Channel Opener Cromakalim Research Articles

Evidence of the involvement of K+ channels and PPARγ receptors in the antidepressant-like activity of diphenyl diselenide in mice

This study investigated the involvement of different types of K(+) channels and PPARgamma receptors in the antidepressant-like effect of diphenyl diselenide in mice. Mice were pretreated with subeffective doses of K(+) channel inhibitors (tetraethylammonium, glibenclamide, charybdotoxin and apamin), openers (cromakalim, minoxidil), GW 9662 (a PPARgamma antagonist) or vehicle. Thirty minutes later the mice received diphenyl diselenide in either an effective or a subeffective dose, 30 min before a tail-suspension test. Pre-treatment with tetraethylammonium, charybdotoxin or apamin combined with a subeffective dose of diphenyl diselenide was effective in decreasing the immobility time in the mouse tail-suspension test. The reduction in the immobility time elicited by an effective dose of diphenyl diselenide in this test was prevented by the pretreatment of mice with minoxidil and GW 9662. Diphenyl diselenide elicited an antidepressant-like effect and this action was mediated, at least in part, by modulation of K(+) channels and PPARgamma receptors.

Involvement of potassium channels in the antidepressant-like effect of venlafaxine in mice

Aims Studies have shown that the acute administration of venlafaxine elicits an antidepressant-like effect in the mouse forced swim test (FST) by a mechanism dependent on the l-arginine–nitric oxide (NO)–cyclic guanosine monophosphate (cGMP) pathway. Because it has been reported that NO activates different types of potassium (K +) channels in the brain, this study investigated the involvement of K + channels in the antidepressant-like effect of venlafaxine in the mouse FST. Main methods Male adult Swiss mice were pretreated with different K + channel inhibitors or openers 15 min before venlafaxine administration. After 30 min, the open-field test (OFT) and FST were carried out. Key findings Intracerebroventricular (i.c.v.) pretreatment of mice with subeffective doses of tetraethylammonium (TEA, a non-specific inhibitor of K + channels, 25 pg/site), glibenclamide (an ATP-sensitive K + channel inhibitor, 0.5 pg/site), charybdotoxin (a large- and intermediate-conductance calcium-activated K + channel inhibitor, 25 pg/site) or apamin (a small-conductance calcium-activated K + channel inhibitor, 10 pg/site) was able to potentiate the action of a subeffective dose of venlafaxine (2 mg/kg, i.p.). Moreover, the reduction in the immobility time elicited by an effective dose of venlafaxine (8 mg/kg, i.p.) in the FST was prevented by the pretreatment of mice with the K + channel openers cromakalim (10 µg/site, i.c.v.) and minoxidil (10 µg/site, i.c.v.). The drugs used in this study did not produce any change in locomotor activity. Significance The results demonstrate that the neuromodulatory effects of venlafaxine, via the inhibition of K + channels, are possibly involved in its anti-immobility activity in the mouse FST.

ATP-sensitive potassium channels contribute to the time-dependent alteration in the pentylenetetrazole-induced seizure threshold in diabetic mice

Although there is evidence that diabetes affects seizure susceptibility, the underlying mechanism has not been completely understood. Several studies also suggest a pivotal role for K ATP channels in the seizure modulation. The aim of the present study was to evaluate the seizure threshold induced by pentylenetetrazole in diabetic mice at different times (3 days, 1–8 weeks) after induction of diabetes with streptozocin and to examine the possible role of ATP-sensitive potassium (K ATP) channels in this manner. Our data showed a time-dependent alteration in the threshold in diabetic mice, reaching a peak on week 2 after streptozocin injection and declining significantly afterwards. The seizure threshold in 8-week diabetic mice was even lower than control levels, though the difference was not significant. The K ATP channel opener cromakalim (0.1–30 μg/kg, i.p.) significantly increased the seizure threshold in control mice. Although the K ATP channel blocker glibenclamide (0.5, 1 mg/kg) had no effect, it prevented the effects of the potent dose of cromakalim (30 μg/kg) on seizure threshold in control mice. Glibenclamide (1 mg/kg, i.p.) also decreased the seizure threshold in 2-week diabetic mice to the control levels which was blocked by pre-treatment with cromakalim (10 μg/kg, i.p.). Cromakalim (10 μg/kg, i.p.) significantly increased the seizure threshold in 8-week diabetic mice which was inhibited by pre-treatment with glibenclamide (1 mg/kg, i.p.). We demonstrated a time-dependent alteration in the pentylenetetrazole-induced seizure threshold in diabetic mice. This phenomenon might be due to the probable alteration in the K ATP channel functioning during the diabetic condition.

Positive inotropic effects of Tityus cambridgei and T. serrulatus scorpion venoms on skeletal muscle

Toxins that block voltage-dependent K + channels and those that modify Na + channel gating exhibit positive inotropic effect on skeletal muscle. We compared the effect of the venom of Tityus cambridgei (Tc) and Tityus serrulatus (Ts) scorpions on mouse diaphragm force, in vitro. In indirect and direct (using d-tubocurarine 7.3 µM) stimulation, Tc, 10µg/mL, increased the contractile force, an effect prevented by tetrodotoxin (TTX) while Ts, 0.5 µg/mL, potentiated only indirectly stimulated diaphragm, thus indicating its activity is mainly mediated through acetylcholine release from nerve terminal. This effect is prevented by TTX and attenuated by the K + channel opener cromakalim. In conclusion, our data show that while the positive inotropic effect of both venoms appears associated to the activity of Na + and K + channels, only Tc venom acts also directly on skeletal muscle. This finding call for further studies on Tc venom to identify the toxin responsible for its direct inotropic activity as it may have clinical applications.

The synthesis and biological evaluation of a range of novel functionalised benzopyrans as potential potassium channel activators

A range of novel benzopyrans have been synthesised and biologically evaluated for K(ATP) channel activity employing cromakalim 1 as a benchmark K(ATP) channel opener. Although the compounds that were evaluated demonstrated a reduced ability to relax phenylephrine stimulated rat thoracic tissue, we provide evidence that benzopyrans 7a-h may be operating via an alternative mechanism than ATP-sensitive K(+) channel activity.

Role of different types of potassium channels in the antidepressant-like effect of agmatine in the mouse forced swimming test

The administration of agmatine elicits an antidepressant-like effect in the mouse forced swimming test by a mechanism dependent on the inhibition of the NMDA receptors and the l-arginine-nitric oxide (NO) pathway. Since it has been reported that the NO can activate different types of potassium (K +) channels in several tissues, the present study investigates the possibility of synergistic interactions between different types of K + channel inhibitors and agmatine in the forced swimming test. Treatment of mice by i.c.v. route with subeffective doses of tetraethylammonium (a non specific inhibitor of K + channels, 25 pg/site), glibenclamide (an ATP-sensitive K + channels inhibitor, 0.5 pg/site), charybdotoxin (a large- and intermediate-conductance calcium-activated K + channel inhibitor, 25 pg/site) or apamin (a small-conductance calcium-activated K + channel inhibitor, 10 pg/site), augmented the effect of agmatine (0.001 mg/kg, i.p.) in the forced swimming test. Furthermore, the administration of agmatine and the K + channel inhibitors, alone or in combination, did not affect locomotion in the open-field test. Moreover, the reduction in the immobility time elicited by an active dose of agmatine (10 mg/kg, i.p.) in the forced swimming test was prevented by the pre-treatment of mice with the K + channel openers cromakalim (10 μg/site, i.c.v.) and minoxidil (10 μg/site, i.c.v.), without affecting locomotion. Together these data raise the possibility that the antidepressant-like effect of agmatine in the forced swimming test is related to its modulatory effects on neuronal excitability, via inhibition of K + channels.

The Endogenous Cannabinoid Anandamide Inhibits Cromakalim-Activated K+Currents in Follicle-EnclosedXenopusOocytes

The effect of the endogenous cannabinoid anandamide on K(+) currents activated by the ATP-sensitive potassium (K(ATP)) channel opener cromakalim was investigated in follicle-enclosed Xenopus oocytes using the two-electrode voltage-clamp technique. Anandamide (1-90 microM) reversibly inhibited cromakalim-induced K(+) currents, with an IC(50) value of 8.1 +/- 2 microM. Inhibition was noncompetitive and independent of membrane potential. Coapplication of anandamide with the cannabinoid type 1 (CB(1)) receptor antagonist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboximide hydrochloride (SR 141716A) (1 microM), the CB(2) receptor antagonist N-[(1S)endo-1,3,3-trimethyl bicyclo heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide (SR144528) (1 microM), or pertussis toxin (5 microg/ml) did not alter the inhibitory effect of anandamide, suggesting that known cannabinoid receptors are not involved in anandamide inhibition of K(+) currents. Similarly, neither the amidohydrolase inhibitor phenylmethylsulfonyl fluoride (0.2 mM) nor the cyclooxygenase inhibitor indomethacin (5 microM) affected anandamide inhibition of K(+) currents, suggesting that the effects of anandamide are not mediated by its metabolic products. In radioligand binding studies, anandamide inhibited the specific binding of the K(ATP) ligand [(3)H]glibenclamide in the oocyte microsomal fractions, with an IC(50) value of 6.3 +/- 0.4 microM. Gonadotropin-induced oocyte maturation and the cromakalim-acceleration of progesterone-induced oocyte maturation were significantly inhibited in the presence of 10 microM anandamide. Collectively, these results indicate that cromakalim-activated K(+) currents in follicular cells of Xenopus oocytes are modulated by anandamide via a cannabinoid receptor-independent mechanism and that the inhibition of these channels by anandamide alters the responsiveness of oocytes to gonadotropin and progesterone.

Time-dependent alteration in cromakalim-induced relaxation of corpus cavernosum from streptozocin-induced diabetic rats

The purpose of the present study was to investigate the relaxant responses to the ATP-sensitive potassium (K ATP) channel opener cromakalim in corpus cavernosum strips from 1-, 2-, 4-, 6-, and 8-week streptozocin-induced diabetic rats. Cromakalim (1 nM–0.1 mM) produced concentration-dependent relaxation in phenylephrine (7.5 μM)-precontracted isolated rat corporal strips. Compared with age-matched control animals, a significant enhancement in cromakalim-induced relaxation of corpus cavernosum was observed in 2-week diabetic animals, whereas the relaxant responses to cromakalim were decreased in 6-and 8-week diabetic animals. However, the cromakalim-induced relaxation was not altered in either 1-week or 4-week rat corporal strips in comparison with corresponding age-matched non-diabetic groups. Preincubation with the K ATP channel blocker glibenclamide (10 μM) significantly inhibited the cromakalim-induced relaxation in both non-diabetic and diabetic rat corpus cavernosum, but neither the voltage-dependent K + channel (K V) antagonist 4-aminopyridine (1 mM) nor the calcium-activated K + channel (K Ca) antagonist charybdotoxin (0.1 μM) had significant effect on cromakalim-induced relaxation in both control and diabetic rat corporal strips. Relaxation responses to the nitric oxide donor sodium nitroprusside (1 nM–0.1 mM) in diabetic rat corpus cavernosum were similar to that of age-matched controls. These data demonstrated that the relaxant responses to cromakalim were altered in diabetic cavernosal strips in a time dependent manner, suggesting that the period of diabetes mellitus may play a key role in the K ATP channels function in rat corpus cavernosum.

Role of ATP-sensitive potassium channels in the biphasic effects of morphine on pentylenetetrazole-induced seizure threshold in mice

Although several studies have indicated that the opioid receptor agonist morphine exerts biphasic effects on clonic seizure threshold, as yet little is known of the underlying mechanisms in this effect. In the present study, using the specific ATP-sensitive K(+) (K(ATP)) channel blocker glibenclamide and the specific K(ATP) channel opener cromakalim, the possible involvement of K(ATP) channels in the effects of morphine on pentylenetetrazole (PTZ)-induced seizure threshold in mice was investigated. Acute administration of lower doses of morphine (1, 3 and 7.5 mg/kg, i.p.) increased and higher doses of morphine (30 and 60 mg/kg, i.p.) decreased the PTZ-induced seizure threshold. Glibenclamide (2.5-5 mg/kg) increased the PTZ-induced seizure threshold. Non-effective dose of cromakalim (0.1 microg/kg) inhibited anticonvulsant effect of glibenclamide (5 mg/kg). Acute administration of non-effective dose of glibenclamide (1 mg/kg) interestingly inhibited both anticonvulsant and pro-convulsant effects of morphine and this effect was significantly reversed by cromakalim (0.1 microg/kg). These results support the involvement of K(ATP) channels in the modulation of seizure threshold by morphine.

Vardenafil increases coronary flow response to hypercapnic acidosis in isolated guinea pig heart

The hypothesis was tested that vardenafil, a PDE5 inhibitor, specifically enhances coronary vasodilation during acidosis. In isolated constant pressure perfused guinea pig hearts, infusion of vardenafil (<or= 1 microM) increased coronary flow concentration-dependently 34 % above baseline. In parallel, cGMP release increased (0.44 +/- 0.094 vs. 0.14 +/- 0.017 pmol/min x g at 0.5 microM vardenafil vs. baseline). Flow increases occurred in the absence of changes in heart function (LVP, heart rate, dP/dt(max), heart rate - pressure product). Infusion of the NO synthase blocker L-NMMA (100 microM) caused a rightward shift of the dose-response curve of vardenafil. To test whether vardenafil treatment may enhance metabolic coronary vasodilation, arterial pCO(2) was raised from 38 to 61 mmHg, which resulted in a steady state flow increase of 18.8 +/- 4.5%. Infusion of vardenafil, given at a threshold flow enhancing concentration, doubled the coronary flow response during hypercapnic acidosis to 38.4 +/- 4.2 % (p=0.004). This flow amplification during acidosis was not shared by the K(ATP) channel opener cromakalim, indicating a specific effect of vardenafil on flow control during myocardial acidosis. We conclude that vardenafil specifically relaxes coronary resistance vessels through NO/cGMP-dependent pathways and increases the coronary flow response toward hypercapnic acidosis. This finding further supports the importance of the NO-cGMP axis in mediation of this flow response.

A New Insight Into the Pathogenesis of Coronary Vasospasm

See related article, pages 682–689 To elucidate the control mechanisms of coronary vascular tone is one of the central interests in cardiac pathophysiology, because ischemic heart disease is one of the major causes of death in many countries. Although the coronary vascular tone is finally determined by the contractile state of smooth muscle, exo-smooth muscle mechanisms including autonomic nerves, endothelial cells, and blood cells have been shown indispensable for the control. Particular types of receptors, ion channels, and intracellular signal-cascades exist in coronary smooth muscle cells and mediate the controls by exo-smooth muscle elements (Figure). For example, stimulation of α1-adrenergic receptor, which is physiologically achieved by noradrenaline (NA) released from sympathetic presynapses, increases the intracellular Ca2+ (Ca2+i) via phosphatidylinositol (PI)-turnover and also probably via opening of receptor-activated Ca2+-permeable TRP channel. Membrane depolarization does so via opening voltage-dependent Ca2+ channels. The augmentation of Ca2+i results in contraction of the smooth muscle cells and thus constriction of the coronary artery.1 Activation of β2-adrenergic receptor, in turn, inhibits the coronary smooth muscle contraction through protein-kinase A pathway.2 The contraction is also suppressed by protein-kinase G activated by a dilating factor, nitric oxide (NO), which is produced in the endothelial cells. The mechanism for control of vascular tone . Activation of voltage-gated Ca2+ channel at the presynapse of sympathetic nerve increases intracellular Ca2+, which triggers a release of norepinephrine (NE). NE stimulates adrenergic α1 receptor (α1R) in the smooth muscle cells and drives PI-turnover as described in this scheme. The intracellular Ca2+, which flows into the cells via smooth-muscle’s Ca2+ channel or moves from endoplasmic reticulum (ER), contracts the cells. The constriction of smooth muscle cells is prevented by activation …

The diacylglycerol lipase inhibitor RHC-80267 potentiates the relaxation to acetylcholine in rat mesenteric artery by anti-cholinesterase action

The diacylglycerol lipase inhibitor 1,6-bis(cyclohexyloximinocarbonylamino) hexane (RHC-80267) was tested for its effect on acetylcholine-evoked relaxation in rat mesenteric artery. In artery contracted with either noradrenaline or KCl, RHC-80267 (0.1–10 μM) potentiated the relaxation evoked by acetylcholine. The effect of RHC-80267 was not affected by nitric oxide synthase inhibition or by inhibitors of protein kinase C or of phospholipase A 2. The diacylglycerol analogue 1-oleoyl-2-acetyl- sn-glycerol did not change the relaxation to acetylcholine. RHC-80267 did not affect the relaxation evoked by carbachol, by the nitric oxide donor SNAP ( S-nitroso- N-acetylpenicillamine) or by the K + channel opener cromakalim. Neostigmine, a cholinesterase inhibitor, produced the same effect as RHC-80267 on acetylcholine-evoked relaxation. When tested on cholinesterase in brain homogenate, RHC-80267 concentration-dependently inhibited cholinesterase activity with an IC 50 of 4 μM. These results indicate that the potentiation of acetylcholine-evoked responses by RHC-80267 in rat mesenteric artery is caused by the inhibition of the cholinesterase activity in the vascular wall.

α‐Adrenoceptor constrictor responses and their modulation in slow‐twitch and fast‐twitch mouse skeletal muscle

Vasoconstrictor responses to sympathetic nerve stimulation and their sensitivity to metabolic modulation reportedly differ in fast-twitch and slow-twitch muscles, but the underlying mechanisms are not known. Both alpha(1)- and alpha(2)-adrenoceptors mediate these vascular responses in fast-twitch muscle, while their roles in slow-twitch muscle are less well defined. In this study, the phosphorylation of smooth muscle myosin regulatory light chain (smRLC) was measured as an index of vasoconstriction in slow-twitch soleus muscles and fast-twitch extensor digitorum longus (EDL) muscles isolated from C57BL/6J mice. In soleus muscles, incubation with phenylephrine (PE) or UK 14,304 to selectively activate alpha(1)- or alpha(2)-adrenoceptors resulted in concentration-dependent increases in smRLC phosphorylation. To evaluate metabolic modulation of these responses, vasodilator pathways previously implicated in such modulation in fast-twitch muscle were activated in soleus muscles by treatment with the nitric oxide (NO) donor nitroprusside or the ATP-sensitive potassium (K(ATP)) channel opener cromakalim. Both drugs inhibited responses to UK 14,304, but not to PE. The effect of nitroprusside to antagonize UK 14,304 responses was prevented by inhibition of guanylyl cyclase or by blockade of K(ATP) channels, but not by blockade of other potassium channels. Results were similar in EDL muscles. These data provide the first evidence for alpha(2)-adrenoceptor-mediated constriction in slow-twitch muscle, and show that it is sensitive to modulation by NO via a cGMP-dependent mechanism that requires K(ATP) channel activation. Based on the similar findings in soleus and EDL muscles, fibre type does not appear to determine the innate vascular response to alpha(1)- or alpha(2)-adrenoceptor activation.

4,6-Disubstituted 2,2-Dimethylchromans Structurally Related to the KATP Channel Opener Cromakalim: Design, Synthesis, and Effect on Insulin Release and Vascular Tone

Five series (ureas, thioureas, carbamates, sulfonylureas, and amides) of 4,6-disubstituted-2,2-dimethylchromans structurally related to cromakalim were prepared and evaluated, as putative ATP-sensitive potassium channel activators, on rat pancreatic islets and rat aorta rings. The biological data indicate that most compounds were, like the reference molecule cromakalim, more active on the vascular smooth muscle tissue (myorelaxant effect on 30 mM KCl induced contractions of rat aorta rings) than on the pancreatic tissue (inhibition of 16.7 mM glucose induced insulin release from rat pancreatic islets). However, some drugs (8h, 8i, 9f, 9g, 9h, and 9i) markedly inhibited insulin release and exhibited an activity equivalent or greater than that of diazoxide. Compounds 9h and 9i were also found to be more active on pancreatic beta-cells than on vascular smooth muscle cells. Last, the amide 6b was selected in order to examine its mechanism of action on vascular smooth muscle cells. Pharmacological results suggest that the compound acted as a K(ATP) channel opener. In conclusion, the present data indicate that appropriate structural modifications can generate dimethylchromans with pharmacological profiles different from that of cromakalim.

Synthesis by microwave irradiation of a substituted benzoxazine parallel library with preferential relaxant activity for guinea pig trachealis

An efficient, facile, and practical parallel combinatorial synthesis of substituted-benzoxazines under microwave irradiation was described. The procedure involved the use of a microwave oven especially designed for organic synthesis suitable for parallel synthesis of solution libraries. A demonstration 19-membered library of substituted N,N-dimethyl- and N-methyl-benzoxazine amide derivatives, structurally related to the potassium channel opener cromakalim, was generated by both conventional and microwave procedures, achieving a reduction from 7 h to 30–36 min in library generation time for the microwave approach. All the synthesized compounds were tested using the in vitro models of rat aorta and guinea pig trachea rings pre-contracted with phenylephrine and carbachol, respectively. All N,N-dimethyl amide derivatives showed a relaxant activity higher on guinea pig trachea rings than on rat aorta rings.

Role of adenosine triphosphate-dependent potassium channels in canine penile erection

Objectives To define the physiologic role and hemodynamic features of nitric oxide (NO) and the adenosine triphosphate (ATP)-dependent K + (K ATP) channel in canine penile erection. Methods Mongrel dogs were anesthetized, and penile erection was induced by electrical stimulation of the pelvic nerve. Changes in the intracavernous pressure (ICP) were measured with a transducer. Results The basal ICP was 12.8 ± 5.0 mm Hg. Pelvic nerve stimulation (5 to 20 V, 5 to 15 Hz, for 1-minute intervals) significantly increased the ICP to 86.2 ± 11.4 mm Hg (n = 5, P <0.05). Treatment with the NO synthase inhibitor N ω-nitro- l-arginine methyl ester (10 mg/kg intravenously) abolished this increase (15.4 ± 5.0 mm Hg, n = 5). Intracavernous injection of the K ATP channel opener cromakalim (3 and 10 μg) increased the ICP (103 ± 14.4 mm Hg and 106 ± 12.1 mm Hg, respectively; n = 4). This response was abolished by the prior intracavernous injection of the selective K ATP channel-specific blocker glibenclamide (10 mg). Glibenclamide did not affect the increase in ICP induced by electric stimulation of the pelvic nerve (88 ± 24.2 mm Hg). Conclusions Our results indicate that relaxation of canine cavernous smooth muscle and penile tumescence are mediated by NO. The failure of glibenclamide to affect the increase in ICP induced by pelvic nerve stimulation suggests that ATP-dependent K + channels probably do not play a physiologic role in canine penile erection.

Visceral periadventitial adipose tissue regulates arterial tone of mesenteric arteries.

Periadventitial adipose tissue produces vasoactive substances that influence vascular contraction. Earlier studies addressed this issue in aorta, a vessel that does not contribute to peripheral vascular resistance. We tested the hypothesis that periadventitial adipose tissue modulates contraction of smaller arteries more relevant to blood pressure regulation. We studied mesenteric artery rings surrounded by periadventitial adipose tissue from adult male Sprague-Dawley rats. The contractile response to serotonin, phenylephrine, and endothelin I was markedly reduced in intact vessels compared with vessels without periadventitial fat. The contractile response to U46619 or depolarizing high K+-containing solutions (60 mmol/L) was similar in vessels with and without periadventitial fat. The K+ channel opener cromakalim induced relaxation of vessels precontracted by serotonin but not by U46619 or high K+-containing solutions (60 mmol/L), suggesting that K+ channels are involved. The intracellular membrane potential of smooth muscle cells was more hyperpolarized in intact vessels than in vessels without periadventitial fat. Both the anticontractile effect and membrane hyperpolarization of periadventitial fat were abolished by inhibition of delayed-rectifier K+ (K(v)) channels with 4-aminopyridine (2 mmol/L) or 3,4-diaminopyridine (1 mmol/L). Blocking other K+ channels with glibenclamide (3 micromol/L), apamin (1 micromol/L), iberiotoxin (100 nmol/L), tetraethylammonium ions (1 mmol/L), tetrapentylammonium ions (10 micromol/L), or Ba2+ (3 micromol/L) had no effect. Longitudinal removal of half the perivascular tissue reduced the anticontractile effect of fat by almost 50%, whereas removal of the endothelium had no effect. We suggest that visceral periadventitial adipose tissue controls mesenteric arterial tone by inducing vasorelaxation via K(v) channel activation in vascular smooth muscle cells.

Cromakalim inhibits transmitter acetylcholine release in rat trachea by an action on epithelial cells and a diffusible factor.

The present study was undertaken to investigate further the effects of the potassium channel opening drug cromakalim on the release of transmitter acetylcholine from cholinergic nerves of rat isolated trachea by using two tracheal preparations superfused in series. In all experiments, the lower chamber contained an epithelium-denuded preparation which had been incubated with [3H]-choline to incorporate [3H]-acetylcholine into cholinergic transmitter stores, whereas the upper chamber contained an unlabelled, epithelium-intact or epithelium-denuded preparation. When the upper chamber contained an epithelium-intact tracheal preparation, cromakalim (1-100 micro M) significantly reduced the stimulation-induced (S-I) efflux of [3H]-acetylcholine from the radiolabelled, epithelium-denuded tracheal preparation in the lower flow chamber. In contrast, when the upper flow chamber contained an epithelium-denuded preparation, cromakalim (10 micro M) was without effect on the S-I efflux. Glibenclamide (1 micro M), an ATP-sensitive potassium channel blocker, was without effect on the S-I efflux when the upper chamber contained an unlabelled, epithelium-intact tracheal preparation. However, glibenclamide (1 micro M) prevented the inhibition of the S-I efflux by cromakalim (10 micro M). When the upper chamber contained an epithelium-intact tracheal preparation and the lower chamber contained an epithelium-denuded tracheal preparation, cromakalim (10 micro M), when infused through the side-arm of the T-piece, such that only the lower radiolabelled epithelium-denuded tracheal preparation was exposed to the drug, was without effect on the S-I efflux. The findings of the present study have provided evidence of an inhibitory action of the potassium channel opener cromakalim on transmitter acetylcholine release in rat trachea which is dependent on the functional integrity of the tracheal epithelium. The findings suggest that cromakalim may inhibit transmitter acetylcholine release by opening ATP-sensitive potassium channels, probably, on cells in the epithelial layer to release a putative epithelial factor, which in turn acts prejunctionally to mediate the inhibitory effect of cromakalim.

Visceral perivascular adipose tissue regulates arterial tone of small mesenteric arteries

Increased visceral adipose tissue in obesity is associated with adverse cardiovascular events and hypertension. Visceral adipose tissue surrounds mesenteric arteries and may produce vasoactive substances that influence vascular contraction. We tested the hypothesis that perivascular adipose tissue modulates contraction of small, resistance-sized mesenteric artery ring preparations. We studied mesenteric rings surrounded by periadventitial adipose tissue from adult male Sprague-Dawley rats. The contractile response to serotonin, phenylephrine, and endothelin I was markedly reduced in intact vessels compared to vessels without periadventitial fat. The contractile response to U46619 or depolarizing high K+ containing solutions (60 mM) was similar in vessels with and without periadventitial fat. The K+ channel opener cromakalim induced relaxation of vessels precontracted by serotonin but not by U46619 or high K+ containing solutions (60 mM), suggesting that K+ channels are involved. The intracellular membrane potential of smooth muscle cells was more hyperpolarized in intact vessels than in vessels without periadventitial fat. Both the anti-contractile effect and membrane hyperpolarization of periadventitial fat were abolished by inhibition of delayed-rectifier K+ channels with 4-aminopyridine (2 mM). Blocking other K+ channels with glibenclamide (3 mM), apamin (1 μM), TEA (1 mM), TPeA (10 μM) did not restore the vascular response in intact vessels. Longitudinal removal of 50% of perivascular tissue reduced the anti-contractile effect to serotonin by almost 50% while removal of the endothelium did not affect the anti-contractile effect. Using Vivaspin filters, we separated and isolated the visceral adventitia-derived relaxing factor. We suggest that visceral perivascular adipose tissue controls mesenteric arterial tone locally. It induces vasorelaxation by activating delayed-rectifier K+ channels in vascular smooth muscle cells.

Inhibition of cromakalim-activated K+ current by ethanol in follicle-enclosed Xenopus oocytes

Ethanol has been reported to modulate arterial dilation and insulin secretion. ATP-inhibited K+ channels (K(ATP)) are reported to have regulatory roles during these events. In the present study, the effect of ethanol on K+ currents activated by the K(ATP) channel opener cromakalim was investigated in follicular cells of Xenopus oocytes. The results indicate that ethanol in the concentration range of 10-300 mM (approximately 0.06%-2% v/v) reversibly inhibits the cromakalim-induced K+ currents. The 50% of maximal ethanol effect was obtained at 53 mM. Inhibition of cromakalim-activated K+ current by ethanol was non-competitive. In oocytes treated with the Ca2+-chelator BAPTA, inhibition of cromakalim-induced K+ currents by ethanol was not altered, suggesting that Ca2+-activated second messenger pathways were not involved in the actions of ethanol. Similarly, currents activated by 8-Br-cAMP were also inhibited by ethanol, but the ethanol inhibition of 8-Br-cAMP-activated currents was significantly less than inhibition of cromakalim-activated currents by ethanol. These results indicate that cromakalim-activated K+ currents in follicular cells of Xenopus oocytes were modulated by ethanol.