Allicin relaxes isolated mesenteric arteries through activation of PKA-KATP channel in rat

Allicin is a natural effective organosulfur compound isolated from garlic, which possesses many beneficial properties, such as antibacterial, anti-inflammatory, antimicrobial, hypotensive and hypolipidemic. In the present study, we investigated the effects and the underlying mechanisms of allicin on isolated mesenteric arteries (MAs). We examined MAs relaxation induced by allicin on rat-isolated mesenteric artery (MA) rings, the K_ATP channels with patch, and the expression of Kir6.1 and SUR2B with western blotting and NO production with Diaminofluorescein-FM diacetate (DAF-FMDA) in rat mesenteric artery smooth muscle cells (MASMCs). The results showed that allicin elicited the dose-dependent vasorelaxation effect with phenylephrine (PE) precontracted rat MA rings. The vasorelaxation effect was endothelium and NO independent but could be diminished by inhibition of PKA and K_ATP channels in the vascular smooth muscle. Allicin activated K_ATP channels in rat MASMCs, and the activation of K_ATP channels was inhibited by the inhibitors of PKA and K_ATP channels. But allicin had no effect on the expression of K_ATP subtypes Kir6.1 and SUR2B. These observations suggest that allicin exerts vasorelaxation effect through activation of PKA-K_ATP^-signaling pathway.     

PKA-dependent activation of the vascular smooth muscle isoform of KATP channels by vasoactive intestinal polypeptide and its effect on relaxation of the mesenteric resistance artery

Vasoactive intestinal polypeptide (VIP) is a potent vasodilator and has been successfully used to alleviate hypertension. Consistently, disruption of VIP gene in mice leads to hypertension. However, its downstream targets in the vascular regulation are still not well demonstrated. To test the hypothesis that the vascular smooth muscle isoform of K_ATP channels is a downstream target of the VIP signaling, we performed the studies on the Kir6.1/SUR2B channel expressed in HEK293 cells. We found that the channel was strongly activated by VIP. Through endogenous VIP receptors, the channel activation was reversible and dependent on VIP concentrations with the midpoint-activation concentration ∼ 10 nM. The channel activation was voltage-independent and could be blocked by K_ATP channel blocker glibenclamide. In cell-attached patches, VIP augmented the channel open-state probability with modest suppression of the single channel conductance. The VIP-induced Kir6.1/SUR2B channel activation was blocked by PKA inhibitor RP-cAMP. Forskolin, an adenylyl cyclase activator, activated the channel similarly as VIP. The effect of VIP was further evident in the native tissues. In acutely dissociated mesenteric vascular smooth myocytes, VIP activated the K_ATP currents in a similar manner as in HEK293 cells. In endothelium-free mesenteric artery rings, VIP produced concentration-dependent vasorelaxation that was attenuated by glibenclamide. These results therefore indicate that the vascular isoform (Kir6.1/SUR2B) of K_ATP channels is a target of VIP. The channel activation relies on the PKA pathway and produces mesenteric arterial relaxation.     

Steady-State Modulation of Voltage-Gated K+ Channels in Rat Arterial Smooth Muscle by Cyclic AMP-Dependent Protein Kinase and Protein Phosphatase 2B

Voltage-gated potassium channels (K_v) are important regulators of membrane potential in vascular smooth muscle cells, which is integral to controlling intracellular Ca2+ concentration and regulating vascular tone. Previous work indicates that K_v channels can be modulated by receptor-driven alterations of cyclic AMP-dependent protein kinase (PKA) activity. Here, we demonstrate that K_v channel activity is maintained by tonic activity of PKA. Whole-cell recording was used to assess the effect of manipulating PKA signalling on K_v and ATP-dependent K+ channels of rat mesenteric artery smooth muscle cells. Application of PKA inhibitors, KT5720 or H89, caused a significant inhibition of K_v currents. Tonic PKA-mediated activation of K_v appears maximal as application of isoprenaline (a β-adrenoceptor agonist) or dibutyryl-cAMP failed to enhance K_v currents. We also show that this modulation of K_v by PKA can be reversed by protein phosphatase 2B/calcineurin (PP2B). PKA-dependent inhibition of K_v by KT5720 can be abrogated by pre-treatment with the PP2B inhibitor cyclosporin A, or inclusion of a PP2B auto-inhibitory peptide in the pipette solution. Finally, we demonstrate that tonic PKA-mediated modulation of K_v requires intact caveolae. Pre-treatment of the cells with methyl-β-cyclodextrin to deplete cellular cholesterol, or adding caveolin-scaffolding domain peptide to the pipette solution to disrupt caveolae-dependent signalling each attenuated PKA-mediated modulation of the K_v current. These findings highlight a novel, caveolae-dependent, tonic modulatory role of PKA on K_v channels providing new insight into mechanisms and the potential for pharmacological manipulation of vascular tone.     

Pharmacology and modulation of K(ATP) channels by protein kinase C and phosphatases in gallbladder smooth muscle

ATP-sensitiveK⁺ (K_ATP) channels exhibit pharmacologicaldiversity, which is critical for the development of novel therapeutic agents. We have characterized K_ATP channels in gallbladdersmooth muscle to determine how their pharmacological properties compare to K_ATP channels in other types of smooth muscle.K_ATP currents were measured in myocytes isolated fromgallbladder and mesenteric artery. The potencies of pinacidil,diazoxide, and glibenclamide were similar in gallbladder and vascularsmooth muscle, suggesting that the regions of the channel conferringsensitivity to these agents are conserved among smooth muscle types.Activators of protein kinase C (PKC), however, were less effective atinhibiting K_ATP currents in myocytes from gallbladder thanmesenteric artery. The phosphatase inhibitor okadaic acid increased theefficacy of PKC activators and revealed ongoing basal activation ofK_ATP channels by protein kinase A in gallbladder. Theseresults suggest that phosphatases and basal kinase activity play animportant role in controlling K_ATP channel activity.

     

Cerebral vasodilator properties of Danshen and Gegen: A study of their combined efficacy and mechanisms of actions

Danshen and Gegen are two commonly used Chinese herbal medicines for treatment of cardiovascular diseases. The aim of the present study was to elucidate the combination effects of these two herbs on cerebral vascular tone and their underlying mechanisms of actions. Basilar artery rings were obtained from rats and precontracted with U46619. Cumulative administrations of aqueous extracts of Danshen, Gegen, or the two herbs combined (DG; ratio 7:3) produced concentration-dependent relaxation of the artery rings. Statistical analysis on these findings produced a combination index (CI) of 1.041 at ED₅₀, which indicates the two herbs produced additive vasodilator effects when used as a combined decoction. Removal of the endothelium had no effect on the vasodilator properties of Danshen, Gegen, and DG. However, their maximum effects (Imax) were significantly blunted by a K_ATP channel inhibitor glibenclamide, a non-selective K⁺ channel inhibitor tetraethylammonium (TEA), and by a combination of K⁺ channel inhibitors (glibenclamide + TEA + iberiotoxin + 4-aminopyridine + barium chloride). In addition, Danshen, Gegen, and DG produced augmentation of K_ATP currents and inhibited Ca²⁺ influx in vascular smooth muscle cells isolated from rat basilar arteries. Furthermore, these agents inhibited CaCl₂-induced contraction in the artery rings. In conclusion, the present study showed that Danshen and Gegen produced additive vasodilator effects on rat cerebral basilar arteries. These effects were independent of endothelium-derived relaxant factors (EDRF), but required the opening of K_ATP channels and inhibition of Ca²⁺ influx in the vascular smooth muscle cells. It is suspected that the cerebral vasodilator effects of Danshen and Gegen produced either on their own or in combination, can help patients with obstructive cerebrovascular diseases.     

Insulin-like growth factor-I inhibits rat arterial K(ATP) channels through pI 3-kinase

Since, in addition to its growth-promoting actions, insulin-like growth factor-I (IGF-I) has rapid vasoactive actions, we investigated the effects of IGF-I on whole-cell ATP-sensitive K⁺ (K_ATP) currents of rat mesenteric arterial smooth muscle cells. IGF-I (10 or 30 nM) reduced K_ATP currents activated by pinacidil or a membrane permeant cAMP analogue. Inhibition of phospholipase C, protein kinase C, protein kinase A, mitogen-activated protein kinase or mammalian target of rapamycin (mTOR) did not prevent the action of IGF-I. However, inhibition of K_ATP currents by IGF-I was abolished by the tyrosine kinase inhibitor genistein or the phosphoinositide 3-kinase inhibitors, LY 294002 and wortmannin. Intracellular application of either phosphatidylinositol 4,5-bisphosphate (PIP₂) or phosphatidylinositol 3,4,5-trisphosphate (PIP₃) increased the K_ATP current activated by pinacidil and abolished the inhibitory effect of IGF-I. Thus, we show regulation of arterial K_ATP channels by polyphosphoinositides and report for the first time that IGF-I inhibits these channels via a phosphoinositide 3-kinase-dependent pathway.     

Activation of rat mesenteric arterial KATP channels by 11,12-epoxyeicosatrienoic acid

Epoxyeicosatrienoic acids (EETs), the cytochrome P-450 epoxygenase metabolites of arachidonic acid, are candidates of endothelium-derived hyperpolarizing factors. We have previously reported that EETs are potent activators of cardiac ATP-sensitive K(+) (K(ATP)) channels, but their effects on the vascular K(ATP) channels are unknown. With the use of whole cell patch-clamp techniques with 0.1 mM ATP in the pipette and holding at -60 mV, freshly isolated smooth muscle cells from rat mesenteric arteries had small glibenclamide-sensitive currents at baseline (13.1 +/- 3.9 pA, n = 5) that showed a 7.2-fold activation by 10 microM pinacidil (94.1 +/- 21.9 pA, n = 7, P < 0.05). 11,12-EET dose dependently activated the K(ATP) current with an apparent EC(50) of 87 nM. Activation of the K(ATP) channels by 500 nM 11,12-EET was inhibited by inclusion of the PKA inhibitor peptide (5 microM) but not by the inclusion of the PKC inhibitor peptide (100 microM) in the pipette solution. These results were corroborated by vasoreactivity studies. 11,12-EET produced dose-dependent vasorelaxation in isolated small mesenteric arteries, and this effect was reduced by 50% with glibenclamide (1 microM) preincubation. The 11,12-EET effects on vasorelaxation were also significantly attenuated by preincubation with cell-permeant PKA inhibitor myristoylated PKI(14-22), and, in the presence of PKA inhibitor, glibenclamide had no additional effects. These results suggest that 11,12-EET is a potent activator of the vascular K(ATP) channels, and its effects are dependent on PKA activities.     

PKA-dependent activation of the vascular smooth muscle isoform of KATP channels by vasoactive intestinal polypeptide and its effect on relaxation of the mesenteric resistance artery

Vasoactive intestinal polypeptide (VIP) is a potent vasodilator and has been successfully used to alleviate hypertension. Consistently, disruption of VIP gene in mice leads to hypertension. However, its downstream targets in the vascular regulation are still not well demonstrated. To test the hypothesis that the vascular smooth muscle isoform of KATP channels is a downstream target of the VIP signaling, we performed the studies on the Kir6.1/SUR2B channel expressed in HEK293 cells. We found that the channel was strongly activated by VIP. Through endogenous VIP receptors, the channel activation was reversible and dependent on VIP concentrations with the midpoint-activation concentration approximately 10 nM. The channel activation was voltage-independent and could be blocked by KATP channel blocker glibenclamide. In cell-attached patches, VIP augmented the channel open-state probability with modest suppression of the single channel conductance. The VIP-induced Kir6.1/SUR2B channel activation was blocked by PKA inhibitor RP-cAMP. Forskolin, an adenylyl cyclase activator, activated the channel similarly as VIP. The effect of VIP was further evident in the native tissues. In acutely dissociated mesenteric vascular smooth myocytes, VIP activated the KATP currents in a similar manner as in HEK293 cells. In endothelium-free mesenteric artery rings, VIP produced concentration-dependent vasorelaxation that was attenuated by glibenclamide. These results therefore indicate that the vascular isoform (Kir6.1/SUR2B) of KATP channels is a target of VIP. The channel activation relies on the PKA pathway and produces mesenteric arterial relaxation.     

Role of Potassium Channels in the Effects of Hydrogen Sulfide on Contractility of Gastric Smooth Muscle Cells in Rats

The effect of sodium hydrosulfide (NaHS), a hydrogen sulfide (H₂S) donor, on spontaneous contractile activity of rat gastric smooth muscle cells was analyzed. Experiments were conducted on gastric stripes under conditions of isometric contraction. It was shown that NaHS has a biphasic effect on spontaneous contractile activity, increasing tonic tension and the amplitude of phasic contractions within the first minutes since application. This initial phase is followed by a decrease in amplitude, basal tone, and frequency of spontaneous contractions. The inhibitory effect of NaHS was dose-dependent at concentrations from 10 to 600 μM. Preliminary application of tetraethylammonium and 4-aminopirydine, inhibitors of voltage-gated and calciumactivated potassium channels, prevented a NaHS-induced initial increase in basal tone and phasic contraction amplitude. Activation of ATP-dependent potassium channels (KATP-channels) by diazoxide prevented in part a NaHS-induced decrease in basal tone and amplitude of spontaneous contractions. Glibenclamide, an inhibitor of K_ATP-channels, decreased the inhibitory effect of NaHS on amplitude, basal tone and frequency of spontaneous contractions. It was concluded that in rat gastric smooth muscles the excitatory effect of H₂S is mediated by the inhibition of voltagegated and calcium-activated potassium channels, while its inhibitory effect involves the activation of K_ATP-channels.     

cGMP-Dependent Protein Kinase Contributes to Hydrogen Sulfide-Stimulated Vasorelaxation

A growing body of evidence suggests that hydrogen sulfide (H₂S) is a signaling molecule in mammalian cells. In the cardiovascular system, H₂S enhances vasodilation and angiogenesis. H₂S-induced vasodilation is hypothesized to occur through ATP-sensitive potassium channels (K_ATP); however, we recently demonstrated that it also increases cGMP levels in tissues. Herein, we studied the involvement of cGMP-dependent protein kinase-I in H₂S-induced vasorelaxation. The effect of H₂S on vessel tone was studied in phenylephrine-contracted aortic rings with or without endothelium. cGMP levels were determined in cultured cells or isolated vessel by enzyme immunoassay. Pretreatment of aortic rings with sildenafil attenuated NaHS-induced relaxation, confirming previous findings that H₂S is a phosphodiesterase inhibitor. In addition, vascular tissue levels of cGMP in cystathionine gamma lyase knockouts were lower than those in wild-type control mice. Treatment of aortic rings with NaHS, a fast releasing H₂S donor, enhanced phosphorylation of vasodilator-stimulated phosphoprotein in a time-dependent manner, suggesting that cGMP-dependent protein kinase (PKG) is activated after exposure to H₂S. Incubation of aortic rings with a PKG-I inhibitor (DT-2) attenuated NaHS-stimulated relaxation. Interestingly, vasodilatory responses to a slowly releasing H₂S donor (GYY 4137) were unaffected by DT-2, suggesting that this donor dilates mouse aorta through PKG-independent pathways. Dilatory responses to NaHS and L-cysteine (a substrate for H₂S production) were reduced in vessels of PKG-I knockout mice (PKG-I−/−). Moreover, glibenclamide inhibited NaHS-induced vasorelaxation in vessels from wild-type animals, but not PKG-I−/−, suggesting that there is a cross-talk between K_ATP and PKG. Our results confirm the role of cGMP in the vascular responses to NaHS and demonstrate that genetic deletion of PKG-I attenuates NaHS and L-cysteine-stimulated vasodilation.     

Formononetin,an isoflavone,relaxes rat isolated aorta through endothelium-dependent and endothelium-independent pathways

We evaluated the vasorelaxation effects of formononetin, an isoflavone/phytoestrogen found abundantly in Astragalus mongholicus Bunge, on rat isolated aorta and the underlying mechanisms involved. Cumulative administration of formononetin, genistein, daidzein and biochanin A relaxed phenylephrine-preconstricted aorta. Formononetin and biochanin A caused a similar magnitude of relaxation whereas daidzein was least potent. Mechanical removal of endothelium, L-NAME (100 μM) and methylene blue (10 μM) suppressed formononetin-induced relaxation. Formononetin increased endothelial nitric oxide (NO) synthase (eNOS), but not inducible NO synthase, activity with an up-regulation of eNOS mRNA and p-eNOS^Ser1177 protein expression. In endothelium-denuded preparations, formononetin-induced vasorelaxation was significantly reduced by glibenclamide (3 μM) and iberiotoxin (100 nM), and a combination of glibenclamide (3 μM) plus iberiotoxin (100 nM) abolished the relaxation. In contrast, formononetin-elicited endothelium-independent relaxation was not altered by ICI 182,780 (10 μM, an estrogen receptor (ERα/ERβ) antagonist) or mifepristone (10 μM, a progesterone receptor antagonist). In single aortic smooth muscle cells, formononetin caused opening of iberiotoxin-sensitive Ca²⁺-activated K⁺ (BK_Ca) channels and glibenclamide-sensitive adenosine triphosphate (ATP)-dependent K⁺ (K_ATP) channels. Thus, our results suggest that formononetin caused vascular relaxation via endothelium/NO-dependent mechanism and endothelium-independent mechanism which involves the activation of BK_Ca and K_ATP channels.     

ATP-sensitive potassium channels mediate hyperosmotic stimulation of NKCC in slow-twitch muscle

In mildly hyperosmotic medium, activation of the Na⁺-K⁺-2Cl^- cotransporter (NKCC) counteracts skeletal muscle cell water loss, and compounds that stimulate protein kinase A (PKA) activity inhibit the activation of the NKCC. The aim of this study was to determine the mechanism for PKA inhibition of NKCC activity in resting skeletal muscle. Incubation of rat slow-twitch soleus and fast-twitch plantaris muscles in isosmotic medium with the PKA inhibitors H-89 and KT-5720 caused activation of the NKCC only in the soleus muscle. NKCC activation caused by PKA inhibition was insensitive to MEK MAPK inhibitors and to insulin but was abolished by the PKA stimulators isoproterenol and forskolin. Furthermore, pinacidil [an ATP-sensitive potassium (K_ATP) channel opener] or inhibition of glycolysis increased NKCC activity in the soleus muscle but not in the plantaris muscle. Preincubation of the soleus muscle with glibenclamide (a K_ATP channel inhibitor) prevented the NKCC activation by hyperosmolarity, PKA inhibition, pinacidil, and glycolysis inhibitors. In contrast, glibenclamide stimulated NKCC activity in the plantaris muscle. In cells stably transfected with the Kir6.2 subunit of the of K_ATP channel, inhibition of glycolysis activated potassium current and NKCC activity. We conclude that activation of K_ATP channels in slow-twitch muscle is necessary for activation of the NKCC and cell volume restoration in hyperosmotic conditions. protein kinase A; glibenclamide; glycolysis; Na⁺-K⁺-2Cl^- cotransporter; Kir6.2     

Dexmedetomidine directly inhibits vascular ATP-sensitive potassium channels

AimsDexmedetomidine is reported to have an effect on peripheral vasoconstriction; however, the exact mechanisms underlying this process are unclear. In this study, we hypothesized that dexmedetomidine-induced inhibition of vascular ATP-sensitive K⁺ (K_ATP) channels may be associated with this vasoconstriction. To test this hypothesis, we investigated the effects of dexmedetomidine on vascular K_ATP-channel activity at the single-channel level.Main methodsWe used cell-attached and inside-out patch-clamp configurations to examine the effects of dexmedetomidine on the activities of native rat vascular K_ATP channels, recombinant K_ATP channels with different combinations of various inwardly rectifying potassium channels (Kir6.0 family: Kir6.1, 6.2) and sulfonylurea receptor subunits (SUR1, 2A, 2B), and SUR-deficient channels derived from a truncated isoform of Kir6.2 subunit, namely, Kir6.2ΔC36 channels.Key findingsDexmedetomidine was observed to inhibit the native rat vascular K_ATP channels in both cell-attached and inside-out configurations. This drug also inhibited the activity of all types of recombinant SUR/Kir6.0 K_ATP channels as well as Kir6.2ΔC36 channels with equivalent potency.SignificanceThese results indicate that dexmedetomidine directly inhibits K_ATP channels through the Kir6.0 subunit.     

Dual regulation of the ATP-sensitive potassium channel by caffeine

ATP-sensitive potassium (K_ATP) channels couple cellular metabolic status to changes in membrane electrical properties. Caffeine (1,2,7-trimethylxanthine) has been shown to inhibit several ion channels; however, how caffeine regulates K_ATP channels was not well understood. By performing single-channel recordings in the cell-attached configuration, we found that bath application of caffeine significantly enhanced the currents of Kir6.2/SUR1 channels, a neuronal/pancreatic K_ATP channel isoform, expressed in transfected human embryonic kidney (HEK)293 cells in a concentration-dependent manner. Application of nonselective and selective phosphodiesterase (PDE) inhibitors led to significant enhancement of Kir6.2/SUR1 channel currents. Moreover, the stimulatory action of caffeine was significantly attenuated by KT5823, a specific PKG inhibitor, and, to a weaker extent, by BAPTA/AM, a membrane-permeable Ca²⁺ chelator, but not by H-89, a selective PKA inhibitor. Furthermore, the stimulatory effect was completely abrogated when KT5823 and BAPTA/AM were co-applied with caffeine. In contrast, the activity of Kir6.2/SUR1 channels was decreased rather than increased by caffeine in cell-free inside-out patches, while tetrameric Kir6.2LRKR368/369/370/371AAAA channels were suppressed regardless of patch configurations. Caffeine also enhanced the single-channel currents of recombinant Kir6.2/SUR2B channels, a nonvascular smooth muscle K_ATP channel isoform, although the increase was smaller. Moreover, bidirectional effects of caffeine were reproduced on the K_ATP channel present in the Cambridge rat insulinoma G1 (CRI-G1) cell line. Taken together, our data suggest that caffeine exerts dual regulation on the function of K_ATP channels: an inhibitory regulation that acts directly on Kir6.2 or some closely associated regulatory protein(s), and a sulfonylurea receptor (SUR)-dependent stimulatory regulation that requires cGMP-PKG and intracellular Ca²⁺-dependent signaling. phosphodiesterase; protein kinase; calcium; single channel; patch clamp     

Leptin Regulates KATP Channel Trafficking in Pancreatic β-Cells by a Signaling Mechanism Involving AMP-activated Protein Kinase (AMPK) and cAMP-dependent Protein Kinase (PKA)

Pancreatic β-cells secrete insulin in response to metabolic and hormonal signals to maintain glucose homeostasis. Insulin secretion is under the control of ATP-sensitive potassium (K_ATP) channels that play key roles in setting β-cell membrane potential. Leptin, a hormone secreted by adipocytes, inhibits insulin secretion by increasing K_ATP channel conductance in β-cells. We investigated the mechanism by which leptin increases K_ATP channel conductance. We show that leptin causes a transient increase in surface expression of K_ATP channels without affecting channel gating properties. This increase results primarily from increased channel trafficking to the plasma membrane rather than reduced endocytosis of surface channels. The effect of leptin on K_ATP channels is dependent on the protein kinases AMP-activated protein kinase (AMPK) and PKA. Activation of AMPK or PKA mimics and inhibition of AMPK or PKA abrogates the effect of leptin. Leptin activates AMPK directly by increasing AMPK phosphorylation at threonine 172. Activation of PKA leads to increased channel surface expression even in the presence of AMPK inhibitors, suggesting AMPK lies upstream of PKA in the leptin signaling pathway. Leptin signaling also leads to F-actin depolymerization. Stabilization of F-actin pharmacologically occludes, whereas destabilization of F-actin simulates, the effect of leptin on K_ATP channel trafficking, indicating that leptin-induced actin reorganization underlies enhanced channel trafficking to the plasma membrane. Our study uncovers the signaling and cellular mechanism by which leptin regulates K_ATP channel trafficking to modulate β-cell function and insulin secretion.     

Hydrogen sulfide-induced relaxation of resistance mesenteric artery beds of rats

Hydrogen sulfide (H2S) has been shown recently to function as an important gasotransmitter. The present study investigated the vascular effects of H2S, both exogenously applied and endogenously generated, on resistance mesenteric arteries of rats and the underlying mechanisms. Both H2S and NaHS evoked concentration-dependent relaxation of in vitro perfused rat mesenteric artery beds (MAB). The sensitivity of MAB to H2S (EC50, 25.2 +/- 3.6 microM) was about fivefold higher than that of rat aortic tissues. Removal of endothelium or coapplication of charybdotoxin and apamin to endothelium-intact MAB significantly reduced the vasorelaxation effects of H2S. The H2S-induced relaxation of MAB was partially mediated by ATP-sensitive K+ (KATP) channel activity in vascular smooth muscle cells. Pinacidil (EC50, 1.7 +/- 0.1 microM, n=6) mimicked, but glibenclamide (10 microM, n=6) suppressed, the vasorelaxant effect of H2S. KATP channel currents in isolated mesenteric artery smooth muscle cells were significantly augmented by H2S. L-cysteine, a substrate of cystathionine-gamma-lyase (CSE), at 1 mM increased endogenous H2S production by sixfold in rat mesenteric artery tissues and decreased contractility of MAB. DL-propargylglycine (a blocker of CSE) at 10 microM abolished L-cysteine-dependent increase in H2S production and relaxation of MAB. Our results demonstrated a tissue-specific relaxant response of resistance arteries to H2S. The stimulation of KATP channels in vascular smooth muscle cells and charybdotoxin/apamin-sensitive K+ channels in vascular endothelium by H2S represents important cellular mechanisms for H2S effect on MAB. Our study also demonstrated that endogenous CSE can generate sufficient H2S from exogenous L-cysteine to cause vasodilation. Future studies are merited to investigate direct contribution of endogenous H2S to regulation of vascular tone.     

Exercise responsive genes measured in peripheral blood of women with Chronic Fatigue Syndrome and matched control subjects

Background

Electrophysiological data suggest that cardiac K_ATP channels consist of Kir6.2 and SUR2A subunits, but the distribution of these (and other K_ATP channel subunits) is poorly defined. We examined the localization of each of the K_ATP channel subunits in the mouse and rat heart.

Results

Immunohistochemistry of cardiac cryosections demonstrate Kir6.1 protein to be expressed in ventricular myocytes, as well as in the smooth muscle and endothelial cells of coronary resistance vessels. Endothelial capillaries also stained positive for Kir6.1 protein. Kir6.2 protein expression was found predominantly in ventricular myocytes and also in endothelial cells, but not in smooth muscle cells. SUR1 subunits are strongly expressed at the sarcolemmal surface of ventricular myocytes (but not in the coronary vasculature), whereas SUR2 protein was found to be localized predominantly in cardiac myocytes and coronary vessels (mostly in smaller vessels). Immunocytochemistry of isolated ventricular myocytes shows co-localization of Kir6.2 and SUR2 proteins in a striated sarcomeric pattern, suggesting t-tubular expression of these proteins. Both Kir6.1 and SUR1 subunits were found to express strongly at the sarcolemma. The role(s) of these subunits in cardiomyocytes remain to be defined and may require a reassessment of the molecular nature of ventricular K_ATP channels.

Conclusions

Collectively, our data demonstrate unique cellular and subcellular K_ATP channel subunit expression patterns in the heart. These results suggest distinct roles for K_ATP channel subunits in diverse cardiac structures.     

Protein phosphatase 2A and Ca2+-activated K+ channels contribute to 11,12-epoxyeicosatrienoic acid analog mediated mesenteric arterial relaxation

Epoxyeicosatrienoic acids (EETs) are considered to be endothelium-derived hyperpolarizing factors, and are potent activators of the large-conductance, Ca(2+)-activated K(+) (BK(Ca)) channel in vascular smooth muscle. Here, we investigate the signal transduction pathway involved in the activation of BK(Ca) channels by 11,12-EET and 11,12-EET stable analogs in rat mesenteric vascular smooth muscle cells. 11,12-EET and the 11,12-EET analogs, 11-nonyloxy-undec-8(Z)-enoic acid (11,12-ether-EET-8-ZE), 11-(9-hydroxy-nonyloxy)-undec-8(Z)-enoic acid (11,12-ether-EET-8-ZE-OH) and 11,12-trans-oxidoeicosa-8(Z)-enoic acid (11,12-tetra-EET-8-ZE), caused vasorelaxation of mesenteric resistance arteries. Mesenteric myocyte whole-cell (perforated-patch) currents were substantially (approximately 150%) increased by 11,12-EET and 11,12-EET analogs. Single-channel recordings were conducted to identify the target for 11,12-EET. 11,12-EET and 11,12-EET analogs also increased mesenteric myocyte BK(Ca) channel activity in cell-attached patches. Similar results were obtained in cell-free patches. Baseline mesenteric myocyte BK(Ca) channel activity (NPo) in cell-free patches averaged less than 0.001 at +50 mV and 11,12-EET (1 micromol/L) increased NPo to 0.03+/-0.02 and 11,12-EET analogs (1 micromol/L) increased NPo to 0.09+/-0.006. Inhibition of protein phosphatase 2A (PP2A) activity with okadaic acid (10 nmol/L) completely reversed 11,12-EET stimulated BK(Ca) channel activity and greatly attenuated 11,12-ether-EET-8-ZE mesenteric resistance artery vasorelaxation. 11,12-EET and 11,12-EET analogs increased mesenteric myocyte PP2A activity by 3.5-fold. Okadaic acid and the EET inhibitor, 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE) inhibited the 11,12-EET mediated increase in PP2A activity. These findings provide initial evidence that PP2A activity contributes to 11,12-EET and 11,12-EET analog activation of mesenteric resistant artery BK(Ca) channels and vasorelaxation.     

Ovariectomy-Induced Reductions in Endothelial SK3 Channel Activity and Endothelium-Dependent Vasorelaxation in Murine Mesenteric Arteries

Mesenteric artery endothelium expresses both small (SK3)- and intermediate (IK1)-conductance Ca²⁺-activated K⁺ (K_Ca) channels whose activity modulates vascular tone via endothelium-dependent hyperpolarization (EDH). Two other major endothelium-dependent vasodilation pathways utilize nitric oxide (NO) and prostacyclin (PGI₂). To examine how ovariectomy (ovx) affects the basal activity and acetylcholine (ACh)-induced activity of each of these three pathways to vasorelaxation, we used wire myograph and electrophysiological recordings. The results from functional studies using isolated murine mesenteric arteries show that ovx reduces ACh-induced endothelium-dependent vasodilation due to decreased EDH and NO contributions, although the contribution of PGI₂ is upregulated. Both endothelial SK3 and IK1 channels are functionally coupled to TRPV4 (transient receptor potential, vanilloid type 4) channels: the activation of TRPV4 channels activates SK3 and IK1 channels, leading to EDH-mediated vascular relaxation. The decreased EDH-mediated vasorelaxation in ovx vessels is due to reduced SK3 channel contribution to the pathway. Further, whole-cell recordings using dispersed endothelial cells also show reduced SK3 current density in ovx endothelial cells. Consequently, activation of TRPV4 channels induces smaller changes in whole-cell current density. Thus, ovariectomy leads to a reduction in endothelial SK3 channel activity thereby reducing the SK3 contribution to EDH vasorelaxation.