Propafenone modulates potassium channel activities of vascular smooth muscle from rat portal veins

We have studied the effects of the class Ic antiarrhythmic propafenone on K+ currents in freshly isolated smooth muscle cells from rat portal veins and on the spontaneous contractions in whole tissues. Under Ca2+-free conditions, when cells were clamped at -80 mV (whole-cell configuration) depolarizing steps from -80 to +50 mV induced a family of K+ currents (I(Ktotal)) that mainly comprised the delayed rectifier current [I(K(V))], whereas when held at -10 mV only small-amplitude, noninactivating, currents (I(NI)) were recorded. Propafenone (10 microM) markedly inhibited I(Ktotal), but at potentials positive to +30 mV it also induced a noisy outwardly rectifying current [I(BK(Ca))] that was abolished by iberiotoxin (0.1 microM). Inhibition of I(Ktotal) by propafenone was concentration-dependent (EC50 = 0.059 +/- 0.009 microM). Propafenone also inhibited the transient outward current [I(K(A))] and ATP-sensitive potassium current [I(K(ATP))] induced by levcromakalim (10 microM). Inhibition of I(K(V)), I(K(A)), and I(K(ATP)) by propafenone was voltage-independent. In Ca(2+)-containing conditions propafenone inhibited I(K(V)) and I(BK(Ca)) and immediately abolished spontaneous outward transient K+ currents. In whole veins, propafenone behaved as the K(V) inhibitor 4-aminopyridine, increasing the amplitude and duration of spontaneous contractions. Propafenone also inhibited the inhibitory effects of the K(ATP) channel opener levcromakalim on spontaneous contractions. These results indicate that in vascular smooth muscle cells, propafenone inhibits K(V), K(A), BK(Ca), and K(ATP) channels. These actions correlated with its effects on mechanical activity in whole portal veins.     

Role of KATP channels in the regulation of rat dura and pia artery diameter

The aim of the present study was to examine the effect of K(ATP) channel openers pinacidil and levcromakalim on rat dural and pial arteries as well as their inhibition by glibenclamide. We used an in-vivo genuine closed cranial window model and an in-vitro organ bath. Glibenclamide alone reduced the dural but not the pial artery diameter compared with controls. Intravenous pinacidil and levcromakalim induced dural and pial artery dilation that was significantly attenuated by glibenclamide. In the organ bath pinacidil and levcromakalim induced dural and middle cerebral artery relaxation that was significantly attenuated by glibenclamide. In conclusion, K(ATP) channel openers induce increasing diameter/relaxation of dural and pial arteries after intravenous infusion in vivo and on isolated arteries in vitro. Furthermore, dural arteries were more sensitive to K(ATP) channel openers than pial arteries.     

The relaxation induced by S-nitroso-glutathione and S-nitroso-N-acetylcysteine in rat aorta is not related to nitric oxide production.

S-nitroso-glutathione (GSNO) and S-nitroso-N-acetylcysteine (NACysNO) are nitrosothiols that release nitric oxide (NO) and mimic the effects of endogenous NO. This study investigated the relaxation induced by GSNO and NACysNO in rat aorta and the relation between relaxation and NO formation. Both compounds at concentrations from 10(-9) M to 10(-4) M relaxed the rat aorta in a concentration-dependent manner. However, NO production depended on the concentration of nitrosothiols present and was detected only above 100 microM GSNO or NACysNO. To determine whether K+ channels are involved in the relaxation induced by nitrosothiols, the contractions were induced with KCl at concentrations of 30, 60, or 90 mM. The concentration-effect curves for the relaxation induced by nitrosothiols were shifted to the right for all the K+ concentrations compared with aortas precontracted with phenylephrine. These results indicate the participation of K+ channels in the relaxation induced by GSNO and NACysNO. A selective inhibitor of soluble guanylyl cyclase, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, significantly inhibited the relaxation induced by the nitrosothiols. The relaxation induced by GSNO and NACysNO was inhibited by the K+ channel blockers glibenclamide, selective K(ATP) channels, and apamin, selective for low-conductance Ca2+-activated K+ channels in rat aorta, but was not inhibited by charybdotoxin, a potent and selective Ca2+-activated K+ channel blocker, or by 4-aminopyridine, a voltage-gated K+ channel blocker. These results indicate that relaxation induced by GSNO and NACysNO is partially due to activation of K(ATP) channels and partially due to activation of low-conductance Ca2+-activated K+ channels. However, the ability of the nitrosothiol compounds to overcome the inhibitory effect of high extracellular K+ concentrations suggests another mechanism of relaxation contributing to the nitrosothiol response. The most intriguing finding is that relaxation is not related to the NO produced in rat aorta.     

A xanthine-based epithelium-dependent airway relaxant KMUP-3 (7-[2-[4-(4-nitrobenzene)piperazinyl]ethyl]-1,3-dimethylxanthine) increases respiratory performance and protects against tumor necrosis factor-alpha-induced tracheal contraction, involving nitric oxide release and expression of cGMP and protein kinase G

KMUP-3 (7-[2-[4-(4-nitrobenzene)piperazinyl]ethyl]-1,3-dimethylxanthine) was investigated in guinea pig tracheal smooth muscle. Intratracheal instillation of tumor necrosis factor (TNF)-alpha (0.01 mg/kg/300 microl) induced bronchoconstriction, increases of lung resistance, and decreases of dynamic lung compliance. Instillation of KMUP-3 (0.5-2.0 mg/kg) reversed this situation. In isolated trachea precontracted with carbachol, KMUP-3 (10-100 microM)-caused relaxations were attenuated by epithelium removal and by pretreatments with an inhibitor of K(+) channel, tetraethylammonium (10 mm); K(ATP) channel, glibenclamide (1 microM); voltage-dependent K(+) channel, 4-aminopyridine (100 microM); Ca(2+)-dependent K(+) channel, charybdotoxin (0.1 microM) or apamin (1 microM); soluble guanylate cyclase (sGC), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1one (ODQ, 1 microM); nitric-oxide (NO) synthase, N(omega)-nitro-L-arginine methyl ester (L-NAME, 100 microM); and adenylate cyclase, SQ 22536 [9-(terahydro-2-furanyl)-9H-purin-6-amine] (100 microM). KMUP-3 (0.01-100 microM) induced increases of cGMP and cAMP in primary culture of tracheal smooth muscle cells (TSMCs). The increase in cGMP by KMUP-3 was reduced by ODQ and L-NAME; the increase in cAMP was reduced by SQ 22536. Western blot analysis indicated that KMUP-3 (1 microM) induced expression of protein kinase A (PKA)(ri) and protein kinase G (PKG)(1alpha 1beta) in TSMCs.SQ 22536 inhibited KMUP-3-induced expression of (PKA)(ri).On the contrary, ODQ inhibited KMUP-3-induced expression of PKG(1alpha 1beta) In epithelium-intact trachea, KMUP-3 increased the NO release. Activation of sGC, NO release, and inhibition of phosphodiesterases in TSMCs by KMUP-3 may result in increases of intracellular cGMP and cAMP, which subsequently activate PKG and PKA, efflux of K(+) ion, and associated reduction in Ca(2+) influx in vitro, indicating the action mechanism to protect against TNF-alpha-induced airway dysfunction in vivo.     

Involvement of charybdotoxin-sensitive K+ channel in the relaxation of bovine tracheal smooth muscle by glyceryl trinitrate and sodium nitroprusside.

To elucidate the involvement of K+ channels in the smooth muscle relaxation by glyceryl trinitrate (GTN) and sodium nitroprusside (SNP), effects of several K+ channel antagonists on the relaxant responses to GTN, SNP and 8-bromoguanosine 3',5'-cyclic monophosphate (8-Br-cGMP) were studied in bovine tracheal smooth muscle. Although an antagonist of large conductance Ca(++)-activated K+ channel, charybdotoxin, produced no definite effect on the relaxation induced by GTN, SNP and atriopeptin in the rabbit aortic ring preparation, this antagonist inhibited the relaxation by GTN, SNP, atriopeptin and 8-Br-cGMP in the bovine tracheal smooth muscle. Methylene blue, a soluble guanylate cyclase inhibitor, also had an inhibitory effect on the relaxation by GTN and SNP. Both apamin, a small conductance Ca(++)-activated K+ channel antagonist, and glibenclamide, an ATP-sensitive K+ channel antagonist, did not exhibit any inhibitory effect on the relaxant responses to GTN and SNP. GTN and SNP increased cGMP content. The increment was attenuated by methylene blue, whereas it was unaffected by charybdotoxin. These results indicate the involvement of large conductance Ca(++)-activated K+ channel in the relaxation of bovine tracheal smooth muscle by GTN, SNP and 8-Br-cGMP. The activation of K+ channel by GTN and SNP is thought to occur via increases in cGMP content.     

The endogenous brain constituent N-arachidonoyl L-serine is an activator of large conductance Ca2+-activated K+ channels

The novel endocannabinoid-like lipid N-arachidonoyl L-serine (ARA-S) causes vasodilation through both endothelium-dependent and -independent mechanisms. We have analyzed the vasorelaxant effect of ARA-S in isolated vascular preparations and its effects on Ca(2+)-activated K(+) currents in human embryonic kidney cells stably transfected with the alpha-subunit of the human, large conductance Ca(+)-activated K(+) (BK(Ca)) channel [human embryonic kidney (HEK) 293hSlo cells]. ARA-S caused relaxation of rat isolated, intact and denuded, small mesenteric arteries preconstricted with (R)-(-)-1-(3-hydroxyphenyl)-2-methylaminoethanol hydrochloride (pEC(50), 5.49 and 5.14, respectively), whereas it caused further contraction of vessels preconstricted with KCl (pEC(50), 5.48 and 4.82, respectively). Vasorelaxation by ARA-S was inhibited by 100 nM iberiotoxin. In human embryonic kidney cells stably transfected with the alpha-subunit of the human BK(Ca) channel cells, ARA-S and its enantiomer, N-arachidonoyl-D-serine, enhanced the whole-cell outward K(+) current with similar potency (pEC(50), 5.63 and 5.32, respectively). The potentiation was not altered by the beta(1) subunit or mediated by ARA-S metabolites, stimulation of known cannabinoid receptors, G proteins, protein kinases, or Ca(2+)-dependent processes; it was lost after patch excision or after membrane cholesterol depletion but was restored after cholesterol reconstitution. BK(Ca) currents were also enhanced by N-arachidonoyl ethanolamide (pEC(50), 5.27) but inhibited by another endocannabinoid, O-arachidonoyl ethanolamine (pIC(50), 6.35), or by the synthetic cannabinoid O-1918 [(-)-1,3-dimethoxy-2-(3-3,4-trans-p-menthadien-(1,8)-yl)-orcinol] (pIC(50), 6.59), which blocks ARA-S-induced vasodilation. We conclude the following. 1) ARA-S directly activates BK(Ca) channels. 2) This interaction does not involve cannabinoid receptors or cytosolic factors but is dependent on the presence of membrane cholesterol. 3) Direct BK(Ca) channel activation probably contributes to the endothelium-independent component of ARA-S-induced mesenteric vasorelaxation. 4) O-1918 is a BK(Ca) channel inhibitor.     

The inhibitory effect of adrenomedullin in the rat ileum: cross-talk with beta3-adrenoceptor in the serotonin-induced muscle contraction

In contrast to vascular muscles, the contribution of a hypotensive peptide adrenomedullin (AM) to the regulation of visceral smooth muscles is obscure. The content, synthesis, and effects of AM on the muscular tone in rat ileum were explored. It was found that there was immunoreactive AM (301 pg/mg of protein) and AM mRNA expression (162 fg/pg actin mRNA) in the ileum and that AM evoked relaxation in ileal strips (Ki = 0.85 nM) precontracted with serotonin. Antagonists of both AM (AM(22-52)) and calcitonin gene-related peptide (CGRP(8-37)) receptors did not affect this AM-induced relaxation, whereas it was suppressed by a selective blocker of beta3-adrenoreceptor (SR 59230A). The AM-induced relaxation was accompanied by a production of cAMP. Antagonists of protein kinases A (KT 5720 and H-7) and an inhibitor of the ATP-dependent K(+)-channels (glibenclamide) attenuated the effect of AM. We suggest that AM is a local regulator of the ileal tone, with an inhibitory action on muscle contraction. AM may activate the beta3-adrenoceptors, resulting in protein kinase A activation, which in turn opens the ATP-dependent K(+)-channels.     

Ca2+-independent, inhibitory effects of cyclic adenosine 5'-monophosphate on Ca2+ regulation of phosphoinositide 3-kinase C2alpha, Rho, and myosin phosphatase in vascular smooth muscle

We have recently demonstrated in vascular smooth muscle (VSM) that membrane depolarization by high KCl induces Ca(2+)-dependent Rho activation and myosin phosphatase (MLCP) inhibition (Ca(2+)-induced Ca(2+)-sensitization) through the mechanisms involving phosphorylation of myosin-targeting protein 1 (MYPT1) and 17-kDa protein kinase C (PKC)-potentiated inhibitory protein of PP1 (CPI-17). In the present study, we investigated whether and how cAMP affected Ca(2+)-dependent MLCP inhibition by examining the effects of forskolin, cell-permeable dibutyryl cAMP (dbcAMP), and isoproterenol. Forskolin, but not its inactive analog 1,9-dideoxyforskolin, inhibited KCl-induced contraction and the 20-kDa myosin light chain (MLC) phosphorylation without inhibiting Ca(2+) mobilization in rabbit aortic VSM. dbcAMP mimicked these forskolin effects. We recently suggested that Ca(2+)-mediated Rho activation is dependent on class II alpha-isoform of phosphoinositide 3-kinase (PI3K-C2alpha). Forskolin inhibited KCl-induced stimulation of PI3K-C2alpha activity. KCl-induced membrane depolarization stimulated Rho in a manner dependent on a PI3K but not PKC and stimulated phosphorylation of MYPT1 at Thr(850) and CPI-17 at Thr(38) in manners dependent on both PI3K and Rho kinase, but not PKC. Forskolin, dbcAMP, and isoproterenol inhibited KCl-induced Rho activation and phosphorylation of MYPT1 and CPI-17. Consistent with these data, forskolin, isoproterenol, a PI3K inhibitor, or a Rho kinase inhibitor, but not a PKC inhibitor, abolished KCl-induced diphosphorylation of MLC. These observations indicate that cAMP inhibits Ca(2+)-mediated activation of the MLCP-regulating signaling pathway comprising PI3K-C2alpha, Rho, and Rho kinase in a manner independent of Ca(2+) and point to the novel mechanism of the cAMP actions in the regulation of vascular smooth muscle contraction.     

Comparison of effects of cromakalim and pinacidil on mechanical activity and 86Rb efflux in dog coronary arteries

Effects of two K+ channel openers, cromakalim and pinacidil, on mechanical activity and on 86Rb efflux were compared in strips of dog coronary arteries. Cromakalim and pinacidil produced the relaxation in 20.9 mM K(+)-contracted strips with a pD2 of 6.53 and 5.95, respectively. In 65.9 mM K(+)-contracted strips, high concentrations of pinacidil, but not cromakalim, produced relaxation. Ca+(+)-induced contractions in 80 mM K(+)-depolarized strips were also inhibited by pinacidil but not by cromakalim. Glibenclamide, a blocker of ATP-regulated K+ (KATP) channels, competitively antagonized the relaxant responses to cromakalim with a pA2 value of 7.62. However, the antagonism by glibenclamide of the relaxant responses to pinacidil was not a typical competitive type, suggesting the contribution of other effects than the KATP channel opening activity to the relaxant effects of pinacidil. In resting strips preloaded with 86Rb, cromakalim and pinacidil increased the basal 86Rb efflux in a dose-dependent manner. The increase in the 86Rb efflux induced by cromakalim was greater than that by pinacidil. When the effects of cromakalim and pinacidil on the 86Rb efflux were determined in the 20.9 or 65.9 mM K(+)-contracted strips, both drugs increased the 86Rb efflux. Under the same conditions nifedipine, a Ca(+)+ channel blocker, produced the relaxation that is accompanied by the decrease in 86Rb efflux. The increase in the 86Rb efflux induced by cromakalim was much greater than that by pinacidil.(ABSTRACT TRUNCATED AT 250 WORDS)     

Does cyclic AMP mediate rat urinary bladder relaxation by isoproterenol?

Cyclic AMP is the prototypical second messenger of beta-adrenergic receptors, but recent findings have questioned its role in mediating smooth muscle relaxation upon beta-adrenergic receptor stimulation. We have investigated the signaling mechanisms underlying beta-adrenergic receptor-mediated relaxation of rat urinary bladder. Concentration-response curves for isoproterenol-induced bladder relaxation were generated in the presence or absence of inhibitors, with concomitant experiments using passive tension and KCl-induced precontraction. The adenylyl cyclase inhibitor 9-(tetrahydro-2-furanyl)-9H-purin-6-amine (SQ 22,536; 1 microM), the protein kinase A inhibitors 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7; 10 microM), N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H89; 1 microM), and Rp-adenosine 3',5'-cyclic monophosphorothioate (Rp-cAMPS; 30 microM), and the guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ; 3 microM) produced only minor if any inhibition of relaxation against passive tension or KCl-induced precontraction. Among various potassium channel inhibitors, BaCl2 (10 microM), tetraethylammonium (3 microM), apamin (300 nM), and glibenclamide (10 microM) did not inhibit isoproterenol-induced relaxation. Some inhibition of the isoproterenol effects against KCl-induced tone but not against passive tension was seen with inhibitors of calcium-dependent potassium channels such as charybdotoxin and iberiotoxin (30 nM each). A combination of SQ 22,536 and ODQ significantly inhibited relaxation against passive tension by about half, but not that against KCl-induced tone. Moreover, the combination failed to enhance inhibition by charybdotoxin against KCl-induced tone. We conclude that cAMP and cGMP each play a minor role in beta-adrenergic receptor-mediated relaxation against passive tension, and calcium-dependent potassium channels play a minor role against active tension.     

O2 deprivation inhibits Ca2+-activated K+ channels via cytosolic factors in mice neocortical neurons

O(2) deprivation induces membrane depolarization in mammalian central neurons. It is possible that this anoxia-induced depolarization is partly mediated by an inhibition of K(+) channels. We therefore performed experiments using patch-clamp techniques and dissociated neurons from mice neocortex. Three types of K(+) channels were observed in both cell-attached and inside-out configurations, but only one of them was sensitive to lack of O(2). This O(2)-sensitive K(+) channel was identified as a large-conductance Ca(2+)-activated K(+) channel (BK(Ca)), as it exhibited a large conductance of 210 pS under symmetrical K(+) (140 mM) conditions, a strong voltage-dependence of activation, and a marked sensitivity to Ca(2+). A low-O(2) medium (PO(2) = 10-20 mmHg) markedly inhibited this BK(Ca) channel open probability in a voltage-dependent manner in cell-attached patches, but not in inside-out patches, indicating that the effect of O(2) deprivation on BK(Ca) channels of mice neocortical neurons was mediated via cytosol-dependent processes. Lowering intracellular pH (pH(i)), or cytosolic addition of the catalytic subunit of a cAMP-dependent protein kinase A in the presence of Mg-ATP, caused a decrease in BK(Ca) channel activity by reducing the sensitivity of this channel to Ca(2+). In contrast, the reducing agents glutathione and DTT increased single BK(Ca) channel open probability without affecting unitary conductance. We suggest that in neocortical neurons, (a) BK(Ca) is modulated by O(2) deprivation via cytosolic factors and cytosol-dependent processes, and (b) the reduction in channel activity during hypoxia is likely due to reduced Ca(2+) sensitivity resulting from cytosolic alternations such as in pH(i) and phosphorylation. Because of their large conductance and prevalence in the neocortex, BK(Ca) channels may be considered as a target for pharmacological intervention in conditions of acute anoxia or ischemia.     

Nitric oxide from enteric nerves acts by a different mechanism from myogenic nitric oxide in canine lower esophageal sphincter

In canine lower esophageal sphincter, myogenic constitutive nitric-oxide (NO) synthase (NOS) in plasma membrane limits tone by opening large conductance Ca(2+)-dependent K(+) channels (BK(Ca) channels) and hyperpolarizing the membrane. We examined whether K(V) channels were involved and whether NO from enteric nerves and from NO donors used the same mechanisms. With nerves inactive, 100 nM iberiotoxin, like N-nitro-L-arginine (L-NOARG), increased tone but less. 4-Aminopyridine (4-AP) at 5 mM behaved similarly. Tetraethyl ammonium (TEA) at 20 mM equaled the effect of L-NOARG and occluded any tone increase from any combination of these agents. More than iberiotoxin or 4-AP, TEA decreased relaxations in response to sodium nitroprusside (SNP) or 3-morpholino-sydnonimine (Sin-1) by approximately 50%. In whole-cell patch-clamp recordings, TEA and 4-AP reduced outward K(+) currents additively by >90% at depolarization of +90 mV. Thus, K(+) channels in addition to BK(Ca) channels are opened by myogenic NO, and exogenous NO had relaxing effects both related and unrelated to K(+) channel openings. TEA (20 mM) increased tone but did not inhibit relaxations to electrical field stimulation (EFS) of enteric nerves. 4-AP relaxed tone, an effect that was abolished and reversed by L-NOARG. 4-AP apparently released NO and acetylcholine from nerves. The putative Cl(-) channel blocker niflumic acid (NFA; 30-100 microM) dose dependently reduced tone, but tone, restored by 10(-6) M carbachol or 20 mM TEA, was still relaxed by EFS and by SNP. 4,4'-Diisothiocyanatostilbene-2, 2'-disulfonic acid (DIDS) at 500 to 1000 microM did not inhibit relaxation to EFS or SNP. The addition of TEA (20 mM) to DIDS (1000 microM) induced tonic and phasic activity and markedly inhibited relaxations to EFS. DIDS plus TEA reduced the relaxations to SNP like TEA alone. Reduction in extracellular ?Cl(-) by isethionate substitution reduced tone but did not reduce relaxations when tone was restored. The combination of reduced extracellular ?Cl(-) and TEA did not abolish relaxation to EFS until DIDS was added. Thus, multiple K(+) channels are opened by myogenic NO, and openings of these channels, as well as DIDS-sensitive, undefined mechanisms, are induced when NO is released from nerves or SNP.     

Characterization of the relaxant response to N,N'-dipropyl-1,2-bis(2,6-dichloro-4-hydroxyphenyl)ethylenediamine in porcine coronary arteries

N,N'-Dialkyl-1,2-bis(2,6-dichloro-4-hydroxyphenyl)ethylenediamines show structural analogy with estrogens and selective estrogen receptor modulators. Because the vasodilator properties of these compounds are unknown, we investigated their potential to relax porcine coronary arteries and determined the mechanism(s) of relaxation. Isolated porcine coronary arterial rings were suspended in organ chambers, precontracted with KCl (30 mM), and the relaxant response was determined by measurement of changes in isometric force. Dependent on the chemical structure, the drugs induced concentration-dependent relaxation in rings with and without endothelium. N,N'-Dipropyl-1,2-bis(2,6-dichloro-4-hydroxyphenyl)ethylenediamine (8) was most potent and showed a 12- to 15-fold higher vasodilatory effect than 17beta-estradiol (E2). The vasorelaxation was independent of endothelium. Calcium concentration-dependent contractions in high-potassium depolarizing medium were insurmountably inhibited by 8. The effect of the L-type Ca2+ channel activator (S)-(-)-Bay K 8644 [(S)-(-)-1,4-dihydro-2,6-dimethyl-5-nitro-4-[2-(trifluoromethyl)phenyl]-3-pyridine-carboxylic acid methyl ester], which induced a leftward shift of Ca2+ contraction, was blocked by 8. The relaxant response to 8 was unaffected by the estrogen receptor antagonist ICI 182,780 (7alpha-[9-[(4,4,5,5,5-pentafluoropentyl]-sulfinyl]nonyl]-estra-1,3,5(10)-triene-3,17beta-diol) and K+ channel blockers, i.e., TEA, glibenclamide, and 4-aminopyridine. Furthermore, the vasodilatory effect of 8 was unaffected by the adenylyl cyclase inhibitor SQ 22536 [9-(tetrahydro-2-furanyl)-9H-purin-6-amine], the guanylyl cyclase inhibitor ODQ [1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one], the protein kinase A inhibitor KT 5720 [(9S,10S,12R)-2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg: 3',2',1'-kl]pyrrolo[3,4-i][1,6]benzodiazocine-10-carboxylic acid hexyl ester], the protein kinase G inhibitor KT 5823 [(9S,10R,12R)-2,3,9,10,11,12-hexahydro-10-methoxy-2,9-dimethyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-i][1,6]benzodiazocine-10-carboxylic acid methyl ester], and the p38 mitogen-activated protein kinase (MAPK) inhibitor SB 203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole]. Western blot analysis demonstrated that 8, unlike E2, raloxifene, and tamoxifen, failed to stimulate p38 MAPK. It is concluded that N,N'-dipropyl-1,2-bis(2,6-dichloro-4-hydroxyphenyl)ethylenediamine induces endothelium-independent relaxation of coronary arteries; the mechanism apparently involves inhibition of L-type Ca2+ channels. The drug may be protective against cardiovascular diseases.     

Characterization of tetrandrine-induced inhibition of large-conductance calcium-activated potassium channels in a human endothelial cell line (HUV-EC-C)

The effects of tetrandrine, a blocker of voltage-dependent Ca(2+) channels, on ionic currents were investigated in an endothelial cell line (HUV-EC-C) originally derived from human umbilical vein. In whole-cell configuration, tetrandrine (0.5-50 microM) reversibly decreased the amplitude of K(+) outward currents. The IC(50) value of tetrandrine-induced decrease in outward current was 5 microM. The K(+) outward current in response to depolarizing voltage pulses was also inhibited by iberiotoxin (200 nM), yet not by glibenclamide (10 microM) or apamin (200 nM). The reduced amplitude of outward current by tetrandrine can be reversed by the further addition of Evans' blue (30 microM) or niflumic acid (30 microM). Thus, the tetrandrine-sensitive component of outward current is believed to be Ca(2+)-activated K(+) current. Pretreatment with thapsigargin (1 microM) or sodium nitroprusside (10 microM) for 5 h did not prevent tetrandrine-mediated inhibition of outward current. In outside-out configuration, bath application of tetrandrine (5 microM) did not change the single-channel conductance but significantly reduced the opening probability of large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels. The tetrandrine-mediated decrease in the channel activity was independent on internal Ca(2+) concentration. Tetrandrine (5 microM) can also shift the activation curve of BK(Ca) channels to more positive potentials by approximately 20 mV. The change in the kinetic behavior of BK(Ca) channels caused by tetrandrine is due to a decrease in mean open time and an increase in mean closed time. The present study provides substantial evidence that tetrandrine is capable of suppressing the activity of BK(Ca) channels in endothelial cells. The direct inhibition of these channels by tetrandrine should contribute to its effect on the functional activities of endothelial cells.     

The novel and specific Rho-kinase inhibitor (S)-(+)-2-methyl-1-[(4-methyl-5-isoquinoline)sulfonyl]-homopiperazine as a probing molecule for Rho-kinase-involved pathway

We have developed several kinds of protein kinase inhibitors, which are classified as isoquinolinesulfonamides and characterized as ATP competitive inhibitors of Ser/Thr protein kinases. These include H9, H89, KN62, and 1-(5-isoquinolinesulfonyl)-homopiperazine (HA-1077) against protein kinase C (PKC), protein kinase A, Ca(2+)/calmodulin-dependent protein kinase II, and Rho-kinase, respectively, and they have been used widely to confirm the involvement of the target protein kinase in biological or physiological reaction(s). In some cases, inhibitors have predicted the involvement of the target protein kinase in cell or tissue before its precise mechanism or its effector was defined. On a clinical level, we developed the Rho-kinase inhibitor HA-1077 as an anti-spastic that effectively suppresses the spasm of cerebral arteries after subarachnoid hemorrhage. We have improved HA-1077 to obtain (S)-(+)-2-methyl-1-[(4-methyl-5-isoquinoline)sulfonyl]-homopiperazine (H-1152P), which is a more selective inhibitor of Rho-kinase, with a K(i) value of 1.6 nM for Rho-kinase, 630 nM for protein kinase A, and 9270 nM for PKC. This inhibitor suppressed the phosphorylation of myristoylated alanine-rich C-kinase substance (MARCKS) in neuronal cells stimulated with lysophosphatidic acid, whose phosphorylation site was confirmed to be the Ser159 residue, using a phosphorylation site-specific antibody. In contrast, phorbol 12-myristate 13-acetate-induced phosphorylation of MARCKS was scarcely inhibited by H-1152P. Furthermore, lysophosphatidic acid-stimulated phosphorylation in neuronal cells was characterized as a C3 toxin-sensitive event. Our results show that the Rho-kinase inhibitor targets a protein with a well-known function, MARCKS in neuronal cells. Although MARCKS is widely recognized as a substrate of PKC, our results raise the possibility that MARCKS is a target protein of Rho-kinase in neuronal cells. In this review, we address the possible role of Rho-kinase in neuronal functions, using the Rho-kinase specific inhibitor H-1152P.     

K(+)-induced neurogenic relaxation of rat distal colon.

Relaxations of segments of rat distal colon were elicited by hypertonic solutions of potassium (K(+); final concentration, 20.8 or 50.8 mM). The initial part of the response to K(+) was antagonized by the nerve blocker tetrodotoxin. This effect could, moreover, be significantly antagonized by apamin (a blocker of K(+) channels), reactive blue 2 (a P(2y)-purinoceptor antagonist), N(G)-nitro-L-arginine (an inhibitor of NO synthase), 1H-[1,2,4]- oxadiazolo[4,3-a]quinoxaline-1-one (ODQ; an inhibitor of soluble guanylyl cyclase), or N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89; an inhibitor of cAMP-dependent protein kinase). Sodium nitroprusside (a donor of NO) and vasoactive intestinal peptide (VIP) both relaxed the tissues. The response to sodium nitroprusside was abolished by ODQ and unaffected by H-89, and that to VIP was partially inhibited by VIP(10-28) (a VIP receptor antagonist), ODQ, or H-89. When combining reactive blue 2 and N(G)-nitro-L-arginine, the response to 50.8 mM K(+) was reduced by approximately 70% and was abolished by the concomitant administration of these antagonists and VIP(10-28). ATP, NO, and VIP may, thus, be inhibitory neurotransmitters in rat distal colon.     

Ca2+-activated K+ channels underlying the impaired acetylcholine-induced vasodilation in 2K-1C hypertensive rats

We tested the hypothesis that an abnormal function of K(+) channels in vascular smooth muscle cells plays a key role in the impaired acetylcholine (ACh) vasodilation in aortas from two kidney-one clip (2K-1C) hypertensive rats and further investigated the K(+) channel subtype involved in this altered response. ACh-induced endothelium-dependent relaxation was assessed in aortic rings from 2K-1C and normotensive two kidney (2K) rats. Glibenclamide, an ATP-sensitive K(+) channel blocker, did not inhibit ACh-induced relaxation in aortic rings from 2K or 2K-1C rats. The voltage-dependent K(+) channels inhibitor 4-aminopyridine attenuated ACh-induced relaxation in both groups. Charybdotoxin and iberiotoxin, blockers of Ca(2+)-sensitive (K(Ca)) and large-conductance K(Ca) (BK(Ca)) channels, respectively, reduced ACh-induced relaxation in aortic rings from 2K rats without affecting this response in those from 2K-1C rats, abolishing the differences between groups. ACh-induced relaxation in vessels from both 2K and 2K-1C rats was unaffected by apamin, a small-conductance K(Ca) blocker. NS1619 [1,3-dihydro-1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-2H-benzimidazol-2-one], an activator of K(Ca), induced a smaller vasodilation in endothelium-denuded aortic rings from 2K-1C rats compared with those from 2K rats. Iberiotoxin reduced sodium nitroprusside-induced relaxation in endothelium-denuded aortic rings from 2K without affecting this response in those from 2K-1C rats. The inhibition of Na(+),K(+)-ATPase with ouabain had no effects on ACh-induced relaxation in aortic rings from 2K-1C or 2K rats. These data indicate that a deficient functional activity of BK(Ca) channels plays a key role in the impaired ACh vasodilation in aortas from 2K-1C rats.     

Neuronally released acetylcholine acts on the M2 muscarinic receptor to oppose the relaxant effect of isoproterenol on cholinergic contractions in mouse urinary bladder

We investigated whether M(2) muscarinic receptor activation opposes isoproterenol-induced relaxation in mouse urinary bladder and whether endogenous acetylcholine acts through a similar M(2) mechanism. When measured in urinary bladder from M(3) receptor knockout mice, the muscarinic agonist oxotremorine-M elicited only very weak contractions. In the presence of alpha,beta-methylene ATP (30 microM) and isoproterenol (1 microM), however, oxotremorine-M elicited a robust contractile response. This response was completely absent in bladder from M(2)/M(3) double knockout mice, indicating that activation of the M(2) receptor inhibits the relaxant effect of isoproterenol on the contraction to alpha,beta-methylene ATP. Similar results were obtained when prostaglandin F(2alpha) (5 microM) was used as the contractile agent but not when serotonin was used. Electrical field stimulation of the urinary bladder from wild-type mouse elicited contractions that were inhibited 20% by atropine and 40% by desensitization with alpha,beta-methylene ATP. When measured in the presence of alpha,beta-methylene ATP to desensitize the purinergic component of contraction, isoproterenol exhibited moderately greater relaxant activity in field-stimulated bladder from the M(2) knockout mouse compared with that observed in wild-type bladder. This differential relaxant effect of isoproterenol was greatly increased in the presence of physostigmine. In contrast, no differential effects were noted for isoproterenol in similar experiments on bladders from M(3) knockout and M(2)/M(3) double knockout mice in the presence of physostigmine. Our results suggest that neuronally released acetylcholine acts on the M(2) muscarinic receptor to inhibit the relaxant effect of isoproterenol on the minor, cholinergic component of contraction in the field-stimulated mouse urinary bladder.     

Mechanisms of beta-adrenergic receptor-mediated venodilation in humans

OBJECTIVES: Recent studies suggest that stimulation of beta-adrenergic receptors results in both endothelium-dependent and endothelium-independent venodilation, but results of former studies are inconsistent. This study was designed to elucidate the underlying mechanisms of isoproterenol (INN, isoprenaline)-induced venodilation by investigation of dorsal hand vein responses. METHODS: In phenylephrine-constricted veins, isoproterenol (2-514 ng/min) was infused with and without oral pretreatment with 1 g acetylsalicylic acid (n = 7) or 5 mg of the selective beta(1)-adrenergic receptor antagonist bisoprolol (n = 7). In addition, isoproterenol was coinfused with the nitric oxide inhibitor N(G)-monomethyl-l-arginine (l-NMMA) (6.3 micromol/min [n = 6]), with selective blockers of calcium (Ca(++))-dependent potassium (K(+)) channels (tetraethylammonium, 300 microg/min [n = 6]) and adenosine triphosphate (ATP)-sensitive K(+) channels (glyburide [INN, glibenclamide], 20 microg/min [n = 6]) or with the cyclic guanosine monophosphate inhibitor methylene blue (13 microg/min [n = 6]). Finally, L-NMMA was coinfused with potassium chloride (20 mmol/L) to inhibit hyperpolarization (n = 6). RESULTS: Isoproterenol induced dose-dependent venodilation to 67.4% +/- 6.8%. Oral pretreatment with bisoprolol (P =.340) or acetylsalicylic acid (P =.760) did not affect isoproterenol-induced venodilation. Coinfusion of isoproterenol and L-NMMA relaxed the veins to the same extent as in the presence of isoproterenol alone. Neither inhibition of ATP-sensitive K(+) channels (P =.196) nor blockade of Ca(++)-dependent K(+) channels (P =.640) modulated isoproterenol-induced venodilation. In contrast, methylene blue reduced the maximum response to isoproterenol by about one third (68.5% +/- 4.3% versus 41.7% +/- 5.5%, P =.001). Infusion of L-NMMA alone raised vein size to 38.8% +/- 6.5%, yielding an L-NMMA-sensitive increase of 20% (P =.001), which was antagonized by coinfusion of potassium chloride to 17.1% +/- 6.7% (P =.02). CONCLUSIONS: Isoproterenol dilates human hand veins exclusively via beta(2)-adrenergic receptors without involvement of endothelium-derived epoprostenol. Although a contribution of endothelium-derived nitric oxide appears unlikely, the venodilating effect of L-NMMA could have obscured the nitric oxide component of isoproterenol. beta(2)-Adrenergic receptor-mediated dilation is mediated in part by cyclic guanosine monophosphate-dependent mechanisms, whereas ATP- and Ca(++)-dependent K(+) channels are not involved, excluding a significant contribution of smooth muscle cell hyperpolarization. In addition, high concentrations of the nitric oxide synthase blocker L-NMMA dilate human hand veins via activation of endothelium-derived hyperpolarizing factors.