Description and role in proliferation of iberiotoxin-sensitive currents in different human mammary epithelial normal and cancerous cells

Several studies suggested that potassium channels are involved in the proliferation of cancer cells but the involvement of the large conductance Ca2+-activated K+ channels (BKCa) in the cancerous phenomenon is still controversial. In the present study, we used iberiotoxin, a specific blocker of BKCa, and report the activity of an iberiotoxin-sensitive current in various human breast cancer cell lines (MCF-7, MDA-MB-231, MDA-MB-468 and MDA-MB-435s) as well as in normal mammary epithelial cells (HME). Iberiotoxin and NS1619, an activator of BKCa, did not interfere with either cell proliferation or with the invasive properties of the cells, under normal culture conditions. However, extracellular pulses of ATP, which induced transient increases in intracellular Ca2+ concentration, revealed a significant reduction effect of iberiotoxin on cell proliferation. We conclude that the iberiotoxin-sensitive current is not involved in cell proliferation in basal conditions but participates when the intracellular Ca2+ concentration is increased. These experiments also suggest that BKCa channels are not involved in the cancerous transformation and are probably a relic from normal cells.     

Nitric oxide-dependent and -independent mechanisms in the relaxation elicited by acetylcholine in fetal rat aorta

The aim of the present study was to analyze the mechanisms involved in the relaxation induced by 1 microM acetylcholine (ACh) in aortic segments from fetal rats at term precontracted with 3 microM prostaglandin F2alpha (PGF2alpha) and incubated with 1 microM indomethacin. The endothelium-dependent relaxation caused by ACh was reduced by the nitric oxide (NO) synthase inhibitor NG-monomethyl-L-arginine (L-NMMA, 0.1 mM), such an effect was reversed by 0.1 mM L-arginine (L-Arg). After precontraction of segments with 50 mM KCl the relaxant response to ACh was smaller than that after precontraction with PGF2alpha; this reduction was increased by L-NMMA, whereas L-NMMA plus L-Arg potentiated the relaxation. Thiopentone sodium (0. 1 mM), ouabain (10 microM), tetraethylammonium (TEA, 0.5 mM) and apamin (1 microM), inhibitors of cytochrome P450 monooxygenases, Na+ pump, Ca2+-activated (KCa) and small-conductance (SKCa) K+ channels, respectively, reduced the relaxation to ACh, which was unaffected by charybdotoxin (0.1 microM) and glibenclamide (1 microM), inhibitors of large-conductance BKCa and ATP-sensitive K+ channels. The L-NMMA/indomethacin-resistant relaxation to ACh was markedly reduced by thiopentone sodium, and similarly decreased by either ouabain or TEA. The endothelium-independent relaxation induced by exogenous NO (10 microM) in segments precontracted with PGF2alpha was unaltered by ouabain, glibenclamide, TEA and after precontraction with 50 mM KCl, and potentiated by L-NMMA. The potentiation of NO responses by L-NMMA was also observed in segments precontracted with KCl. These results suggest that ACh relaxes the fetal rat aorta by endothelial release of both NO and endothelium-derived hyperpolarizing factor (EDHF), a metabolite derived from cytochrome P450 monooxygenases, that hyperpolarizes smooth muscle cells by activation of KCa, essentially SKCa channels, and Na+ pump. It seems that when the effect of EDHF is abolished, the formation of NO could be increased.     

Glucocorticoids stimulate the activity of large-conductance Ca2+ -activated K+ channels in pituitary GH3 and AtT-20 cells via a non-genomic mechanism

The effects of glucocorticoids on ion currents were investigated in pituitary GH3 and AtT-20 cells. In whole-cell configuration, dexamethasone, a synthetic glucocorticoid, reversibly increased the density of Ca2+ -activated K+ current (IK(Ca)) with an EC50 value of 21 +/- 5 microM. Dexamethasone-induced increase in IK(Ca) density was suppressed by paxilline (1 microM), yet not by glibenclamide (10 microM), pandinotoxin-Kalpha (1 microM) or mifepristone (10 microM). Paxilline is a blocker of large-conductance Ca2+ -activated K+ (BKCa) channels, while glibenclamide and pandinotoxin-Kalpha are blockers of ATP-sensitive and A-type K+ channels, respectively. Mifepristone can block cytosolic glucocorticoid receptors. In inside-out configuration, the application of dexamethasone (30 microM) into the intracellular surface caused no change in single-channel conductance; however, it did increase BKCa -channel activity. Its effect was associated with a negative shift of the activation curve. However, no Ca2+ -sensitiviy of these channels was altered by dexamethasone. Dexamethasone-stimulated channel activity involves an increase in mean open time and a decrease in mean closed time. Under current-clamp configuration, dexamethasone decreased the firing frequency of action potentials. In pituitary AtT-20 cells, dexamethasone (30 microM) also increased BKCa -channel activity. Dexamethasone-mediated stimulation of IK(Ca) presented here that is likely pharmacological, seems to be not linked to a genomic mechanism. The non-genomic, channel-stimulating properties of dexamethasone may partly contribute to the underlying mechanisms by which glucocorticoids affect neuroendocrine function.     

Diclofenac-induced peripheral antinociception is associated with ATP-sensitive K+ channels activation

In order to investigate to the contribution of K+ channels on the peripheral antinociception induced by diclofenac, we evaluated the effect of several K+ channel blockers, using the rat paw pressure test, in which sensitivity is increased by intraplantar injection (2 microg) of prostaglandin E2. Diclofenac administered locally into the right hindpaw (25, 50, 100 and 200 microg) elicited a dose-dependent antinociceptive effect which was demonstrated to be local, since only higher doses produced an effect when injected in the contralateral paw. This blockade of PGE2 mechanical hyperalgesia induced by diclofenac (100 microg/paw) was antagonized in a dose-dependent manner by intraplantar administration of the sulphonylureas glibenclamide (40, 80 and 160 microg) and tolbutamide (80, 160 and 320 microg), specific blockers of ATP-sensitive K+ channels, and it was observed even when the hyperalgesic agent used was carrageenin, while the antinociceptive action of indomethacin (200 microg/paw), a typical cyclo-oxygenase inhibitor, over carrageenin-induced hyperalgesia was not affected by this treatment. Charybdotoxin (2 microg/paw), a blocker of large conductance Ca2+-activated K+ channels and dequalinium (50 microg/paw), a selective blocker of small conductance Ca2+-activated K+ channels, did not modify the effect of diclofenac. This effect was also unaffected by intraplantar administration of non-specific voltage-dependent K+ channel blockers tetraethylammonium (1700 microg) and 4-aminopyridine (100 microg) or cesium (500 microg), a non-specific K+ channel blocker. The peripheral antinociceptive effect induced by diclofenac was antagonized by NG-Nitro L-arginine (NOarg, 50 microg/paw), a NO synthase inhibitor and methylene blue (MB, 500 microg/paw), a guanylate cyclase inhibitor, and this antagonism was reversed by diazoxide (300 microg/paw), an ATP-sensitive K+ channel opener. We also suggest that an endogenous opioid system may not be involved since naloxone (50 microg/paw) did not affect diclofenac-induced antinociception in the PGE2-induced hyperalgesia model. This study provides evidence that the peripheral antinociceptive effect of diclofenac may result from activation of ATP-sensitive K+ channels, possible involving stimulation of L-arginine/NO/cGMP pathway, while Ca2+-activated K+ channels, voltage-dependent K+ channels as well as endogenous opioids appear not to be involved in the process.     

Expression and function of potassium channels in the human placental vasculature

In the placental vasculature, where oxygenation may be an important regulator of vascular reactivity, there is a paucity of data on the expression of potassium (K) channels, which are important mediators of vascular smooth muscle tone. We therefore addressed the expression and function of several K channel subtypes in human placentas. The expression of voltage-gated (Kv)2.1, KV9.3, large-conductance Ca2+-activated K channel (BKCa), inward-rectified K+ channel (KIR)6.1, and two-pore domain inwardly rectifying potassium channel-related acid-sensitive K channels (TASK)1 in chorionic plate arteries, veins, and placental homogenate was assessed by RT-PCR and Western blot analysis. Functional activity of K channels was assessed pharmacologically in small chorionic plate arteries and veins by wire myography using 4-aminopyridine, iberiotoxin, pinacidil, and anandamide. Experiments were performed at 20, 7, and 2% oxygen to assess the effect of oxygenation on the efficacy of K channel modulators. KV2.1, KV9.3, BKCa, KIR6.1, and TASK1 channels were all demonstrated to be expressed at the message level. KV2.1, BKCa, KIR6.1, and TASK1 were all demonstrated at the protein level. Pharmacological manipulation of voltage-gated and ATP-sensitive channels produced the most marked modifications in vascular tone, in both arteries and veins. We conclude that K channels play an important role in controlling placental vascular function.     

Low-affinity state beta1-adrenoceptor-induced vasodilation in SHR

Low-affinity state beta1-adrenoceptor (beta1-AR) was functionally expressed in some blood vessels and was different from beta1, beta2 and beta3-AR. In rat aorta, low-affinity state beta1-AR activation produced an endothelium-independent relaxation which was impaired in spontaneously hypertensive rats (SHRs). In the present work, we investigated whether renin-angiotensin system was involved in this alteration by evaluating the effects of enalapril, an angiotensin converting enzyme (ACE) inhibitor or losartan, an AT1 angiotensin receptor antagonist. Cumulative concentration-response curves to low-affinity state beta1-AR agonists (CGP 12177, cyanopindolol or alprenolol) and to NS 1619, a large conductance Ca2+-activated K+ channels (BK) agonist were performed in denuded aortic rings isolated from control or treated Wistar Kyoto (WKY) rats or SHRs in different experimental conditions. The low-affinity state beta1-AR-mediated aortic vasodilation was impaired in 5 and 12 weeks old SHRs when compared to age-matched WKY. Twelve days enalapril (5 mg/kg/day) or losartan (15 mg/kg/day) treatments reduced systolic blood pressure (SBP) only in 12 weeks old SHRs whereas no significant change was observed in other groups. These treatments improved low-affinity state beta1-AR effect only in SHRs groups. In 12 weeks old WKY rats, CGP 12177-induced relaxation was insensitive to glibenclamide, a K(ATP)+ channel blocker, but was reduced by TEA or iberiotoxin, two large conductance Ca2+-activated K+ channel (BK) blockers. The impairment of NS 1619-induced vasodilation in both 5 and 12 weeks old SHRs was restored by enalapril or losartan. These results suggested that improvement of the low-affinity state beta1-AR-mediated vasodilation in 5 and 12 weeks old SHRs could be attributed to enhanced BK channels-induced hyperpolarization in SHRs independently of lowering of SBP.     

Bradykinin activates calcium-dependent potassium channels in cultured human airway smooth muscle cells

Bradykinin (BK) is an inflammatory mediator that can cause bronchoconstriction. In this study, we investigated the membrane currents induced by BK in cultured human airway smooth muscle (ASM) cells. Depolarization of the cells induced outward currents, which were inhibited by tetraethylammonium (TEA) in a concentration-dependent manner with an IC50 of 0.33 microM. The currents were increased by elevating intracellular free Ca2+ concentration, suggesting they are calcium-activated potassium channels [I(K(Ca))]. Preexposure to inhibitor of I(K(Ca)) of large conductance (BKCa), iberiotoxin, and small conductance (SKCa), apamin, inhibited the increase of outward current induced by BK. The relative contribution of BKCa was greatest in early passage cells. Both nickel and SKF-96365 (10 microM) inhibited the increase of the I(K(Ca)) induced by BK; however, the l-type Ca2+ channel blocker, nifedipine, had no effect. Activation of the BK-induced current was inhibited by heparin, indicating dependence on intact inositol 1,4,5-triphosphate (IP3)-sensitive intracellular Ca2+ stores. BK also increased inositol phosphate accumulation and induced a transient Ca2+-activated chloride current (CACC) and a sustained nonselective cation current (I(CAT)). In summary, BK activates BKCa, SKCa, CACC, and I(CAT) via IP3-sensitive stores in human ASM.     

Nitric oxide opens ATP-sensitive K+ channels through suppression of phosphofructokinase activity and inhibits glucose-induced insulin release in pancreatic beta cells

Nitric oxide (NO) is known to be a potent messenger in the intracellular signal transduction system in many tissues. In pancreatic beta cells, NO has been reported to be formed from L-arginine through NO synthase. To elucidate the effect of NO on insulin secretion and to investigate the intracellular mechanism of its effect, we have used sodium nitroprusside (SNP) as a NO donor. SNP inhibited glucose-induced insulin secretion in a dose-dependent manner, and its effect was reversed by hemoglobin, a known NO scavenger. However, glyceraldehyde- induced insulin secretion was not affected by SNP. Since the closure of ATP-sensitive K+ channels (KATP channel) has been established as a key step in glucose-induced insulin secretion, we have directly assessed the effect of SNP on KATP channel activity using the patch clamp technique. The KATP channel activity reduced by glucose was found to be reversibly activated by the addition of SNP, and this activation was able to be similarly reproduced by applying S-Nitroso-N-acetyl-DL- penicillamine (SNAP), another NO generator. Furthermore, these activating effects were completely eliminated by hemoglobin, in accordance with the reversibility in inhibition of glucose-induced insulin release. However, SNP could not affect the KATP channel suppression by ATP applied to the inside of the plasma membrane. The activation of the KATP channel by NO, therefore, seems to be due to the decreased ATP production attributable to impairment of glucose metabolism in beta cells. Since SNP exhibited no effect on glyceraldehyde-induced KATP channel inhibition, NO may disturb a glycolytic step before glyceraldehyde-3-phosphate. The KATP channel activation by 2-deoxyglucose through presumable ATP consumption due to its phosphorylation by glucokinase was, however, not affected even in the presence of SNP. But in the permeabilized beta cells made by exposure to a low concentration (0.02 U/ml) of streptolysin O (open cell-attached configuration), SNP reopens KATP channels which have been eliminated by fructose-6-phosphate, while this effect was not observed in the KATP channels inhibited by fructose-1,6-bisphosphate. On the other hand, in rat ventricular myocyte KATP channels were not activated by SNP even under a low concentration of glucose. From these observations, the inhibition of phosphofructokinase activity is probably the site responsible for the impairment of glucose metabolism induced by NO in pancreatic beta cells. NO, therefore, seems to be a factor in the deterioration of glucose-induced insulin secretion from pancreatic beta cells through a unique intracellular mechanism.     

Coronary blood flow regulation in exercising swine involves parallel rather than redundant vasodilator pathways

In dogs, only combined blockade of vasodilator pathways [via adenosine receptors, nitric oxide synthase (NOS) and ATP-sensitive K+ (KATP) channels] results in impairment of metabolic vasodilation, which suggests a redundancy design of coronary flow regulation. Conversely, in swine and humans, blocking KATP channels, adenosine receptors, or NOS each impairs coronary blood flow (CBF) at rest and during exercise. Consequently, we hypothesized that these vasodilators act in parallel rather than in redundancy to regulate CBF in swine. Swine exercised on a treadmill (0-5 km/h), during control and after blockade of KATP channels (with glibenclamide), adenosine receptors [with 8-phenyltheophylline (8-PT)], and/or NOS [with Nomega-nitro-l-arginine (l-NNA)]. l-NNA, 8-PT, and glibenclamide each reduced myocardial O2 delivery and coronary venous O2 tension. These effects of l-NNA, 8-PT, and glibenclamide were not modified by simultaneous blockade of the other vasodilators. Combined blockade of KATP channels and adenosine receptors with or without NOS inhibition was associated with increased H+ production and impaired myocardial function. However, despite an increase in O2 extraction to >90% during administration of l-NNA + 8-PT + glibenclamide, vasodilator reserve could still be recruited during exercise. Thus in awake swine, loss of KATP channels, adenosine, or NO is not compensated for by increased participation of the other two vasodilator mechanisms. These findings suggest a parallel rather than a redundancy design of CBF regulation in the porcine circulation.     

Ischemic shortening of action potential duration as a result of KATP channel opening attenuates myocardial stunning by reducing calcium influx

Action potential duration (APD) shortening due to opening of sarcolemmal ATP-dependent potassium (KATP) channels has been postulated to protect the myocardium against postischemic damage by reducing Ca²⁺ influx. This hypothesis was assessed, assuming that increased postischemic stunning due to KATP channel inhibition with glibenclamide could be reverted by the addition of the Ca²⁺ channel blocker diltiazem. Percent wall thickening fraction (% WTh, conscious sheep) and APD (open-chest sheep) were obtained from the following groups: control: 12 min ischemia by anterior descending coronary artery occlusion followed by 2 h reperfusion; glibenclamide: same as control, with glibenclamide (0.4 mg/kg) infused 30 min before ischemia; diltiazem: same as control, with diltiazem (100 g/kg) administered prior to ischemia; glibenclamide+diltiazem: both drugs infused as in glibenclamide and diltiazem groups. APD was reduced in control ischemia. Conversely, KATP-channel blockade by glibenclamide lengthened APD and increased postischemic stunning (p < 0.01 vs. control); glibenclamide+diltiazem did not shorten APD but enhanced functional recovery (p < 0.01 vs. glibenclamide). Ca²⁺ channel blockade improvement of increased stunning provoked by KATP channel inhibition supports the hypothesis that APD shortening due to opening of KATP channels protects against postischemic stunning by limiting Ca²⁺ influx.     

Endothelium-dependent and -independent vasorelaxation by a theophylline derivative MCPT: roles of cyclic nucleotides, potassium channel opening and phosphodiesterase inhibition

The vasorelaxation activities of MCPT, a newly synthesized xanthine derivative, were investigated in this study. In phenylephrine (PE)-precontracted rat aortic rings with intact endothelium, MCPT caused a concentration-dependent relaxation, which was inhibited by endothelium removed. This relaxation was also reduced by the presence of nitric oxide synthase inhibitor Nomega-nitro-L-arginine methylester (L-NAME, 100 microM), soluble guanylyl cyclase (sGC) inhibitors methylene blue (10 microM), 1 H-[1,2,4] oxidazolol [4,3-a] quinoxalin-1-one (ODQ, 1 microM), adenylyl cyclase (AC) blocker SQ 22536 (100 microM), ATP-sensitive K+ channel blocker (KATP) glibenclamide (1 microM), a Ca2+ activated K+ channels blocker tetraethylammonium (TEA, 10 mM) and a voltage-dependent potassium channels blocker 4-aminopyridine (4-AP, 100 microM). The vasorelaxant effects of MCPT together with IBMX (0.5 microM) had an additive action. In PE-preconstricted endothelium-denuded aortic rings, the vasorelaxant effects of MCPT were attenuated by pretreatments with glibenclamide (1 microM), SQ 22536 (100 microM) or ODQ (1 microM), respectively. MCPT enhanced cAMP-dependent vasodilator isoprenaline- and NO donor/cGMP-dependent vasodilator sodium nitroprusside-induced relaxation activities in endothelium-denuded aortic rings. In A-10 cell and washed human platelets, MCPT induced a concentration-dependent increase in intracellular cyclic GMP and cyclic AMP levels. In phosphodiesterase assay, MCPT displayed inhibition effects on PDE 3, PDE 4 and PDE 5. The inhibition % were 52 +/- 3.9, 32 +/- 2.6 and 8 +/- 1.1 respectively. The Western blot analysis on HUVEC indicated that MCPT increased the expression of eNOS. It is concluded that the vasorelaxation by MCPT may be mediated by the inhibition of phosphodiesterase, stimulation of NO/sGC/ cGMP and AC/cAMP pathways, and the opening of K+ channels.     

Noradrenaline activates the NO/cGMP/ATP-sensitive K(+) channels pathway to induce peripheral antinociception in rats

Despite the classical peripheral pronociceptive effect of noradrenaline (NA), recently studies showed the involvement of NA in antinociceptive effect under immune system interaction. In addition, the participation of the NO/cGMP/KATP pathway in the peripheral antinociception has been established by our group as the molecular mechanism of another adrenoceptor agonist xylazine. Thus the aim of this study was to obtain pharmacological evidences for the involvement of the NO/cGMP/KATP pathway in the peripheral antinociceptive effect induced by exogenous noradrenaline. The rat paw pressure test was used, with hyperalgesia induced by intraplantar injection of prostaglandin E(2) (2μg/paw). All drugs were locally administered into the right hind paw of male Wistar rats. NA (5, 20 and 80ng/paw) elicited a local inhibition of hyperalgesia. The non-selective NO synthase inhibitor l-NOarg (12, 18 and 24μg/paw) antagonized the antinociception effect induced by the highest dose of NA. The soluble guanylyl cyclase inhibitor ODQ (25, 50 and 100μg/paw) antagonized the NA-induced effect; and cGMP-phosphodiesterase inhibitor zaprinast (50μg/paw) potentiated the antinociceptive effect of NA low dose (5ng/paw). In addition, the local effect of NA was antagonized by a selective blocker of an ATP-sensitive K(+) channel, glibenclamide (20, 40 and 80μg/paw). On the other hand, the specifically voltage-dependent K(+) channel blocker, tetraethylammonium (30μg/paw), Ca(2+)-activated K(+) channel blockers of small and large conductance types dequalinium (50μg/paw) and paxilline (20μg/paw), respectively, were not able to block local antinociceptive effect of NA. The results provide evidences that NA probably induces peripheral antinociceptive effects by activation of the NO/cGMP/KATP pathway.     

Hypercontractility and impaired sildenafil relaxations in the BKCa channel deletion model of erectile dysfunction

Erectile dysfunction (ED) can be elicited by a variety of pathogenic factors, particularly impaired formation of and responsiveness to nitric oxide (NO) and the downstream effectors soluble guanylate cyclase (sGC) and cGMP-dependent protein kinase I (PKGI). One important target of PKGI in smooth muscle is the large-conductance, Ca2+ -activated potassium (BKCa) channel. In our previous report (42), we demonstrated that deletion of the BKCa channel in mice induced force oscillations and led to reduced nerve-evoked relaxations and ED. In the current study, we used this ED model to explore the role of the BKCa channel in the NO/sGC/PKGI pathway. Electrical field stimulation (EFS)-induced contractions of corpus cavernosum smooth muscle strips were significantly enhanced in the absence of BKCa channel function. In strips precontracted with phenylephrine, EFS-induced relaxations were converted to contractions by inhibition of sGC, and this was further enhanced by loss of BK channel function. Sildenafil-induced relaxations were decreased to a similar extent by inhibition of sGC or BKCa channels. At concentrations >1 microM, sildenafil caused relaxations independent of inhibition of sGC or BKCa channels. Sildenafil did not affect the enhanced force oscillations that were induced by the loss of BKCa channel function. Yet, these oscillations could be completely eliminated by blocking L-type voltage-dependent Ca2+ channels (VDCCs). These results suggest that therapeutically relevant concentrations of sildenafil act through cGMP and BKCa channels, and loss of BKCa channel function leads to hypercontractility, which depends on VDCCs and cannot be modified by the cGMP pathway.     

Nitric oxide-epoxygenase interactions and arachidonate-induced dilation of rat renal microvessels

Nitric oxide (NO) is an inhibitor of hemoproteins including cytochrome P-450 enzymes. This study tested the hypothesis that NO inhibits cytochrome P-450 epoxygenase-dependent vascular responses in kidneys. In rat renal pressurized microvessels, arachidonic acid (AA, 0.03-1 microM) or bradykinin (BK, 0.1-3 microM) elicited NO- and prostanoid-independent vasodilation. Miconazole (1.5 microM) or 6-(2-propargyloxyphenyl)hexanoic acid (30 microM), both of which are inhibitors of epoxygenase enzymes, or the fixing of epoxide levels with 11,12-epoxyeicosatrienoic acid (11,12-EET; 1 and 3 microM) inhibited these responses. Apamin (1 microM), which is a large-conductance Ca2+-activated K+ (BKCa) channel inhibitor, or 18alpha-glycyrrhetinic acid (30 microM), which is an inhibitor of myoendothelial gap junctional electromechanical coupling, also inhibited these responses. NO donors spermine NONOate (1 and 3 microM) or sodium nitroprusside (0.3 and 3 microM) but not 8-bromo-cGMP (100 microM), which is an analog of cGMP (the second messenger of NO), blunted the dilation produced by AA or BK in a reversible manner without affecting that produced by hydralazine. However, the non-NO donor hydralazine did not affect the dilatory effect of AA or BK. Spermine NONOate did not affect the dilation produced by 11,12-EET, NS-1619 (a BKCa channel opener), or cromakalim (an ATP-sensitive K+ channel opener). AA and BK stimulated EET production, whereas hydralazine had no effect. On the other hand, spermine NONOate (3 microM) attenuated basal (19 +/- 7%; P < 0.05) and AA stimulation (1 microM, 29 +/- 9%; P < 0.05) of renal preglomerular vascular production of all regioisomeric EETs: 5,6-; 8,9-; 11,12-; and 14,15-EET. These results suggest that NO directly and reversibly inhibits epoxygenase-dependent dilation of rat renal microvessels without affecting the actions of epoxides on K+ channels.     

Contributions of prostacyclin and nitric oxide to carbon monoxide-induced cerebrovascular dilation in piglets

Carbon monoxide (CO) is an endogenous dilator in the newborn cerebral microcirculation. Other dilators include prostanoids and nitric oxide (NO), and interactions among the systems are likely. Experiments on anesthetized piglets with cranial windows address the hypothesis that CO-induced dilation of pial arterioles involves interaction with the prostanoid and NO systems. Topical application of CO or the heme oxygenase substrate heme-L-lysinate (HLL) produced dilation. Indomethacin, N(omega)-nitro-L-arginine (L-NNA), and either iberiotoxin or tetraethylammonium chloride (TEA) were used to inhibit prostanoids, NO, and Ca(2+)-activated K(+) (K(Ca)) channels, respectively. Indomethacin, L-NNA, iberiotoxin, or TEA blocked cerebral vasodilation to CO and HLL. Vasodilations to both CO and HLL were returned to indomethacin-treated piglets by topical application of iloprost. Vasodilations to both CO and HLL were returned to L-NNA-treated piglets by sodium nitroprusside but not iloprost. In iberiotoxin- or TEA-treated piglets, dilations to CO and HLL could not be restored by either iloprost or sodium nitroprusside. The dilator actions of CO involve prostacyclin and NO as permissive enablers. The permissive actions of prostacyclin and NO may alter the K(Ca) channel response to CO because neither iloprost nor sodium nitroprusside could restore dilation to CO when these channels were blocked.     

CGRP receptors in the gerbil spiral modiolar artery mediate a sustained vasodilation via a transient cAMP-mediated Ca2+-decrease

Alteration of cochlear blood flow may be involved in the etiology of inner ear disorders like sudden hearing loss, fluctuating hearing loss and tinnitus. The aim of the present study was to localize the vasodilator calcitonin gene-related peptide (CGRP) and to identify CGRP receptors and their signaling pathways in the gerbil spiral modiolar artery (SMA) that provides the main blood supply of the cochlea. CGRP was localized in perivascular nerves by immunocytochemistry. The vascular diameter and cytosolic Ca2+ concentration [Ca2+]i in the smooth muscle cells were measured simultaneously with videomicroscopy and fluo-4-microfluorometry. Calcitonin receptor-like receptor (CRLR) mRNA was identified by RT-PCR as a specific 288 bp fragment in total RNA isolated from the vascular wall. The SMA was preconstricted by a 2-min application of 1 nM endothelin-1 (ET1). CGRP, forskolin, and dibutyryl-cAMP caused a vasodilation (EC50 = 0.1 nM, 0.3 mM, and 20 mM). CGRP and forskolin caused an increase in cAMP production and a transient decrease in the [Ca2+]i. The CGRP-induced vasodilation was antagonized by CGRP8-37 (KDB = 2 mM). The K+-channel blockers iberiotoxin and glibenclamide partially prevented the CGRP- or forskolin-induced vasodilations but failed to reverse these vasodilations. These results demonstrate that CGRP is present in perivascular nerves and causes a vasodilation of the ET1-preconstricted SMA. The data suggest that this vasodilation is mediated by an increase in the cytosolic cAMP concentration, a transient activation of iberiotoxin-sensitive BK and glibenclamide-sensitive KATP K+ channels, a transient decrease in the [Ca2+]i and a long-lasting Ca2+ desensitization.     

Neurally-derived nitric oxide regulates vascular tone in pulmonary and cutaneous arteries of the toad, Bufo marinus

In this study, the role of nitric oxide (NO) in regulation of the pulmocutaneous vasculature of the toad, Bufo marinus was investigated. In vitro myography demonstrated the presence of a neural NO signaling mechanism in both arteries. Vasodilation induced by nicotine was inhibited by the soluble guanylyl cyclase (GC) inhibitor, 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one, and the NO synthase (NOS) inhibitor, N(omega)-nitro-l-arginine (l-NNA). Removal of the endothelium had no significant effect on the vasodilation. Furthermore, pretreatment with N(5)-(1-imino-3-butenyl)-l-ornithine (vinyl-l-NIO), a more specific inhibitor of neural NOS, caused a significant decrease in the nicotine-induced dilation. In the pulmonary artery only, a combination of l-NNA and the calcitonin gene-related peptide (CGRP) receptor antagonist, CGRP((8-37)), completely blocked the nicotine-induced dilation. In both arteries, the vasodilation was also significantly decreased by glibenclamide, an ATP-sensitive K(+) (K(+)(ATP)) channel inhibitor. Levcromakalim, a K(+)(ATP) channel opener, caused a dilation that was blocked by glibenclamide in both arteries. In the pulmonary artery, NO donor-mediated dilation was significantly decreased by pretreatment with glibenclamide. The physiological data were supported by NADPH-diaphorase histochemistry and immunohistochemistry, which demonstrated NOS in perivascular nerve fibers but not the endothelium of the arteries. These results indicate that the pulmonary and cutaneous arteries of B. marinus are regulated by NO from nitrergic nerves rather than NO released from the endothelium. The nitrergic vasodilation in the arteries appears to be caused, in part, via activation of K(+)(ATP) channels. Thus, NO could play an important role in determining pulmocutaneous blood flow and the magnitude of cardiac shunting.     

The potassium channel opener cromakalim (BRL 34915) activates ATP-dependent K+ channels in isolated cardiac myocytes

In cardiac myocytes, cromakalim (BRL 34915), a potassium channel opener, activates a time-independent K+ current exhibiting poor voltage-sensitivity. This effect of cromakalim is antagonized by low concentrations of glibenclamide, a specific blocker of ATP-dependent K+ channels in cardiac cells. Direct recording of the activity of K+ channels in inside-out membrane patches, confirmed that cromakalim is a potent activator of ATP-dependent K+ channels in cardiac myocytes.     

Myelin basic protein as a binding partner and calmodulin adaptor for the BKCa channel

The activity and localization of large-conductance Ca2+ -activated K+ (BKCa) channels are known to be modulated by several different proteins. Although many binding partners have been identified via yeast two-hybrid screening, this method may not detect certain classes of interacting proteins such as low affinity binding proteins or multi-component protein complexes. In this study, we employed mass spectrometry to identify proteins that interact with BKCa channels. We expressed and purified the 'tail domain' of the rat BKCa channel alpha-subunit, a 54-kDa region that is crucial for expression and functional activity of the channel. Using rat brain lysate and purified 'tail domain', we identified several novel proteins that interact with the BKCa channel. These included the myelin basic protein (MBP), upon which we performed subsequent biochemical and electrophysiological studies. Interaction between the BKCa channel and MBP was confirmed in vivo and in vitro. MBP co-expression affected the Ca2+ -dependent activation of the BKCa channel by increasing its Ca2+ sensitivity. Moreover, we showed that calmodulin (CaM) interacts with the BKCa channel via MBP. Since CaM is a key regulator of many Ca2+ -dependent processes, it may be recruited by MBP to the vicinity of the BKCa channel, modulating its functional activity.