Mechanisms of the vasorelaxant effect of 1-hydroxy-2, 3, 5-trimethoxy-xanthone, isolated from a Tibetan herb, Halenia elliptica, on rat coronary artery

1-Hydroxy-2, 3, 5-trimethoxyxanthone (HM-1) is a xanthone isolated from Halenia elliptica, a Tibetan medicinal herb. HM-1 (0.33-42.1 microM) produced a concentration-dependent relaxation in rat coronary artery rings pre-contracted with 1 microM 5-hydroxytryptamine (5-HT), with an EC(50) of 1.67+/-0.27 microM. Removal of the endothelium significantly affected the vasodilator potency of HM-1, resulting in 46% decrease in E(max) value. The endothelium-dependent effects of HM-1 was confirmed when its vasorelaxant effect was inhibited after addition of nitric oxide synthase (NOS) inhibitor N(omega)-nitro-l-arginine methyl ester (100 microM) or the soluble guanylate cyclase inhibitor 1H-[1, 2, 4] oxadiazolo [4,3-alpha] quinoxalin-1-one (ODQ, 10 microM). Atropine (100 nM), flurbiprofen (10 microM), propranolol (100 microM), pyrilamine (10 microM), cimetidine (10 microM) and SQ22536 (100 microM) had no effect on the vasorelaxant activity of HM-1 indicated the non-involvement of other receptor/enzyme systems. In endothelium-denuded coronary artery rings, the vasorelaxant effect of HM-1 was unaffected by potassium channel blockers such as tetraethylammonium (10 mM), iberiotoxin (100 nM), barium chloride (100 microM) and 4-aminopyridine (1 mM). The involvement of Ca(2+) channel in 5-HT-primed artery ring preparations incubated with Ca(2+)-free buffer was confirmed when HM-1 (9.93 microM) partially abolished the CaCl(2)-induced vasoconstriction (87% inhibition in intact-endothelium artery rings; 50% inhibition in endothelium-denuded rings). In the KCl-primed preparations incubated with Ca(2+)-free buffer, HM-1 (9.93 microM) produced a 27.3% inhibition in endothelium-denuded rings. HM-1 (3.31-33.1 microM) had minimal relaxant effects (14.4%-20.3%) on the contractile response generated by 10 microM phorbol 12,13-diacetate (PDA) in Ca(2+)-free solutions, suggesting minimal effects on intracellular Ca(2+) mechanisms. These findings suggest the vasodilator action of HM-1 involved both an endothelium-dependent mechanism involving NO and an endothelium-independent mechanism by inhibiting Ca(2+) influx through L-type voltage-operated Ca(2+) channels; a minor contribution to the effects of HM-1 may be related to inhibition of the protein kinase C-mediated release of intracellular Ca(2+) stores.     

Novel tacrine derivatives that block neuronal calcium channels

A new series of tacrine (9-amino-1,2,3,4-tetrahydroacridine) derivatives were synthesized and their effects on 45Ca(2+) entry into bovine adrenal chromaffin cells stimulated with dimethylphenylpiperazinium (DMPP) or K(+), studied. At 3 microM, compound 1 did not affect (45)Ca(2+) uptake evoked by DMPP. Compounds 14, 15 and 17 inhibited the effects of DMPP by 30%. Compounds 3, 9 and tacrine blocked the DMPP signal by about 50%. Compounds 5 and 12 were the most potent blockers of DMPP-stimulated 45Ca(2+) entry (90%); the rest of the compounds inhibited the effects of DMPP by 70-80%. Compounds 1, 3, 4, 8, 10, 11, 13, 16, 17 and tacrine inhibited 45Ca(2+) uptake induced by K(+) about 20%. Compounds 6, 14 and 15 inhibited the K(+) effects by 10% or less. Compounds 7, 9, 12 and 18 blocked the K(+) signal by 30% and, finally, compounds 2 and 5 inhibited the K(+)-induced 45Ca(2+) entry by 50%. None of the new compounds was as effective as diltiazem (IC(50)=0.03 microM) in causing relaxation of the rat aorta precontracted with 35 mM K(+); the most potent was compound 7 (IC(50)=0.3 microM). Compounds 5, 6, 8, 9, 10 and 13 had IC(50)s around 10 microM and compounds 3, 4, 11 and 12 around 20 microM. Blockade of Ca(2+) entry through neuronal voltage-dependent Ca(2+) channels, without concomitant blockade of vascular Ca(2+) channels, suggests that some of these compounds might exhibit neuroprotectant effects but not undesirable hemodynamic effects.     

Peroxynitrite hyperpolarizes smooth muscle and relaxes internal carotid artery in rabbit via ATP-sensitive K+ channels

The goal of this study was to determine the effects of peroxynitrite (ONOO-) on smooth muscle membrane potential and vasomotor function in rabbit carotid arteries. ONOO- is known to affect vascular tone by several mechanisms, including effects on K+ channels. Xanthine (X, 0.1 mM), xanthine oxidase (XO, 0.01 U/ml), and a low concentration of sodium nitroprusside (SNP, 10 nM) were used to generate ONOO-. In the common carotid artery, X and XO (X/XO) in the presence of SNP tended to increase tension. In contrast, in the internal carotid artery, X/XO in the presence of SNP transiently hyperpolarized the membrane (-8.5 +/- 1.8 mV, mean +/- SE) and decreased tension (by 85 +/- 5.6%). In internal carotid arteries, in the absence of SNP, X/XO did not hyperpolarize the membrane and produced much less relaxation (by 23 +/- 5.6%) than X/XO and SNP. Ebselen (50 microM) inhibited both hyperpolarization and relaxation to X/XO and SNP, and uric acid (100 microM) inhibited relaxation. Glibenclamide (1 microM) abolished hyperpolarization and inhibited relaxation during X/XO and SNP. Charybdotoxin (100 nM) or tetraethylammonium (1 mM) did not affect hyperpolarization or relaxation, respectively. These results suggest that ONOO- hyperpolarizes and relaxes smooth muscle in rabbit internal carotid artery but not in common carotid artery through activation of K(ATP) channels.     

Identification of 30 kDa protein for Ca(2+) releasing action of myotoxin a with a mechanism common to DIDS in skeletal muscle sarcoplasmic reticulum.

The molecular mechanism of Ca(2+) release by myotoxin a (MTYX), a polypeptide toxin isolated from the venom of prairie rattlesnakes (Crotalus viridis viridis), was investigated in the heavy fraction of sarcoplasmic reticulum (HSR) of rabbit skeletal muscles. [(125)I]MYTX bound to four HSR proteins (106, 74, 53 and 30 kDa) on polyvinylidene difluoride (PVDF) membrane. DIDS, 4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid, bound predominantly to 30 kDa protein on the PVDF membrane, the molecular weight of which was similar to one of the MYTX binding proteins. The maximum (45)Ca(2+) release induced by caffeine (30 mM) was further increased in the presence of MYTX (10 microM) or DIDS (30 microM), whereas that induced by DIDS (30 microM) was not affected by MYTX (10 microM). MYTX inhibited [(3)H]DIDS binding to HSR in a concentration-dependent manner. Furthermore, [(125)I]MYTX binding to 30 kDa protein was inhibited by DIDS in a concentration-dependent manner. These results suggest that MYTX and DIDS release Ca(2+) from HSR in a common mechanism. The 30 kDa protein may be a target protein for the Ca(2+) releasing action of MYTX and DIDS.     

The anti-anginal drug fendiline elevates cytosolic Ca(2+) in rabbit corneal epithelial cells

The effect of the anti-anginal drug fendiline on intracellular free Ca(2+) levels ([Ca(2+)](i)) in a rabbit corneal epithelial cell line (SIRC) was explored using fura-2 as a fluorescent Ca(2+) indicator. At a concentration above 1 microM, fendiline increased [Ca(2+)](i) in a concentration-dependent manner with an EC(50) value of 7 microM. The [Ca(2+)](i) response consisted of an immediate rise and an elevated phase. Extracellular Ca(2+) removal decreased half of the [Ca(2+)](i )signal. Fendiline induced quench of fura-2 fluorescence by Mn(2+) (50 microM), suggesting the presence of Ca(2+) influx across the plasma membrane. This Ca(2+) influx was abolished by La(3+) (50 microM), but was insensitive to dihydropyridines, verapamil and diltiazem. Fendiline (10 microM)-induced store Ca(2+) release was largely reduced by pretreatment with thapsigargin (1 microM) (an endoplasmic reticulum Ca(2+) pump inhibitor) to deplete the endoplasmic reticulum Ca(2+). Conversely, pretreatment with 10 microM fendiline abolished thapsigargin-induced Ca(2+) release. Fendiline (10 microM)-induced Ca(2+) release was not altered by inhibiting phospholipase C with 2 microM 1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122). Cumulatively, this study shows that fendiline induced concentration-dependent [Ca(2+)](i )increases in corneal epithelial cells by releasing the endoplasmic reticulum Ca(2+) in a phospholipase C-independent manner, and by causing Ca(2+) influx.     

Effect of diethylstilbestrol (DES) on intracellular Ca(2+) levels in renal tubular cells

The effect of the estrogen diethylstilbestrol (DES) on intracellular Ca(2+) concentrations ([Ca(2+)](i)) in Madin Darby canine kidney (MDCK) cells was investigated, using the fluorescent dye fura-2 as a Ca(2+) indicator. DES (10-50 microM) evoked [Ca(2+)](i) increases in a concentration-dependent manner. Extracellular Ca(2+) removal inhibited 45 +/- 5% of the Ca(2+) response. In Ca(2+)-free medium, pretreatment with 50 microM DES abolished the [Ca(2+)](i) increases induced by 2 microM carbonylcyanide m-chlorophenylhydrazone (CCCP; a mitochondrial uncoupler) and 1 microM thapsigargin (an endoplasmic reticulum Ca(2+) pump inhibitor); and pretreatment with CCCP and thapsigargin partly inhibited DES-induced [Ca(2+)](i) signals. Adding 3 mM Ca(2+) increased [Ca(2+)](i) in cells pretreated with 50 microM DES in Ca(2+)-free medium, suggesting that DES may induce capacitative Ca(2+) entry. 17beta-Estradiol (2-20 microM) increased [Ca(2+)](i), but 100 microM diethylstilbestrol dipropionate had no effect. Pretreatment with the phospholipase C inhibitor U73122 (1 microM) to abolish inositol 1,4,5-trisphosphate formation inhibited 30% of DES-induced Ca(2+) release. DES (20 microM) also increased [Ca(2+)](i) in human normal hepatocytes and osteosarcoma cells. Cumulatively, this study shows that DES induced rapid and sustained [Ca(2+)](i) increases by releasing intracellular Ca(2+) and triggering extracellular Ca(2+) entry in renal tubular cells.     

Cytochrome P-450 metabolites of 2-arachidonoylglycerol play a role in Ca2+-induced relaxation of rat mesenteric arteries

The perivascular sensory nerve (PvN) Ca(2+)-sensing receptor (CaR) is implicated in Ca(2+)-induced relaxation of isolated, phenylephrine (PE)-contracted mesenteric arteries, which involves the vascular endogenous cannabinoid system. We determined the effect of inhibition of diacylglycerol (DAG) lipase (DAGL), phospholipase A(2) (PLA(2)), and cytochrome P-450 (CYP) on Ca(2+)-induced relaxation of PE-contracted rat mesenteric arteries. Our findings indicate that Ca(2+)-induced vasorelaxation is not dependent on the endothelium. The DAGL inhibitor RHC 802675 (1 microM) and the CYP and PLA(2) inhibitors quinacrine (5 microM) (EC(50): RHC 802675 2.8 +/- 0.4 mM vs. control 1.4 +/- 0.3 mM; quinacrine 4.8 +/- 0.4 mM vs. control 2.0 +/- 0.3 mM; n = 5) and arachidonyltrifluoromethyl ketone (AACOCF(3), 1 microM) reduced Ca(2+)-induced relaxation of mesenteric arteries. Synthetic 2-arachidonoylglycerol (2-AG) and glycerated epoxyeicosatrienoic acids (GEETs) induced concentration-dependent relaxation of isolated arteries. 2-AG relaxations were blocked by iberiotoxin (IBTX) (EC(50): control 0.96 +/- 0.14 nM, IBTX 1.3 +/- 0.5 microM) and miconazole (48 +/- 3%), and 11,12-GEET responses were blocked by IBTX (EC(50): control 55 +/- 9 nM, IBTX 690 +/- 96 nM) and SR-141716A. The data suggest that activation of the CaR in the PvN network by Ca(2+) leads to synthesis and/or release of metabolites of the CYP epoxygenase pathway and metabolism of DAG to 2-AG and subsequently to GEETs. The findings indicate a role for 2-AG and its metabolites in Ca(2+)-induced relaxation of resistance arteries; therefore this receptor may be a potential target for the development of new vasodilator compounds for antihypertensive therapy.     

Mechanisms of the vasorelaxant effect of 1, 5-dihydroxy-2, 3-dimethoxy-xanthone, an active metabolite of 1-hydroxy-2, 3, 5-trimethoxy-xanthone isolated from a Tibetan herb, Halenia elliptica, on rat coronary artery

1, 5-Dihydroxy-2, 3-dimethoxy-xanthone (HM-5) is one of the naturally-occurring xanthones of a Tibetan medicinal herb Halenia elliptica. Recently, it has been shown that HM-5 is one of the phase I metabolites of 1-hydroxy-2, 3, 5-trimethoxy-xanthone (HM-1), the major active component of H. elliptica with potent vasorelaxant actions. This study investigated the vasorelaxant effect of HM-5 and its mechanism(s). HM-5 (0.35-21.9 microM) produced a concentration-dependent relaxation in rat coronary artery rings pre-contracted with 1 microM 5-hydroxytryptamine (5-HT), with an EC(50) of 4.40+/-1.08 microM. Unlike HM-1, the effect of HM-5 was endothelial-independent such that removal of the endothelium did not affect its vasodilator potency. Nitric oxide synthase (NOS) inhibitor N(omega)-nitro-l-arginine methyl ester (l-NAME, 100 microM), the soluble guanylate cyclase inhibitor 1H-[1,2,4] oxadiazolo [4,3-alpha] quinoxalin-1-one (ODQ, 10 microM) did not affect the vasodilatory effects of HM-5, thus confirming the non-involvement of endothelium related mechanisms. In endothelium-denuded coronary artery rings, the vasorelaxant effect of HM-5 was inhibited by a potassium channel blocker, TEA (10 mM), and 4-aminopyridine (4-AP, a K(v) blocker; 1 mM) but not by other K+ channel blockers such as iberiotoxin (100 nM), barium chloride (100 microM) and glibenclamide (10 microM). The involvement of Ca2+ channel was studied in artery rings pre-incubated with Ca2+-free buffer (intact endothelium or endothelium-denuded) and primed with 1 microM 5-HT or 60 mM KCl prior to the addition of CaCl2 to elicit contraction. In the 5-HT-primed preparations, HM-5 (34.7 microM) significantly inhibited the CaCl(2)-induced vasoconstriction (89.9% inhibition in intact endothelium artery rings; 83.3% inhibition in endothelium-denuded rings). In the KCl-primed preparations, HM-5 (34.7 microM) produced a 34% inhibition in endothelium-denuded rings. The same concentration of HM-5 inhibited (by 62.3%) the contractile response to 10 microM phorbol 12, 13-diacetate (PDA), a protein kinase C activator, in Ca2+-free solutions. Taken together, this study showed that the mechanisms of the vasorelaxant effects of HM-5 were distinctly different from those of its parent drug HM-1. The vasorelaxant effect of HM-5 was mediated through opening of potassium channel (4-AP) and altering intracellular calcium by partial inhibition of Ca2+ influx through L-type voltage-operated Ca2+ channels and intracellular Ca2+ stores.     

Effect of nordihydroguaiaretic acid on intracellular Ca concentrations in C6 glioma cells

The effect of nordihydroguaiaretic acid (NDGA) on Ca(2+) signaling in C6 glioma cells has been investigated. NDGA (5-100 microM) increased [Ca(2+)]i concentration-dependently. The [Ca(2+)]i increase comprised an initial rise and an elevated phase over a time period of 4 min. Removal of extracellular Ca(2+) reduced NDGA-induced [Ca(2+)]i signals by 52+/-2%. After incubation of cells with NDGA in Ca(2+)-free medium for 4 min, addition of 3 mM CaCl2 induced a concentration-dependent increase in [Ca(2+)]i. NDGA (100 microM)-induced [Ca(2+)]i increases in Ca(2+)-containing medium was not changed by pretreatment with 10 microM nifedipine or verapamil. In Ca(2+)-free medium, pretreatment with the endoplasmic reticulum Ca(2+) pump inhibitor thapsigargin (1 microM) abolished 100 microM NDGA-induced [Ca(2+)]i increases. Inhibition of phospholipase C with 2 microM U73122 had little effect on 100 microM NDGA-induced Ca(2+) release. Several other lipoxygenase inhibitors had no effect on basal [Ca(2+)]i. Collectively, the results suggest that NDGA increased [Ca(2+)]i in glioma cells in a lipoxygenase-independent manner, by releasing Ca(2+) from the endoplasmic reticulum in a manner independent of phospholipase C activity and by causing Ca(2+) influx.     

Direct noncompetitive inhibition of 5-HT(3) receptor-mediated responses by forskolin and steroids

5-HT(3) receptors cloned from NCB-20 cells were expressed in Xenopus oocytes, and the effects of forskolin and steroids on the function of the receptors were investigated using the two-electrode voltage-clamp technique. Forskolin, 17-beta-estradiol, and progesterone inhibited the currents activated by 1 microM 5-HT in a reversible and concentration-dependent manner, with IC(50) values of 12, 33, and 89 microM, respectively. The inhibitory effects of forskolin and 17-beta-estradiol were independent of the membrane potential. Forskolin and 17-beta-estradiol significantly reduced the maximal amplitude of the 5-HT concentration-response curve (E(max)) without significantly affecting the EC(50), indicating that these compounds act as noncompetitive inhibitors of the 5-HT(3) receptor. The cAMP analogue, 8-Br-cAMP (0.2 mM), and the protein kinase A activator, Sp-cAMP (0.1 mM), did not affect the amplitude of 5-HT(3) receptor-mediated currents. The membrane-permeable protein kinase A inhibitor Rp-cAMP (0.1 mM) and the estrogen-receptor antagonist tamoxifen (1 microM) did not affect the inhibition of 5-HT-activated current. In addition, 5-HT(3) receptor-mediated currents were inhibited by both 1,9-dideoxy forskolin (30 microM), which does not activate adenylyl cyclase, and wForskolin (30 microM), a charged hydrophilic analogue of forskolin that is membrane impermeable. These results indicate that both forskolin and 17-beta-estradiol inhibit the function of the 5-HT(3) receptor in a noncompetitive manner and that this inhibition is independent of cAMP levels.     

Protective effect of melatonin against homocysteine-induced vasoconstriction of human umbilical artery

Hyperhomocysteinemia is a major and independent risk factor for vascular disease. Oxidative stress is a possible mechanism for homocysteine (Hcy)-induced endothelial dysfunction. Herein, we evaluated the antioxidant property of melatonin (MLT) in relation to the vasoconstrictive effect of Hcy on the human umbilical artery. In an initial experiment in a cell-free system, a micromolar concentration of iron was found to catalyze oxygen-dependent oxidation of Hcy. MLT (10 or 100 microM) did not affect oxygen-dependent oxidation of Hcy. Next, smooth muscle contraction induced by prostaglandin F(2alpha) (10 microM) was measured in arterial strips. Hcy (10 to 500 microM) increased this vascular tension in a concentration-dependent manner (P < 0.0001). Addition of Fe(2+) (10 microM) significantly potentiated the Hcy effect. Removal of endothelium (P < 0.05), pretreatment with a nitric oxide (NO) synthesis inhibitor (l-N(G)-monomethylarginine, 200 microM, P < 0.001), or pretreatment with a hydroxyl radical ((*)OH) scavenger (mannitol, 10 mM, P < 0.001) significantly attenuated contraction potentiated by Hcy plus Fe(2+). At a much lower concentration than mannitol, MLT (1 to 100 microM) significantly reduced the contractile effect of Hcy and Fe(2+) in a concentration-dependent manner. Hcy plus Fe(2+) significantly impaired calcium ionophore A 23187-induced relaxation (P < 0.0001), while MLT restored this relaxation in a concentration-dependent manner. These findings suggest that Hcy potentiates vascular tension in human umbilical artery, possibly by suppressing bioavailable NO. MLT protects against the vasoconstrictive effect of Hcy, most likely by scavenging (*)OH arising from Hcy autooxidation.     

Melatonin counteracts potentiation by homocysteine of KCL-induced vasoconstriction in human umbilical artery: relation to calcium influx

Homocysteinemia is a major and independent risk factor for vascular disease. Oxidative stress is a possible mechanism for homocysteine (HCY)-induced vascular disease. Herein, we evaluated the antioxidant property of melatonin (MLT) in relation to the vasoconstrictive effect of HCY on the human umbilical artery. Helical umbilical arterial strips without endothelium were obtained at elective Cesarean delivery near term. Changes in potassium chloride (KCl)-induced vasoconstriction were measured. Arterial strips were treated with HCY (10 or 100 microM) plus FeSO(4) (10 microM) alone or pretreated with a hydroxyl radical ((*)OH) scavenger, mannitol (20 mM), or MLT (1 or 10 microM). The effect of HCY on the response of arterial strips to external calcium (Ca(2+)) in the presence of KCl (20 mM) was determined. HCY plus FeSO(4) potentiated KCl-induced vasoconstriction in a concentration-dependent manner; pretreatment with mannitol significantly reduced this vasospastic effect. HCY (100 microM) significantly augmented the contractile response to external Ca(2+). MLT (10 microM) significantly suppressed the contractile response to external Ca(2+). These results suggest that HCY potentiates KCl-induced umbilical artery vasoconstriction, in part by increasing Ca(2+) influx in vascular smooth muscle cells via activation of Ca(2+) channels. MLT significantly suppressed the vasoconstrictive effect of HCY, probably by scavenging (*)OH arising from HCY autooxidation.     

Nongenomic inhibition of catecholamine secretion by 17beta-estradiol in PC12 cells

We investigated the effects of 17beta-estradiol, an estrogen, on [(3)H]norepinephrine ([(3)H]NE) secretion in PC12 cells. Pretreatment with 17beta-estradiol reduced 70 mM K(+)-induced [(3)H]NE secretion in a concentration-dependent manner with a half-maximal inhibitory concentration (IC(50)) of 2 +/- 1 microM. The 70 mM K(+)-induced cytosolic free Ca(2+) concentration ([Ca(2+)](i)) rise was also reduced when the cells were treated with 17beta-estradiol (IC(50) = 15 +/- 2 microM). Studies with voltage-sensitive calcium channel (VSCC) antagonists such as nifedipine and omega-conotoxin GVIA revealed that both L- and N-type VSCCs were affected by 17beta-estradiol treatment. The 17beta-estradiol effect was not changed by pretreatment of the cells with actinomycin D and cycloheximide for 5 h. In addition, treatment with pertussis or cholera toxin did not affect the inhibitory effect of 17beta-estradiol. 17beta-Estradiol also inhibited the ATP-induced [(3)H]NE secretion and [Ca(2+)](i) rise. In PC12 cells, the ATP-induced [Ca(2+)](i) rise is known to occur through P2X(2) receptors, the P2Y(2)-mediated phospholipase C (PLC) pathway, and VSCCs. 17beta-Estradiol pretreatment during complete inhibition of the PLC pathway and VSCCs inhibited the ATP-induced [Ca(2+)](i) rise. Our results suggest that 17beta-estradiol inhibits catecholamine secretion by inhibiting L- and N-type Ca(2+) channels and P2X(2) receptors in a nongenomic manner.     

Actin cytoskeletal modulation of pressure-induced depolarization and Ca(2+) influx in cerebral arteries

The objective of this study was to examine the role of the actin cytoskeleton in the development of pressure-induced membrane depolarization and Ca(2+) influx underlying myogenic constriction in cerebral arteries. Elevating intraluminal pressure from 10 to 60 mmHg induced membrane depolarization, increased intracellular cytosolic Ca(2+) concentration ([Ca(2+)](i)) and elicited myogenic constriction in both intact and denuded rat posterior cerebral arteries. Pretreatment with cytochalasin D (5 microM) or latrunculin A (3 microM) abolished constriction but enhanced the [Ca(2+)](i) response; similarly, acute application of cytochalasin D to vessels with tone, or in the presence of 60 mM K(+), elicited relaxation accompanied by an increase in [Ca(2+)](i). The effects of cytochalasin D were inhibited by nifedipine (3 microM), demonstrating that actin cytoskeletal disruption augments Ca(2+) influx through voltage-sensitive L-type Ca(2+) channels. Finally, pressure-induced depolarization was enhanced in the presence of cytochalasin D, further substantiating a role for the actin cytoskeleton in the modulation of ion channel function. Together, these results implicate vascular smooth muscle actin cytoskeletal dynamics in the control of cerebral artery diameter through their influence on membrane potential as well as via a direct effect on L-type Ca(2+) channels.     

Hormone-specific combinations of isoforms of adenylyl cyclase and phosphodiesterase in the rat liver

Since many isoforms of adenylyl cyclase and adenosine 3', 5'-monophosphate (cAMP) phosphodiesterase have been cloned, it is likely that receptors of each hormone have a specific combination of these isoforms. Types I, III and VIII adenylyl cyclases are reported to be stimulated by Ca(2+)-calmodulin, type I phosphodiesterase by Ca(2+)-calmodulin, but types IV and VII (cAMP-specific) phosphodiesterases by Co2+. In the present study, we examined different effects of Ca2+ and Co2+ on hormone-induced cAMP response in the isolated perfused rat liver.The removal of Ca2+ from the perfusion medium (0 mM CaCl(2 ) + 0.5 mM EGTA) did not affect glucagon (0.1 nM)-responsive cAMP but reduced secretin (1 nM)-, vasoactive intestinal polypeptide (VIP, 1-10 nM)- and forskolin (1 microM)-responsive cAMP considerably. The addition of 1 mM CoCl2 reduced glucagon- and secretin-responsive cAMP considerably, forskolin-responsive cAMP partly, did not affect 1 nM VIP-responsive cAMP, but enhanced 10 nM VIP-responsive cAMP. Forskolin- and VIP-responsive cAMP was greater in the combination (0 mM CaCl(2) + 0.5 mM EGTA + 3 mM CoCl2) than in the Ca(2+)-free perfusion alone.These results suggest that secretin, VIP1 and VIP2 receptors are linked to Ca(2+)-calmodulin-sensitive adenylyl cyclase; glucagon receptor to Ca(2+)-calmodulin-insensitive adenylyl cyclase; VIP1 receptor to Ca(2+)-calmodulin-dependent phosphodiesterase; glucagon, secretin and VIP2 receptors to cAMP-specific phosphodiesterase, respectively, in the rat liver.     

Estrogen modulation of endothelium-derived relaxing factors by human endothelial cells

We report the modulatory effects of estrogen on release of endothelium-derived relaxing factors (EDRFs) in a human endothelial cell line, EA.hy926. Using bioassay, we showed that EA.hy926 released EDRF including nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF) measured by relaxation of pre-contracted endothelium-denuded rabbit aortic rings. This EDRF production was significantly higher in cells treated for 24 h with 17-beta-estradiol (10(-6)mol/L) than control cells. Addition of L-NAME to the perfusate of cells caused the relaxation induced by the endothelial cell perfusate to become transient and abolished the enhancement of relaxation due to estrogen treatment. Addition of K(Ca) channel blockers to the perfusate abolished the L-NAME-resistant relaxation of the bioassay ring. Using real-time PCR, we demonstrated that eNOS expression in estrogen-treated cells was significantly higher than controls. These results show that estrogen exerts a potentially important vasculo-protective effect by stimulating NO but not EDHF production.     

Involvements of calcium channel and potassium channel in Danshen and Gegen decoction induced vasodilation in porcine coronary LAD artery

Danshen (Salviae Miltiorrhizae Radix) and Gegen (Puerariae Lobatae Radix) have been widely used in treating cardiovascular diseases for thousands of years in China. The present study was carried out to evaluate the effects of a Danshen and Gegen decoction (DG) on the vascular reactivity of a porcine isolated coronary artery and the underlying mechanisms involved. Porcine coronary rings were precontracted with 15nM U46619. The involvement of endothelium-dependent mechanisms was explored by removing the endothelium; the involvement of potassium channels was investigated by the pretreatment of the artery rings with various blockers, and the involvement of the calcium channels was investigated by incubating the artery rings with Ca(2+)-free buffer and priming them with high [K(+)] prior to adding CaCl(2) to elicit contraction. The involvement of Ca(2+) sensitization was explored by evaluating the Rho-activity expression. The results revealed that DG elicited a concentration-dependent relaxation on a U46619-precontracted coronary artery ring. These relaxation responses were not altered by the pretreatment of inhibitors of endothelium-related dilator synthases, cGMP and cAMP pathway inhibitors, potassium channel (BK(Ca), SK(Ca), K(V) and K(ATP)) blockers and endothelium removal. The K(IR) channel blocker BaCl(2) only slightly attenuated the DG-induced relaxation. However, the Ca(2+)-induced artery contraction was inhibited by DG. Additionally, the expression of the phosphorylated myosin light chain was inhibited by DG whereas the activity of RhoA was not affected. Therefore, DG could be a useful cardioprotective agent for vasodilation in patients who have hypertension.     

A study of mechanisms involved in vasodilatation induced by resveratrol in isolated porcine coronary artery

The present study was designed to investigate the acute relaxing effect of phytoestrogen resveratrol on isolated porcine coronary arteries and to determine the mechanisms underlying its vasodilatation. Rings of porcine coronary arteries were suspended in organ baths containing Krebs-Henseleit solution, and then isometric tension was measured. Resveratrol concentration-dependently relaxed arterial rings precontracted with 30 mM KCl. The IC(50) value of resveratrol was 38.67+/-3.21 microM. Incubation with N(omega)-L-nitro-arginine (L-NNA), endothelium removal or the presence of a potent inhibitor of protein tyrosine phosphatase sodium orthovanadate partly decreased the relaxation induced by resveratrol. However, the relaxation induced by resveratrol was unaffected by the estrogen receptor antagonist tamoxifen, the inhibitor of prostanoid synthesis indomethacin, the antagonist of beta-adrenoceptors propranolol or the protein synthesis inhibitor, cycloheximide. In addition, resveratrol significantly decreased the contractile responses of 5-HT, KCl and CaCl(2), and shifted their cumulative concentration-response curves to the right. These results suggest that the mechanisms of vasorelaxation induced by resveratrol are heterogeneous, two mechanisms participating partially in the relaxation of porcine coronary artery were detected in the study, one being the nitric oxide released from the endothelium, the other causing inhibition of Ca(2+) influx, but estrogen receptors were not involved in resveratrol-induced relaxation.     

Ryanodine-sensitive Ca(2+) release mechanism of rat pancreatic acinar cells is modulated by calmodulin

The effects of calmodulin (CaM) and CaM antagonists on microsomal Ca(2+) release through a ryanodine-sensitive mechanism were investigated in rat pancreatic acinar cells. When caffeine (10 mM) was added after a steady state of ATP-dependent (45)Ca(2+) uptake into the microsomal vesicles, the caffeine-induced (45)Ca(2+) release was significantly increased by pretreatment with ryanodine (10 microM). The presence of W-7 (60 microM), a potent inhibitor of CaM, strongly inhibited the release, while W-5 (60 microM), an inactive CaM antagonist, showed no inhibition. Inhibition of the release by W-7 was observed at all caffeine concentrations (5-30 mM) tested. The presence of exogenously added CaM (10 microg/ml) markedly increased the caffeine (5-10 mM)-induced (45)Ca(2+) release and shifted the dose-response curve of caffeine-induced (45)Ca(2+) release to the left. Cyclic ADP-ribose (cADPR, 2 microM)-induced (45)Ca(2+) release was enhanced by the presence of ryanodine (10 microM). cADPR (2 microM)- or ryanodine (500 microM)-induced (45)Ca(2+) release was also inhibited by W-7 (60 microM), but not by W-5 (60 microM), and was stimulated by CaM (10 microg/ml). These results suggest that the ryanodine-sensitive Ca(2+) release mechanism of rat pancreatic acinar cells is modulated by CaM.     

EDRF-release and Ca+(+)-channel blockade by magnolol, an antiplatelet agent isolated from Chinese herb Magnolia officinalis, in rat thoracic aorta

Magnolol is an antiplatelet agent isolated from Chinese herb Magnolia officinalis. It inhibited norepinephrine (NE, 3 microM)-induced phasic and tonic contractions in rat thoracic aorta. At the plateau of the NE-induced tonic contraction, addition of magnolol caused two phases (fast and slow) of relaxation. These two relaxations were concentration-dependent (10-100 micrograms/ml), and were not inhibited by indomethacin (20 microM). The fast relaxation was completely antagonized by hemoglobin (10 microM) and methylene blue (50 microM), and disappeared in de-endothelialized aorta while the slow relaxation was not affected by the above treatments. Magnolol also inhibited high potassium (60 mM)-induced, calcium-dependent (0.03 to 3 mM) contraction of rat aorta in a concentration-dependent manner. 45Ca(+)+ influx induced by high potassium or NE was markedly inhibited by magnolol. Cyclic GMP, but not PGI2, was increased by magnolol in intact, but not in de-endothelialized aorta. It is concluded that magnolol relaxed vascular smooth muscle by releasing endothelium-derived relaxing factor (EDRF) and by inhibiting calcium influx through voltage-gated calcium channels.