Activation of vascular BK channel by tempol in DOCA-salt hypertensive rats

Large-conductance Ca2+-activated potassium (BK) channels modulate vascular smooth muscle tone. Tempol, a superoxide dismutase (SOD) mimetic, lowers blood pressure and inhibits sympathetic nerve activity in normotensive and hypertensive rats. In the present study, we tested the hypotheses depressor responses caused by tempol are partly mediated by vasodilation. It was found that tempol, but not tiron (a superoxide scavenger), dose-dependently relaxed mesenteric arteries (MA) in anesthetized sham and deoxycorticosterone acetate (DOCA)-salt hypertensive rats. Tempol also reduced perfusion pressure in isolated, norepinephrine (NE) preconstricted MA from sham and DOCA-salt hypertensive rats. Maximal responses in DOCA-salt rats were twice as large as those in sham rats. The vasodilation caused by tempol was blocked by iberiotoxin (IBTX, BK channel antagonist, 0.1 micromol/L) and tetraethylammonium chloride (TEA) (1 mmol/L). Tempol did not relax KCl preconstricted arteries in sham or DOCA-salt rats, and Nomega-nitro-L-arginine methyl ester (L-NAME), apamin, or glibenclamide did not alter tempol-induced vasodilation. IBTX constricted MA and this response was larger in DOCA-salt compared with sham rats. Western blots and immunohistochemical analysis revealed increased expression of BK channel alpha subunit protein in DOCA-salt arteries compared with sham arteries. Whole-cell patch clamp studies revealed that tempol enhanced BK channel currents in HEK-293 cells transiently transfected with mslo, the murine BK channel a subunit. These currents were blocked by IBTX. The data indicate that tempol activates BK channels and this effect contributes to depressor responses caused by tempol. Upregulation of the BK channel alpha subunit contributes to the enhanced depressor response caused by tempol in DOCA-salt hypertension.     

Acute effects of the superoxide dismutase mimetic tempol on split kidney function in two-kidney one-clip hypertensive rats

OBJECTIVE: To investigate the acute effects of the superoxide dismutase mimetic 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (tempol) on split kidney function, and renal haemodynamics, in two-kidney, one-clip (2K1C) hypertensive rats. METHODS: Three weeks after clipping, or the sham procedure, the effects of intravenous tempol (200 micromol/kg per h) were evaluated on thiobutabarbital anaesthetized Sprague-Dawley rats. RESULTS: Mean arterial pressure (MAP; 152 +/- 3 versus 122 +/- 3 mmHg, P < 0.001), plasma renin activity (28.7 +/- 3.0 versus 9.5 +/- 0.6 ng/ml per h, P < 0.001) and urinary 8-iso-prostaglandin F2alpha excretion (124 +/- 4 versus 92 +/- 10 pmol/24 h, P = 0.003) were significantly elevated in 2K1C rats compared with sham. Tempol reduced MAP by 15 +/- 1% compared with baseline (P < 0.001) in 2K1C rats. In clipped kidneys, tempol increased the glomerular filtration rate (GFR; +50 +/- 15% from baseline) and the effective renal plasma flow (ERPF; +37 +/- 13%, from baseline), and reduced renal vascular resistance (RVR; -32 +/- 6% from baseline) compared with saline-treated controls (P < 0.05). In non-clipped kidneys, tempol reduced RVR (-24 +/- 5% from baseline) compared with saline-treated controls (P = 0.001). In sham-operated rats, tempol produced a modest reduction in MAP (-8 +/- 2% from baseline, P = 0.003), but did not significantly affect renal haemodynamics or function. CONCLUSION: Tempol reduced MAP and RVR in both clipped and non-clipped kidneys of 2K1C hypertensive rats. In addition, tempol increased ERPF and GFR in the clipped kidney. These findings suggest important roles for superoxide in the regulation of renal haemodynamics during the early maintenance phase of renovascular hypertension.     

Tempol lowers blood pressure and sympathetic nerve activity but not vascular O2- in DOCA-salt rats

This study tested the hypothesis that depressor responses caused by tempol are not associated with reductions in vascular O2- levels in urethane-anesthetized deoxycorticosterone acetate (DOCA)-salt hypertensive rats. We compared the effects of intravenous (IV) administration of tempol, apocynin, superoxide dismutase-polyethylene glycol (PEG-SOD), and SOD on mean arterial blood pressure (MAP), heart rate (HR), and renal sympathetic nerve activity (RSNA). In DOCA-salt rats, tempol (30 to 300 micromol/kg) dose-dependently decreased RSNA, MAP, and HR. Tempol (300 micromol/kg) decreased MAP from 140+/-5 to 83+/-4 mm Hg (P<0.05). HR decreased from 435+/-15 to 390+/-12 bpm (P<0.05). RSNA was reduced by 54%+/-6% from baseline. However, in the same rats, tempol did not reduce dihydroethidium-induced fluorescent signals in the aorta and vena cava. Apocynin (200 micromol/kg) did not lower MAP (142+/-5 mm Hg versus 140+/-6 mm Hg) or HR (428+/-15 bpm versus 420+/-13 bpm) and apocynin did not potentiate depressor responses caused by tempol. PEG-SOD (10 000 U/kg, bolus or 5000 U/kg bolus followed by a 30-minutes infusion of 500 U/kg/min) or SOD (25 000 U/kg, bolus or 10 000 U/kg bolus followed by a 30-minutes infusion of 1000 U/kg per minute) did not alter MAP or HR. It is concluded that depressor responses and decreases in HR and RSNA caused by acute tempol treatment are caused by direct sympathetic nerve activity inhibition that is not accompanied by SOD-mimetic action in the aorta or vena cava.     

NADPH oxidase-derived superoxide augments endothelin-1-induced venoconstriction in mineralocorticoid hypertension

Deoxycorticosterone acetate (DOCA)-salt hypertension is characterized by low renin/angiotensin but increased arterial superoxide levels. We have recently reported that the arterial endothelin-1 (ET-1) level is increased, resulting in NADPH oxidase activation and superoxide generation. However, the effect of ET-1 on venous superoxide production and its relation to venoconstriction are unknown. The present study tested the hypotheses that ET-1 stimulates venous NADPH oxidase and superoxide via its ET(A) receptors, resulting in enhanced venoconstriction in DOCA-salt hypertensive rats. Treatment with ET-1 (0.01 to 1 nmol/L), but not the selective ET(B) receptor agonist sarafotoxin s6c, of vena cavas of normal rats concentration-dependently increased superoxide levels, an effect that was abolished by the selective ET(A) receptor antagonist ABT-627. Although the ET-1 level was not increased in the vena cava and plasma, both venous NADPH oxidase activity and superoxide levels were significantly higher in DOCA-salt compared with sham rats. Moreover, ET-1 treatment (10(-9) mol/L, 10 minutes) of isolated vena cavas further elevated superoxide levels in DOCA-salt rats only but not sham rats, an effect that was abrogated by the superoxide scavenger tempol. Similarly, ET-1-induced contractions of isolated vena cavas of DOCA-salt but not sham rats were significantly inhibited by tempol. The NADPH oxidase inhibitor apocynin significantly reduced superoxide levels in vena cavas of DOCA-salt rats and in ET-1-treated vena cavas of normal rats. Finally, in vivo ET(A) receptor blockade by ABT-627 significantly lowered venous superoxide levels and blood pressure in DOCA-salt but not sham rats. These results suggest that superoxide contributes to ET-1-induced venoconstriction through an elevated venous NADPH oxidase activity in mineralocorticoid hypertension.     

Calcium exerts a larger regulatory effect on potassium+ channels in small mesenteric artery mycoytes from spontaneously hypertensive rats compared to Wistar-Kyoto rats

Hypertension is associated with a remodeling of arterial smooth muscle K(+) channels with Ca(2+)-gated K(+) channel (BK(Ca)) activity being enhanced and voltage-gated K(+) channel (K(v)) activity depressed. Because both of these channel types are modulated by intracellular Ca(2+), we tested the hypothesis that Ca(2+) had a larger effect on both BK(Ca) and K(v) channels in arterial myocytes from hypertensive animals. Myocytes were enzymatically dispersed from small mesenteric arteries (SMA) of 12-week-old Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). Using whole cell patch clamp methods, BK(Ca) and K(v) current components were determined as iberiotoxin-sensitive and -insensitive currents, respectively. The effects of Ca(2+) on these K(+) current components were determined from measurements made with 0.2 and 2 mmol/L external Ca(2+). Increasing external Ca(2+) from 0.2 to 2 mmol/L Ca(2+) increased BK(Ca) currents recorded using myocytes from both WKY rats and SHR with a larger effect in SHR. Increasing external Ca(2+) decreased K(v) currents recorded using myocytes from both WKY and SHR also with a larger effect in SHR. In other experiments, currents through voltage-gated Ca(2+) channels (Ca(v)) measured at 0.2 mmol/L external Ca(2+) were 12 +/- 2% (n = 12) of those recorded at 2 mmol/L Ca(2+) with no differences in percent effect between WKY and SHR. In isolated SMA segments, isometric force development in response to 140 mmol/L KCl at 0.2 mmol/L external Ca(2+) was about 23 +/- 6% (n = 8) of that measured at 2 mmol/L external Ca(2+). These results suggest that an increase in Ca(2+) influx through Ca(v) or in intracellular Ca(2+) secondary to an increase in external Ca(2+) augments BK(Ca) currents and inhibits K(v) currents in SMA myocytes with a larger effect in SHR compared to WKY. This mechanism may contribute to the functional remodeling of K(+) currents of arterial myocytes in hypertensive animals.     

Distinct stoichiometry of BKCa channel tetramer phosphorylation specifies channel activation and inhibition by cAMP-dependent protein kinase

Large conductance voltage- and calcium-activated potassium (BK(Ca)) channels are important signaling molecules that are regulated by multiple protein kinases and protein phosphatases at multiple sites. The pore-forming alpha-subunits, derived from a single gene that undergoes extensive alternative pre-mRNA splicing, assemble as tetramers. Although consensus phosphorylation sites have been identified within the C-terminal domain of alpha-subunits, it is not known whether phosphorylation of all or single alpha-subunits within the tetramer is required for functional regulation of the channel. Here, we have exploited a strategy to study single-ion channels in which both the alpha-subunit splice-variant composition is defined and the number of consensus phosphorylation sites available within each tetramer is known. We have used this approach to demonstrate that cAMP-dependent protein kinase (PKA) phosphorylation of the conserved C-terminal PKA consensus site (S899) in all four alpha-subunits is required for channel activation. In contrast, inhibition of BK(Ca) channel activity requires phosphorylation of only a single alpha-subunit at a splice insert (STREX)-specific PKA consensus site (S4(STREX)). Thus, distinct modes of BK(Ca) channel regulation by PKA phosphorylation exist: an "all-or-nothing" rule for activation and a "single-subunit" rule for inhibition. This essentially digital regulation has important implications for the combinatorial and conditional regulation of BK(Ca) channels by reversible protein phosphorylation.     

Downregulation of the BK channel beta1 subunit in genetic hypertension

The molecular mechanisms underlying increased arterial tone during hypertension are unclear. In vascular smooth muscle, localized Ca2+ release events through ryanodine-sensitive channels located in the sarcoplasmic reticulum (Ca2+ sparks) activate large-conductance, Ca2+-sensitive K+ (BK) channels. Ca2+ sparks and BK channels provide a negative feedback mechanism that hyperpolarizes smooth muscle and thereby opposes vasoconstriction. In this study, we examined Ca2+ sparks and BK channel function in Wistar-Kyoto (WKY) rats with borderline hypertension and in spontaneously hypertensive rats (SHR), a widely used genetic model of severe hypertension. We found that the amplitude of spontaneous BK currents in WKY and SHR cells were smaller than in normotensive cells even though Ca2+ sparks were of similar magnitude. BK channels in WKY and SHR cells were less sensitive to physiological changes in intracellular Ca2+ than normotensive cells. Our data indicate that decreased expression of the BK channel beta1 subunit underlies the lower Ca2+ sensitivity of BK channels in SHR and WKY myocytes. We conclude that the lower expression of the beta1 subunit during genetic borderline and severe hypertension reduced BK channel activity by decreasing the sensitivity of these channels to physiological changes in Ca2+. These results support the view that changes in the molecular composition of BK channels may be a fundamental event contributing to the development of vascular dysfunction during hypertension.     

Nitric oxide may prevent hypertension early in diabetes by counteracting renal actions of superoxide

The dependence of blood pressure on a balance between superoxide and nitric oxide may be amplified in diabetes. We have shown that the first occurrence of sustained hyperglycemia in type I diabetes causes hypertension when induced in rats that have had nitric oxide synthesis blocked chronically (L-NAME, 10 microg/kg per minute IV). This study used tempol (18 micromol/kg per hour IV) to test the hypothesis that superoxide mediates that hypertensive response. Induction of diabetes in untreated rats had no significant effect on mean arterial pressure (MAP, measured 18 h/d), and glomerular filtration rate (GFR) increased significantly during the 2 weeks of diabetes. Chronic infusion of L-NAME in a separate group of rats increased baseline MAP from approximately 90 mm Hg to a stable level of approximately 120 mm Hg after 6 days of infusion, and induction of diabetes (streptozotocin, 40 mg/kg IV) in those rats caused a rapid, progressive increase in MAP that averaged 156+/-5 mm Hg by day 14 of diabetes that was associated with a decrease in GFR and 4-fold increase in isoprostane excretion. Tempol infusion was begun on day 2 of diabetes in a subgroup of those rats, and the progressive hypertensive response was prevented, with MAP averaging 134+/-10 mm Hg by day 14. In addition, the normal renal hyperfiltration response was restored by tempol and the increase in isoprostane did not occur. Thus, the hypertension and decrease in GFR caused by onset of diabetes in rats without a functioning nitric oxide system was prevented by chronic administration of the superoxide dismutase mimetic tempol.     

Effects of local administrations of tempol and diethyldithio-carbamic on peripheral nerve activity

We have recently shown that systemic administration of a superoxide dismutase mimetic, tempol, resulted in decreases in mean arterial pressure and heart rate along with a reduction in renal sympathetic nerve activity (RSNA). It has also been shown that these parameters are significantly increased by systemic administration of a superoxide dismutase inhibitor, diethyldithio-carbamic (DETC), indicating a potential role of reactive oxygen species in the regulation of RSNA. In this study, we examined the effects of local administrations of 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (tempol) and DETC on RSNA in anesthetized rats. Either tempol or DETC was directly administered onto the renal sympathetic nerves located between the electrode and ganglion. Local application of tempol (10 microL, 0.17 to 1.7 mol/L, n=6) resulted in dose-dependent decreases in integrated RSNA (by -81+/-6% at 1.7 mol/L) without alterations in mean arterial pressure and heart rate. In contrast, DETC (10 microL, 0.17 to 1.7 mol/L, n=6) increased RSNA dose-dependently. The responses of RSNA to tempol and DETC were significantly greater in spontaneously hypertensive rats than in normotensive rats (n=6, respectively). Local application of sodium nitroprusside (1 mmol/L) or N(G)-nitro-L-arginine methyl ester (0.11 mol/L) altered neither basal RSNA nor tempol-induced reductions in RSNA (n=6 and 5, respectively). A voltage-gated potassium channel blocker, 4-aminopyridine (0.1 mol/L), significantly decreased basal RSNA (by -81+/-1%) and completely prevented DETC-induced increases in RSNA (n=5). These results suggest that reactive oxygen species play a role in the regulation of peripheral sympathetic nerve activity, and that at least part of this mechanism is mediated through voltage-gated potassium channels.     

Effects of chronic dietary lithium on phosphatidylinositol pathway in heart and arteries of DOCA-salt hypertensive rats

The sensitivity of the phosphatidylinositol (PI) pathway was evaluated in slices of atria (A), ventricles (V), and mesenteric artery (MA) in normotensive (NT) and DOCA-salt hypertensive (DOCA-HT) rats. During norepinephrine (NE) activation, the PI reactivity was two to three times greater in A, V, and MA of HT rats compared to NT rats. The long-term (2 weeks) administration of dietary lithium (Li) reduced the activation of PI by NE in left A and right V but caused no changes in MA of HT rats. The Li-treated hypertensive rats were also characterized by a lower systolic blood pressure and a lower ratio of ventricular weight/body weight. Plasma epinephrine (E) levels that were higher in HT rats were normalized in DOCA-HT + Li-treated rats, while the NE levels remained elevated in the DOCA-HT + Li group.     

Phosphoinositide 3-kinase mediates enhanced spontaneous and agonist-induced contraction in aorta of deoxycorticosterone acetate-salt hypertensive rats

Arteries from deoxycorticosterone acetate (DOCA)-salt and N(omega)-nitro-L-arginine (L-NNA) hypertensive but not normotensive rats develop spontaneous tone. LY294002 and wortmannin, phosphoinositide 3-kinase (PI3-kinase) inhibitors, eliminate spontaneous tone. We hypothesized that PI3-kinase protein and/or activity was increased in hypertension and contributed to the observed enhanced contractility. PI3-kinase activity assays revealed 2-fold higher activity in thoracic aorta from DOCA-salt [systolic blood pressure (SBP)=184+/-5 mm Hg] compared with sham rats (SBP=111+/-2 mm Hg). Western analyses of aortic homogenates revealed the presence of p85alpha, p110alpha, p110beta, and p110delta but not p110gamma PI3-kinase subunits; p110delta protein was elevated in aorta of hypertensive rats as compared with sham. Aortic homogenates from L-NNA rats also had elevated p110beta protein density, but neither L-NNA nor DOCA-salt had differences in p85alpha and p110alpha. Total Akt density was unaltered, but pAkt was significantly lower in homogenates from DOCA-salt rats. LY294002 (20 micromol/L) and nifedipine (50 nmol/L) abolished Ca2+-induced spontaneous tone in aorta from DOCA-salt rats. However, LY294002 did not alter BayK8644-induced contraction, indicating that LY294002 does not inhibit L-type Ca2+ channels directly. PTEN (phosphatase and tensin homolog) and pPTEN were expressed but not different in aorta from DOCA-salt and sham rats. LY294002 corrected the enhanced contraction to KCl and norepinephrine in aorta from DOCA-salt rats. These data support an increase in PI3-kinase activity and p110delta density in aorta from L-NNA and DOCA-salt rats. Importantly, this increase contributes to the enhanced contractility observed in two models of hypertension.     

Essential role for smooth muscle BK channels in alcohol-induced cerebrovascular constriction

Binge drinking is associated with increased risk for cerebrovascular spasm and stroke. Acute exposure to ethanol at concentrations obtained during binge drinking constricts cerebral arteries in several species, including humans, but the mechanisms underlying this action are largely unknown. In a rodent model, we used fluorescence microscopy, patch-clamp electrophysiology, and pharmacological studies in intact cerebral arteries to pinpoint the molecular effectors of ethanol cerebrovascular constriction. Clinically relevant concentrations of ethanol elevated wall intracellular Ca(2+) concentration and caused a reversible constriction of cerebral arteries (EC(50) = 27 mM; E(max) = 100 mM) that depended on voltage-gated Ca(2+) entry into myocytes. However, ethanol did not directly increase voltage-dependent Ca(2+) currents in isolated myocytes. Constriction occurred because of an ethanol reduction in the frequency (-53%) and amplitude (-32%) of transient Ca(2+)-activated K(+) (BK) currents. Ethanol inhibition of BK transients was caused by a reduction in Ca(2+) spark frequency (-49%), a subsarcolemmal Ca(2+) signal that evokes the BK transients, and a direct inhibition of BK channel steady-state activity (-44%). In contrast, ethanol failed to modify Ca(2+) waves, a major vasoconstrictor mechanism. Selective block of BK channels largely prevented ethanol constriction in pressurized arteries. This study pinpoints the Ca(2+) spark/BK channel negative-feedback mechanism as the primary effector of ethanol vasoconstriction.     

Human chorionic gonadotrophin relaxation of human pregnant myometrium and activation of the BKCa channel

The uterorelaxant effect of human chorionic gonadotropin (hCG) is regarded as an important mediator in maintenance of uterine quiescence during pregnancy with clinical potential for tocolysis, the mechanisms of which are unknown. The large conductance calcium-activated K(+) channel (BK(Ca)) is ubiquitously encountered in human uterine tissue and plays a significant role in modulating myometrial cell membrane potential and excitability. The objective of this study was to investigate the involvement of BK(Ca) channel function in the response of human myometrial cells to hCG. Single electrophysiological BK(Ca) channel recordings from freshly dispersed myocytes were obtained in the presence and absence of increasing hCG concentrations. Isometric tension studies, investigating the effects of hCG on isolated myometrial contractions, in the presence and absence of the BK(Ca) channel blocker, iberiotoxin, were performed. The hCG significantly increased the open-state probability of these channels in a concentration-dependent manner [control 0.036 +/- 0.01; 1 IU/ml hCG 0.065 +/- 0.014 (P = 0.262); 10 IU/ml hCG 0.111 +/- 0.009 (P = 0.001); and 100 IU/ml hCG 0.098 +/- 0.004 (P = 0.007)]. In vitro functional studies demonstrated that hCG exerted a significant concentration-dependent relaxant effect on human myometrial tissue. This effect was significantly attenuated by preincubation with iberiotoxin (P < 0.05). These findings outline that activation of BK(Ca) channel activity may explain the potent uterorelaxant effect of hCG.     

Defining the BK channel domains required for beta1-subunit modulation

In a wide variety of cell types, including neurons and smooth muscle cells, activation of the large-conductance voltage- and Ca(2+)-activated K(+) (BK) channels causes transient membrane hyperpolarization, thereby regulating cellular excitability. Similar to other voltage-gated ion channels, BK channels, a tetramer of alpha-subunits, associate with auxiliary beta-subunits in a tissue-specific manner, modifying the channel's gating properties. The BK beta1-subunit, which is expressed in smooth muscle, increases the apparent Ca(2+) sensitivity (marked by a hyperpolarizing shift in the conductance-voltage relationship at a given Ca(2+) concentration), slows macroscopic activation and deactivation, and is required for channel activation by 17beta-estradiol. The beta1-subunit is essential for normal regulation of vascular smooth muscle contractility and blood pressure. Little is known, however, about the molecular mechanisms of beta1-subunit modulation of alpha-subunits. Here we show that the beta1-subunit's modulation of the Ca(2+) and 17beta-estradiol sensitivities can be dissociated from its effects on gating kinetics by truncation of the alpha-subunit's extracellular N-terminal residues. The BK alpha-subunit N terminus interacts uniquely with the beta1-subunit: beta2 regulation of the alpha-subunit is unaltered by truncation of the N terminus. Although the functional interaction of alpha and beta1 requires the N-terminal tail of alpha, the physical association requires the S1, S2, and S3 transmembrane helices of alpha.     

Intracellular free calcium concentration, Ca++ channel and calmodulin level in experimental hypertension in rats

Using fluorescent calcium indicator quin2, we studied intracellular free calcium concentration in platelets that have a number of features similar to vascular smooth muscle cells. Intracellular free calcium concentration in platelets of male SHR was significantly higher at 4, 11 and 28 weeks old compared with age-matched male WKY. However, no significant difference was observed in platelets cytosolic free calcium level of DOCA-salt hypertensive and two-kidney, one clip hypertensive rats in the chronic stage. Cardiac Ca++ channels were estimated by means of radioligand binding method with [3H]-nimodipine. No significant changes were observed in the concentration and affinity of cardiac Ca++ channel in SHR, DOCA-salt hypertensive and two-kidney, one clip hypertensive rats. Calmodulin levels in mesenteric arteries of SHR were significantly decreased in comparison with those of WKY. However no significant differences were observed in DOCA-salt hypertensive rats in the chronic stage. These results indicate that the increase in intracellular free calcium concentration of SHR is not the secondary change caused by high blood pressure. It is impossible to detect the ratio of the three states (open, resting and inactivated) of Ca++ channel. Therefore, there remains a possibility of the changes in the ratio of the states of Ca++ channel. The observed abnormalities of Ca++ regulation may contribute to the pathogenesis of hypertension.     

Cilostazol, an inhibitor of type 3 phosphodiesterase, stimulates large-conductance, calcium-activated potassium channels in pituitary GH3 cells and pheochromocytoma PC12 cells

The effects of cilostazol, a dual inhibitor of type 3 phosphodiesterase and adenosine uptake, on ion currents were investigated in pituitary GH(3) cells and pheochromocytoma PC12 cells. In whole-cell configuration, cilostazol (10 microm) reversibly increased the amplitude of Ca(2+)-activated K(+) current [I(K(Ca))]. Cilostazol-induced increase in I(K(Ca)) was suppressed by paxilline (1 microM) but not glibenclamide (10 microm), dequalinium dichloride (10 microM), or beta-bungarotoxin (200 nM). Pretreatment of adenosine deaminase (1 U/ml) or alpha,beta-methylene-ADP (100 microM) for 5 h did not alter the magnitude of cilostazol-stimulated I(K(Ca)). Cilostazol (30 microM) slightly suppressed voltage-dependent l-type Ca(2+) current. In inside-out configuration, bath application of cilostazol (10 microM) into intracellular surface caused no change in single-channel conductance; however, it did increase the activity of large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels. Cilostazol enhanced the channel activity in a concentration-dependent manner with an EC(50) value of 3.5 microM. Cilostazol (10 microM) shifted the activation curve of BK(Ca) channels to less positive membrane potentials. Changes in the kinetic behavior of BK(Ca) channels caused by cilostazol were related to an increase in mean open time and a decrease in mean closed time. Under current-clamp configuration, cilostazol decreased the firing frequency of action potentials. In pheochromocytoma PC12 cells, cilostazol (10 microM) also increased BK(Ca) channel activity. Cilostazol-mediated stimulation of I(K(Ca)) appeared to be not linked to its inhibition of adenosine uptake or phosphodiesterase. The channel-stimulating properties of cilostazol may, at least in part, contribute to the underlying mechanisms by which it affects neuroendocrine function.     

Tempol prevents altered K(+) channel regulation of afferent arteriolar tone in diabetic rat kidney

Experiments were performed to test the hypothesis that oxidative stress underlies the enhanced tonic dilator impact of inward-rectifier K(+) channels on renal afferent arterioles of rats with streptozotocin-induced diabetes mellitus. Sham and diabetic rats were left untreated or provided Tempol in their drinking water for 26±1 days, after which afferent arteriolar lumen diameter and its responsiveness to K(+) channel blockade were measured using the in vitro blood-perfused juxtamedullary nephron technique. Afferent diameter averaged 19.4±0.8 μm in sham rats and 24.4±0.8 μm in diabetic rats (P<0.05). The decrease in diameter evoked by Ba(2+) (inward-rectifier K(+) channel blocker) was 3 times greater in diabetic rats than in sham rats. Glibenclamide (K(ATP) channel blocker) and tertiapin-Q (Kir1.1/Kir3.x channel blocker) decreased afferent diameter in diabetic rats but had no effect on arterioles from sham rats. Chronic Tempol treatment prevented diabetes mellitus-induced increases in both renal vascular dihydroethidium staining and baseline afferent arteriolar diameter. Moreover, Tempol prevented the exaggeration of afferent arteriolar responses to Ba(2+), tertiapin-Q, and glibenclamide otherwise evident in diabetic rats. Preglomerular microvascular smooth muscle cells expressed mRNA encoding Kir1.1, Kir2.1, and Kir6.1. Neither diabetes mellitus nor Tempol altered Kir1.1, Kir2.1, Kir6.1, or SUR2B protein levels in renal cortical microvessels. To the extent that the effects of Tempol reflect its antioxidant actions, our observations indicate that oxidative stress contributes to the exaggerated impact of Kir1.1, Kir2.1, and K(ATP) channels on afferent arteriolar tone during diabetes mellitus and that this phenomenon involves posttranslational modulation of channel function.     

cAMP-dependent vasodilators cross-activate the cGMP-dependent protein kinase to stimulate BK(Ca) channel activity in coronary artery smooth muscle cells

cAMP-dependent vasodilators are used to treat a variety of cardiovascular disorders; however, the signal transduction pathways and effector mechanisms stimulated by these agents are not fully understood. In the present study we demonstrate that cAMP-stimulating agents enhance the activity of the large-conductance, calcium-activated potassium (BK(Ca)) channel in single myocytes from coronary arteries by "cross-activation" of the cGMP-dependent protein kinase (protein kinase G, PKG). Single-channel patch-clamp data revealed that 10 micromol/L isoproterenol, forskolin, or dopamine opens BK(Ca) channels in coronary myocytes and that this effect is attenuated by inhibitors of PKG (KT5823; Rp-8-pCPT-cGMPS), but not by inhibiting the cAMP-dependent protein kinase (protein kinase A, PKA). In addition, a membrane-permeable analog, CPT-cAMP, also opened BK(Ca) channels in these myocytes, and this effect was reversed by KT5823. Direct biochemical measurement confirmed that dopamine or forskolin stimulates PKG activity in coronary arteries but does not elevate cGMP. Finally, the stimulatory effect of cAMP on BK(Ca) channels was reconstituted in a cell-free, inside-out patch by addition of purified PKG activated by either cGMP or cAMP. In contrast, channel gating was unaffected by exposure to the purified catalytic subunit of PKA. In summary, findings from on-cell and cell-free patch-clamp experiments provide direct evidence that cAMP-dependent vasodilators open BK(Ca) channels in coronary myocytes by cross-activation of PKG (but not via PKA). Biochemical assay confirmed this cross-activation mechanism of cAMP action in these arteries. This signaling pathway is a novel mechanism for regulation of potassium channel activity in vascular smooth muscle and other cells.     

Voltage gated K+ channel expression in arteries of Wistar-Kyoto and spontaneously hypertensive rats

BACKGROUND: We have previously demonstrated differences in the gene expression of voltage-gated K v1.X channel alpha-subunits in arteries from Wistar-Kyoto rats (WKYs) and spontaneously hypertensive rats (SHRs). The purpose of this study was to test the hypothesis that these differences are also present at the protein level. METHODS: Proteins were isolated from the aorta, mesenteric (MAs) and tail arteries (TAs) of 12- to 15-week-old male WKY and SHR, and analyzed by immunoblotting. K(v) currents were recorded from MA myocytes by patch clamp methods. RESULTS: Expression of Kv1.2, Kv1.5, and Kv2.1 was higher in MAs but was not different in aortas of SHRs as compared to WKYs. In the TA, expression of Kv1.2 and Kv1.5 was higher while that of Kv2.1 was lower in SHR compared to WKY. In the MA, the larger expression of an 80 kDa species of Kv1.2 in SHRs was associated with a lower expression of a 60 kDa species. Kv2.1 gene expression was larger in MAs from SHRs but not different in TAs. K(v) currents associated with Kv1.X and Kv2.1 channels were both larger in MA myocytes from SHRs but less than expected based upon differences in K(v) alpha-subunit protein expression. CONCLUSIONS: For the MA, K(v) protein expression and current components between WKYs and SHRs were qualitatively consistent, but differences in gene and protein expression were not closely correlated. The higher expression of K(v) subunits in small mesenteric arteries (SMAs) of SHR would tend to maintain normal myogenic activity and vasoconstrictor reserve, and could be viewed as a form of homeostatic remodeling.