated (86)Rb(+) influx via the Gardos channel (IK(Ca)) in human erythrocytes. 4. The Na(+)/K(+) ATPase inhibitor ouabain (500 microM) and the K(IR) blocker Ba(2+) (30 microM) neither alone nor in combination inhibited EDHF relaxations. Ba(2+) was also without effect in the presence of either apamin or charybdotoxin. 5. In contrast to EDHF, an increase in extracellular [K(+)] from 4.6 mM to 9.6, 14.6 and 19.6 mM inconsistently relaxed arteries. In K(+)-free physiological salt solution, re-admission of K(+) always caused complete and sustained relaxations which were abolished by ouabain but unaffected by Ba(2+). 6. The present study provides pharmacological evidence for the involvement of SK(Ca) and IK(Ca) in the action of EDHF in the rat hepatic artery. Our results are not consistent with the idea that EDHF is K(+) activating Na(+)/K(+) ATPase and K(IR) in this blood vessel.

Vasodilator action in the nitroxylated cyanoamidine derivative, KRN2391, in rabbit basilar artery.

KRN2391 is a cyanoamidine derivative with a pyridine ring and a nitroxyl group. This gives the molecule a dual pharmacological action as both an ATP-sensitive K channel (K(ATP)) opener and an organic nitrate. In cerebrovascular disease with endothelial dysfunction, such a compound could be advantageous to prevent the negative consequences of a reduced synthesis of endogenous nitric oxide and endothelium-derived hyperpolarizing factor. The objective of this study was to characterise the vasodilator action of KRN2391 in a cerebral artery. As shown in the rabbit basilar artery contracted by endothelin-1 KRN2391 elicited a concentration-dependent relaxation. KRN2391 was unable to relax arteries contracted by a 60 mM K solution. The KRN2391-induced relaxation of endothelin-1-contracted arteries was unaffected by N(G)-nitro-L-arginine (0.1 mM), indomethacin (10 microM) or removal of the endothelium. The guanylate cyclase inhibitors ODQ (10 microM) and LY53583 (10 microM), and the cGMP phosphodiesterase inhibitor zaprinast (10 microM) each had no effect on the KRN2391-induced relaxation. Glibenclamide (1 microM), a blocker of K(ATP), caused a rightward shift of the concentration-response curve for KRN2391. The relaxation induced by the prototype K(ATP) opener levcromakalim was inhibited to a similar extent by glibenclamide. Addition of ODQ or LY53583, or the calcium-sensitive K channel blockers apamin (0.1 microM) and charybdotoxin (0.1 microM) in the presence of glibenclamide did not produce a significant further inhibition of the KRN-induced relaxation. KRN2391 (10 microM) did not influence the content of cGMP in the basilar artery, whereas the nitric oxide donor 3-morpholino-sydnonimine (0.1 mM) increased the cGMP level three-fold. Thus, KRN2391 is an effective vasodilator of the rabbit basilar artery, acting mainly through opening of KATP . The nitro-moiety of the molecule does not seem to contribute to the relaxant effect in this artery.

Endothelial P2Y receptors induce hyperpolarisation of vascular smooth muscle by release of endothelium-derived hyperpolarising factor.

The effects of P2Y receptor agonists on smooth muscle membrane potential in isolated ring segments of rat mesenteric artery were examined by intracellular microelectrodes. In the presence of inhibitors of nitric oxide-synthase and cyclo-oxygenase, the selective P2Y1 receptor agonist adenosine 5'-O-thiodiphosphate (ADPbetaS) induced endothelium-dependent membrane hyperpolarisations, which were abolished by a combination of the K+ channel inhibitors charybdotoxin and apamin, providing direct evidence that ADPbetaS releases endothelium-derived hyperpolarising factor (EDHF). 2-MethylthioATP and ATP, each which stimulates both endothelial P2Y receptors and P2X receptors on the smooth muscle cells, also elicited hyperpolarisation, but only after desensitisation of P2X receptors with alphabeta-methylATP indicating that simultaneous activation of P2X receptors may counteract the action of EDHF. In conclusion, activation of endothelial P2Y receptors induce release of EDHF.

Characterization of the potassium channels involved in EDHF-mediated relaxation in cerebral arteries.

1. In the presence of NG-nitro-L-arginine (L-NOARG, 0.3 mM) and indomethacin (10 microM), the relaxations induced by acetylcholine and the calcium (Ca) ionophore A23187 are considered to be mediated by endothelium-derived hyperpolarizing factor (EDHF) in the guinea-pig basilar artery. 2. Inhibitors of adenosine 5'-triphosphate (ATP)-sensitive potassium (K)-channels (KATP; glibenclamide, 10 microM), voltage-sensitive K-channels (Kv; dendrotoxin-1, 0.1 microM or 4-aminopyridine, 1 mM), small (SKCa; apamin, 0.1 microM) and large (BKCa; iberiotoxin, 0.1 microM) conductance Ca-sensitive K-channels did not affect the L-NOARG/indomethacin-resistant relaxation induced by acetylcholine. 3. Synthetic charybdotoxin (0.1 microM), an inhibitor of BKCa and Kv, caused a rightward shift of the concentration-response curve for acetylcholine and reduced the maximal relaxation in the presence of L-NOARG and indomethacin, whereas the relaxation induced by A23187 was not significantly inhibited. 4. A combination of charybdotoxin (0.1 microM) and apamin (0.1 microM) abolished the L-NOARG/ indomethacin-resistant relaxations induced by acetylcholine and A23187. However, the acetylcholine-induced relaxation was not affected by a combination of iberiotoxin (0.1 microM) and apamin (0.1 microM). 5. Ciclazindol (10 microM), an inhibitor of Kv in rat portal vein smooth muscle, inhibited the L-NOARG/ indomethacin-resistant relaxations induced by acetylcholine and A23187, and the relaxations were abolished when ciclazindol (10 microM) was combined with apamin (0.1 microM). 6. Human pial arteries from two out of four patients displayed an L-NOARG/indomethacin-resistant relaxation in response to substance P. This relaxation was abolished in both cases by pretreatment with the combination of charybdotoxin (0.1 microM) and apamin (0.1 microM), whereas each toxin had little effect alone. 7. The results suggest that Kv, but not KATP and BKCa, is involved in the EDHF-mediated relaxation in the guinea-pig basilar artery. The synergistic action of apamin and charybdotoxin (or ciclazindol) could indicate that both Kv and SKCa are activated by EDHF. However, a single type of K-channel, which may be structurally related to Kv and allosterically regulated by apamin, could also be the target for EDHF.

Effects of potassium channel inhibitors on nitrergic and adrenergic neurotransmission in lamina propria of the female rabbit urethra.

Electrical field stimulation of strip preparations of the female rabbit urethral lamina propria induces a frequency-dependent adrenergic contraction or a non-adrenergic, non-cholinergic (NANC) relaxation, mediated by nitric oxide, depending on the prevailing tension. To study the role of potassium channels in these responses, the effects of inhibitors of voltage-dependent (dendrotoxin I, 4-aminopyridine), low (apamin) and high (iberiotoxin, charybdotoxin) conductance calcium-activated and ATP-sensitive (glibenclamide) potassium channels on the frequency-response relationship were examined. 4-Aminopyridine (1 mM), but none of the other inhibitors, augmented the NANC relaxation. The maximal response was, however, unaffected by 4-aminopyridine. The adrenergic contraction was enhanced by 4-aminopyridine (1 mM), dendrotoxin I (0.1 microM), iberiotoxin (0.1 microM) and charybdotoxin (0.1 microM), but not by apamin (0.1 microM) and glibenclamide (10 microM). Besides reducing the frequency eliciting half maximal contraction dendrotoxin and charybdotoxin also enhanced the maximal response. None of the inhibitors affected the relaxation induced by the nitric oxide donor 3-morpholinosydnonimine or the contraction elicited by noradrenaline. The results suggest that dendrotoxin-sensitive voltage-dependent and high conductance calcium-activated neuronal potassium channels participate in adrenergic, but not in nitrergic, neurotransmission in the lamina propria of the female rabbit urethra. This offers a possibility to selectively interfere with the adrenergic neuroeffector system with drugs acting on these K-channels.

Role of potassium channels in endothelium-dependent relaxation resistant to nitroarginine in the rat hepatic artery.

1. In the presence of indomethacin (IM, 10 microM) and N omega-nitro-L- arginine (L-NOARG, 0.3 mM), acetylcholine (ACh) induces an endothelium-dependent smooth muscle hyperpolarization and relaxation in the rat isolated hepatic artery. The potassium (K) channel inhibitors, tetrabutylammonium (TBA, 1 mM) and to a lesser extent 4-aminopyridine (4-AP, 1 mM) inhibited the L-NOARG/IM-resistant relaxation induced by ACh, whereas apamin (0.1-0.3 microM), charybdotoxin (0.1-0.3 microM), iberiotoxin (0.1 microM) and dendrotoxin (0.1 microM) each had no effect. TBA also inhibited the relaxation induced by the receptor-independent endothelial cell activator, A23187. 2. When combined, apamin (0.1 microM) + charybdotoxin (0.1 microM), but not apamin (0.1 microM) + iberiotoxin (0.1 microM) or a triple combination of 4-AP (1 mM) + apamin (0.1 microM) + iberiotoxin (0.1 microM), inhibited the L-NOARG/IM-resistant relaxation induced by ACh. At a concentration of 0.3 microM, apamin + charybdotoxin completely inhibited the relaxation. This toxin combination also abolished the L-NOARG/ IM-resistant relaxation induced by A23187. 3. In the absence of L-NOARG, TBA (1 mM) inhibited the ACh-induced relaxation, whereas charybdotoxin (0.3 microM) + apamin (0.3 microM) had no effect, indicating that the toxin combination did not interfere with the L-arginine/NO pathway. 4. The gap junction inhibitors halothane (2 mM) and 1-heptanol (2 mM), or replacement of NaCl with sodium propionate did not affect the L-NOARG/IM-resistant relaxation induced by ACh. 5. Inhibition of Na+/K(+)-ATPase by ouabain (1 mM) had no effect on the L-NOARG/IM-resistant relaxation induced by ACh. Exposure to a K(+)-free Krebs solution, however, reduced the maximal relaxation by 13% without affecting the sensitivity to ACh. 6. The results suggest that the L-NOARG/IM-resistant relaxation induced by ACh in the rat hepatic artery is mediated by activation of K-channels sensitive to TBA and a combination of apamin + charybdotoxin. Chloride channels, Na+/K(+)-ATPase and gap junctions are probably not involved in the response. It is proposed that endothelial cell activation induces secretion of an endothelium-derived hyperpolarizing factor(s) (EDHF), distinct from NO and cyclo-oxygenase products, which activates more than one type of K-channel on the smooth muscle cells. Alternatively, a single type of K-channel, to which both apamin and charybdotoxin must bind for inhibition to occur, may be the target for EDHF.

NANC neurotransmission in lamina propria of the rabbit urethra: regulation by different subsets of calcium channels.

1. Electrical field stimulation (EFS) of the rabbit urethral lamina propria elicited a frequency-dependent non-adrenergic, non-cholinergic (NANC) relaxation, which was abolished by N omega-nitro-L-arginine (L-NOARG). 2. omega-Conotoxin GVIA, a selective blocker of N-type voltage-operated calcium channels (VOCCs), and omega-conotoxin MVIIC (blocker of N- and Q-type VOCCs) inhibited the NANC relaxation, and the inhibition was inversely related to the frequency of stimulation. Combined, the two toxins were more effective than omega-contoxin GVIA alone. 3. The relaxation induced by the nitric oxide (NO) donor, 3-morpholino-sydnonimine (SIN-1) was not affected by omega-conotoxin MVIIC. 4. omega-Agatoxin IVA (blocker of P-type VOCCs) did not attenuate the NANC relaxation. 5. Reduction of the calcium concentration from 1.5 to 0.5 mM reduced the NANC relaxation at low but not at high frequencies of stimulation; the relaxation induced by SIN-1 was not affected. 6. EFS (20 Hz, 30 s) increased the cyclic GMP level 3 fold in normal Krebs solutions, but was unable to enhance significantly the cyclic GMP level after calcium omission. L-NOARG reduced the cyclic GMP content in 'calcium-free' medium, indicating an ongoing NO synthesis that was independent of extracellular calcium. 7. Caffeine, ryanodine and thapsigargin (inhibitors of sarcoplasmic calcium release), and CGP 37157 (inhibitor of mitochondrial sodium/calcium exchange) had no effect on the NANC relaxation. 8. It is suggested that nitrergic nerve activation in the rabbit urethral lamina propria is mediated in partby N-type (omega-conotoxin GVIA-sensitive) and in part by Q-type (omega-conotoxin MVIIC-sensitive) VOCCs.With high frequences of stimulation, another mechanism, possibly calcium-independent, appears to become operational.

Endothelium-dependent relaxation resistant to N omega-nitro-L-arginine in the rat hepatic artery and aorta.

Nitric oxide (NO)-independent pathways contributing to acetylcholine (ACh)-induced relaxation were examined in the rat isolated hepatic artery and aorta at low and high levels of precontraction induced by phenylephrine (PhE). In the hepatic artery, the ACh-induced relaxation was unaffected by the NO synthase inhibitors N omega-nitro-L-arginine (L-NOARG, 0.3 mM) and N omega-nitro-L-arginine methyl ester (0.1 mM) at either level of pre-contraction. In the aorta, L-NOARG virtually abolished the ACh-induced relaxation at a high level, but only partially reduced the response at a low level of precontraction. Methylene blue (10 microM) and indomethacin (10 microM) did not affect the ACh-induced relaxation in the hepatic artery. L-NOARG completely inhibited the cGMP and cAMP increases induced by ACh in both types of artery. In the presence of L-NOARG, ACh was unable to relax the hepatic artery precontracted by K+. The sensitivity to PhE was increased less by L-NOARG (threefold) than after endothelial denudation (tenfold) in the hepatic artery, whereas no such difference was observed in the aorta. The relaxation induced by the NO donor 3-morpholino-sydnonimin did not differ between the arteries after endothelial denudation. These results are compatible with the existence of an endothelium-dependent inhibitory pathway distinct from the NO and cyclooxygenase pathways. This pathway seems to contribute more to the ACh-induced relaxation in the hepatic artery than in the aorta, whereas the opposite appears to be true for the NO pathway.

Effects of omega-conotoxin on adrenergic, cholinergic and NANC neurotransmission in the rabbit urethra and detrusor.

1. The effects of omega-conotoxin GVIA (an inhibitor of N-type voltage-operated calcium channels; VOCCs) were compared on adrenergic, cholinergic and non-adrenergic, non-cholinergic (NANC) responses induced by electrical field stimulation (EFS) in the rabbit urethra and detrusor. 2. EFS induced a relaxation in urethral smooth muscle and lamina propria precontracted by arginine vasopressin (AVP). The relaxation was abolished by tetrodotoxin (TTX) or the nitric oxide (NO) synthase inhibitor N omega-nitro-L-arginine. omega-Conotoxin inhibited the relaxation induced by EFS, but not that elicited by the NO donor 3-morpholino-sydnonimin. The inhibition, however, decreased with increasing frequencies of stimulation. Nimodipine, tetramethrin and nickel did not affect the omega-contoxin-resistant relaxation in lamina propria, suggesting that neuronal L or T VOCCs were not involved in the response. 3. EFS contracted urethral smooth muscle at resting tension. The contractions were virtually abolished by TTX or prazosin. omega-Conotoxin effectively inhibited the contractile responses to EFS, but not those to exogenous noradrenaline. An omega-conotoxin-resistant contraction was, however, observed at high frequencies of stimulation. 4. The detrusor responded with frequency-dependent contractions upon EFS. A TTX-resistant contraction less than 10% of controls remained at 30 Hz stimulation. At a stimulation frequency of 10 Hz, scopolamine reduced the EFS-induced contraction by 71%. omega-Conotoxin inhibited the responses in both the absence and presence of scopolamine. The inhibition decreased with increasing frequencies of stimulation (examined in the absence of scopolamine). omega-Conotoxin did not affect the contractile responses to carbachol or adenosine 5'-triphosphate. 5. The adrenergic contraction (25 Hz) and NANC relaxation (10 Hz) in the urethra, and cholinergic and NANC contractions (10 Hz) in the detrusor were inhibited concentration-dependently by omega-conotoxin.The adrenergic contraction in the urethra was 10 times and the cholinergic contraction in the detrusor was three times more sensitive to omega-conotoxin than the NANC responses.6. These results suggest that NANC neurotransmission is less inhibited by omega-conotoxin than transmission mediated by adrenergic and cholinergic nerves in the rabbit lower urinary tract. In the urethra a marked omega-conotoxin-resistant component of the NANC relaxation was observed which increased with increasing stimulation frequencies and was unaffected by inhibitors of L and T type VOCCs. This raises the question whether VOCCs of a type other than L, T, and N is involved in the mediation of this response.

Calcium channels at the adrenergic neuroeffector junction in the rabbit ear artery.

Neurotransmitter release is dependent on influx of Ca2+ through voltage-operated calcium channels (VOCCs). These channels may be divided into L, N, T and P subtypes. To investigate the subtypes of VOCC involved in transmitter release from adrenergic nerves in the isolated rabbit ear artery, the effects of some subtype selective VOCC antagonists were examined on contractile responses induced by electrical field stimulation (EFS), and exposure to an isosmolar (low Na+, normal Cl- content) or a hyperosmolar (normal Na+, high Cl- content) 60 mM K+ solution. Tetrodotoxin (TTX) and the L channel blocker nimodipine were present in the latter experiments to inhibit sodium-dependent action potential discharge and the direct contractile effect of K+ depolarization on the smooth muscle cells. Prazosin abolished the contractile effect of EFS, indicating that the response was elicited by activation of adrenergic nerves. The EFS-induced contractions were concentration-dependently inhibited by the N channel blocker omega-conotoxin (pIC50 = 9.0) and the proposed L channel blocker T-cadinol (pIC50 = 4.5), while nimodipine and the T channel blocker tetramethrin had no effect. The isosmolar and hyperosmolar K+ solutions induced a prazosin-sensitive contraction, amounting to 46% and 10% of the response to 10(-5) M noradrenaline (NA), respectively. omega-Conotoxin inhibited the contractile response to the hyperosmolar K+ solution, but not that to the isosmolar K+ solution. T-cadinol preferentially inhibited the response to the hyperosmolar K+ solution. Tetramethrin had no effect on contractions induced by either type of K+ solution.(ABSTRACT TRUNCATED AT 250 WORDS)

Vascular effects of helodermin, helospectin I and helospectin II: a comparison with vasoactive intestinal peptide (VIP).

1. Helodermin, helospectin I and helospectin II, peptides recently isolated from the salivary gland venom of Heloderma suspectum, were compared to vasoactive intestinal peptide (VIP) with respect to effects on systemic blood pressure and on isolated femoral arteries in the rat. 2. They all reduced blood pressure in a dose-dependent manner; helodermin was less effective than VIP. However, at doses higher than 1 nmol kg-1 all four peptides reduced blood pressure to about the same extent. 3. The half-life of the hypotensive effect of VIP was longer than that of helodermin and the helospectins. 4. VIP and helodermin were equally potent in relaxing femoral arteries precontracted with phenylephrine or prostaglandin F2 alpha. 5. Helospectin I and II relaxed phenylephrine-contracted vessels to the same extent as VIP but with a lower potency. 6. Addition of VIP 1 microM to preparations exposed to helodermin 1 microM or to either of the helospectins did not produce a further relaxation. 7. The findings indicate that VIP, helodermin and helospectin I and II have a similar profile of action and therefore may act on a common receptor.

Actions of some vasodilators on isolated human hand veins.

Vasospasm is a well recognized complication during microvascular surgery of the hand. In the search for new spasmolytic drug therapies, the effects of papaverine, nitroprusside, nimodipine and lidocaine on isolated human hand veins contracted by several postulated mediators of vasospasm were examined. Mechanical activity was recorded isometrically in ring segments of the vessels. Potassium ions, noradrenaline (NA), 5-hydroxytryptamine (5-HT) and prostaglandin F2 alpha (PGF2 alpha) all produced strong contractions that were highly dependent on the presence of extracellular Ca2+. Papaverine acted as a nonselective vasodilator, as it produced an almost identical inhibition of contractile responses to all examined stimulants. Nitroprusside inhibited contractions induced by agonists more than those evoked by K+, whereas the opposite was found for nimodipine. Nitroprusside also seemed to display a certain degree of selectivity among the agonist-induced responses (NA greater than PGF2 alpha greater than 5-HT). Lidocaine increased the contractile response to K+ and at high concentrations (greater than 10(-5) M) produced a contraction per se. The clinical efficacy of lidocaine as a vasodilator after topical application therefore seems to reflect an inhibitory action on vasoconstrictor nerves. Papaverine, nitroprusside, nimodipine and lidocaine differ considerably in their profiles of action and therefore deserve to be further evaluated in the treatment of vasospasm during microvascular surgery.