Ca2+-activated K+ channels in murine endothelial cells: block by intracellular calcium and magnesium

The intermediate (IK(Ca)) and small (SK(Ca)) conductance Ca(2+)-sensitive K(+) channels in endothelial cells (ECs) modulate vascular diameter through regulation of EC membrane potential. However, contribution of IK(Ca) and SK(Ca) channels to membrane current and potential in native endothelial cells remains unclear. In freshly isolated endothelial cells from mouse aorta dialyzed with 3 microM free [Ca(2+)](i) and 1 mM free [Mg(2+)](i), membrane currents reversed at the potassium equilibrium potential and exhibited an inward rectification at positive membrane potentials. Blockers of large-conductance, Ca(2+)-sensitive potassium (BK(Ca)) and strong inward rectifier potassium (K(ir)) channels did not affect the membrane current. However, blockers of IK(Ca) channels, charybdotoxin (ChTX), and of SK(Ca) channels, apamin (Ap), significantly reduced the whole-cell current. Although IK(Ca) and SK(Ca) channels are intrinsically voltage independent, ChTX- and Ap-sensitive currents decreased steeply with membrane potential depolarization. Removal of intracellular Mg(2+) significantly increased these currents. Moreover, concomitant reduction of the [Ca(2+)](i) to 1 microM caused an additional increase in ChTX- and Ap-sensitive currents so that the currents exhibited theoretical outward rectification. Block of IK(Ca) and SK(Ca) channels caused a significant endothelial membrane potential depolarization (approximately 11 mV) and decrease in [Ca(2+)](i) in mesenteric arteries in the absence of an agonist. These results indicate that [Ca(2+)](i) can both activate and block IK(Ca) and SK(Ca) channels in endothelial cells, and that these channels regulate the resting membrane potential and intracellular calcium in native endothelium.     

Endothelial cell Ca2+ increases are independent of membrane potential in pressurized rat mesenteric arteries

In rat mesenteric arteries, the ability of ACh to evoke hyperpolarization of smooth muscle cells and consummate dilatation relies on an increase in endothelial cell cytosolic free [Ca2+] and activation of Ca2+-activated K+ channels (KCa). The time course of average and spatially organized rises in endothelial cell [Ca2+]i and concomitant effects on membrane potential were investigated in individual cells of pressurized arteries and isolated sheets of native cells stimulated with ACh. In both cases, ACh stimulated a sustained and oscillating rise in endothelial cell [Ca2+]i. Overall, the oscillations remained asynchronous between cells, yet occasionally localized intercellular coordination became evident. In pressurized arteries, repetitive waves of Ca2+ moved longitudinally across endothelial cells, and depended on Ca2+-store refilling. The rise in endothelial cell Ca2+ was associated with sustained hyperpolarization of endothelial cells in both preparations. This hyperpolarization was also evident when recording from smooth muscle cells in pressurized arteries, and from resting membrane potential, selective inhibition of small-conductance K Ca (SK Ca) with apamin (50 nM) was sufficient to inhibit this response. In the presence of phenylephrine-tone, both apamin and the selective inhibitor of intermediate conductance K Ca (IK Ca) TRAM-34 (1 microM) were required to inhibit the non-nitric oxide-mediated dilatation to ACh. When hyperpolarization of endothelial cells was fully prevented either with inhibitors of K Ca or in KCl (35 mM)-depolarized cells, both the time course and frequency of oscillations in endothelial cell [Ca2+]i to ACh were unaffected. Together, these data show that although a rise in endothelial cell [Ca2+]i stimulates hyperpolarization, depletion of intracellular stores with ACh stimulates Ca2+-influx which is not significantly influenced by the increase in cellular electrochemical gradient for Ca2+ caused by that hyperpolarization.     

Role of Ca2+-activated K+ channels in duodenal mucosal ion transport and bicarbonate secretion

Stimulation of muscarinic receptors in the duodenal mucosa raises cytosolic free Ca(2+) concentration ([Ca(2+)](cyt)), thereby regulating duodenal epithelial ion transport. However, little is known about the downstream molecular targets that account for this Ca(2+)-mediated biological action. Ca(2+)-activated K(+) (K(Ca)) channels are candidates, but the expression and function of duodenal K(Ca) channels are poorly understood. Therefore, we determined whether K(Ca) channels are expressed in the duodenal mucosa and investigated their involvement in Ca(2+)-mediated duodenal epithelial ion transport. Two selective blockers of intermediate-conductance Ca(2+)-activated K(+) (IK(Ca)) channels, clotrimazole (30 muM) and 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34; 10 muM), significantly inhibited carbachol (CCh)-induced duodenal short-circuit current (I(sc)) and duodenal mucosal bicarbonate secretion (DMBS) in mice but did not affect responses to forskolin and heat-stable enterotoxin of Escherichia coli. Tetraethylammonium, 4-aminopyridine, and BaCl(2) failed to inhibit CCh-induced I(sc) and DMBS. A-23187 (10 muM), a Ca(2+) ionophore, and 1-ethyl-2-benzimidazolinone (1-EBIO; 1 mM), a selective opener of K(Ca) channels, increased both I(sc) and DMBS. The effect of 1-EBIO was more pronounced with serosal than mucosal addition. Again, both clotrimazole and TRAM-34 significantly reduced A23187- or 1-EBIO-induced I(sc) and DMBS. Moreover, clotrimazole (20 mg/kg ip) significantly attenuated acid-stimulated DMBS of mice in vivo. Finally, the molecular identity of IK(Ca) channels was verified as KCNN4 (SK4) in freshly isolated murine duodenal mucosae by RT-PCR and Western blotting. Together, our results suggest that the IK(Ca) channel is one of the downstream molecular targets for [Ca(2+)](cyt) to mediate duodenal epithelial ion transport.     

Reduced expression of SKCa and IKCa channel proteins in rat small mesenteric arteries during angiotensin II-induced hypertension

Ca(2+)-activated K(+) channels (K(Ca)), in particular, the small and intermediate K(Ca) (SK(Ca) and IK(Ca), respectively) channels, are key players in endothelium-derived hyperpolarizing factor (EDHF)-mediated relaxation in small arteries. Hypertension is characterized by an endothelial dysfunction, possibly via reduced EDHF release and/or function. We hypothesize that during angiotensin II (14 days)-induced hypertension (ANG II-14d), the contribution of SK(Ca) and IK(Ca) channels in ACh-induced relaxations is reduced due to decreased expression of SK(Ca) and IK(Ca) channel proteins in rat small mesenteric arteries (MAs). Nitric oxide- and prostacyclin-independent vasorelaxation to ACh was similar in small MAs of sham-operated and ANG II-14d rats. Catalase had no inhibitory effects on these relaxations. The highly selective SK(Ca) channel blocker UCL-1684 almost completely blocked these responses in MAs of sham-operated rats but partially in MAs of ANG II-14d rats. These changes were pressure dependent since UCL-1684 caused a greater inhibition in MAs of 1-day ANG II-treated normotensive rats compared with ANG II-14d rats. Expression levels of both mRNA and protein SK3 were significantly reduced in MAs of ANG II-14d rats. The IK(Ca) channel blocker 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34) resulted in comparable reductions in the relaxation responses to ACh in MAs of sham-operated and ANG II-14d rats. Relative mRNA expression levels of IK1 were significantly reduced in MAs of ANG II-14d rats, whereas protein levels of IK1 were not but tended to be lower in MAs of ANG II-14d rats. The findings demonstrate that EDHF-like responses are not compromised in a situation of reduced functional activity and expression of SK3 channels in small MAs of ANG II-induced hypertensive rats. The role of IK1 channels is less clear but might compensate for reduced SK3 activity.     

Role of 5,6-epoxyeicosatrienoic acid in the regulation of newborn piglet pulmonary vascular tone

We examined the responses of newborn piglet pulmonary resistance arteries (PRAs) to 5,6-epoxyeicosatrienoic acid (5,6-EET), a cytochrome P-450 metabolite of arachidonic acid. In PRAs preconstricted with a thromboxane A(2) mimetic, 5,6-EET caused a concentration-dependent dilation. This dilation was partially inhibited by the combination of charybdotoxin (CTX) and apamin, inhibitors of large and small conductance calcium-dependent potassium (K(Ca)) channels, and was abolished by depolarization of vascular smooth muscle with KCl. Disruption of the endothelium significantly attenuated the dilation, suggesting involvement of one or more endothelium-derived vasodilator pathways in this response. The dilation was partially inhibited by nitro-L-arginine (L-NA), an inhibitor of nitric oxide synthase (NOS), but was unaffected by indomethacin, a cyclooxygenase (COX) inhibitor. The combined inhibition of NOS and K(Ca) channels with L-NA, CTX, and apamin abolished 5,6-EET-mediated dilation. Similarly, combined inhibition of NOS and COX abolished the response. We conclude that 5,6-EET is a potent vasodilator in newborn piglet PRAs. This dilation is mediated by redundant pathways that include release of nitric oxide (NO) and COX metabolites and activation of K(Ca) channels. The endothelium dependence of this response suggests that 5,6-EET is not itself an endothelium-derived hyperpolarizing factor (EDHF) but may induce the release of one or more endothelium-derived relaxing factors, such as NO and/or EDHF.     

Effect of hypercholesterolemia on Ca(2+)-dependent K(+) channel-mediated vasodilatation in vivo

Nitric oxide (NO)-mediated and NO-independent mechanisms of endothelium-dependent vasodilatation involve Ca(2+)-dependent K(+) (K(Ca)) channels. We examined the role in vivo of K(Ca) channels in NO-independent vasodilatation in hypercholesterolemia. Hindlimb vascular conductance was measured at rest and after aortic injection of ACh, bradykinin (BK), and sodium nitroprusside in anesthetized control and cholesterol-fed rabbits. Conductances were measured before and after treatment with the NO synthase antagonist N(omega)-nitro-L-arginine methyl ester (L-NAME, 10 mg/kg) or K(Ca) blockers tetraethylammonium (30 mg/kg), charybdotoxin (10 microgram/kg), and apamin (50 microgram/kg). The contribution of NO to basal conductance was greater in control than in cholesterol-fed rabbits [2.2 +/- 0.4 vs. 1.1 +/- 0.3 (SE) ml. min(-1). kg(-1). 100 mmHg(-1), P < 0.05], but the NO-independent K(Ca) channel-mediated component was greater in the cholesterol-fed than in the control group (1.1 + 0.4 vs. 0.3 +/- 0.1 ml. min(-1). kg(-1). 100 mmHg(-1), P < 0.05). Maximum conductance response to ACh and BK was less in cholesterol-fed than in control rabbits, and the difference persisted after L-NAME (ACh: 7.7 +/- 0.7 vs. 10.1 +/- 0.5 ml. min(-1). kg(-1). 100 mmHg(-1), P < 0.005). Blockade of K(Ca) channels with tetraethylammonium or charybdotoxin + apamin almost completely abolished L-NAME-resistant vasodilatation after ACh or BK. The magnitude of K(Ca)-mediated vasodilatation after ACh or BK was impaired in hypercholesterolemic rabbits. Vasodilator responses to nitroprusside did not differ between groups. In vivo, hypercholesterolemia is associated with an altered balance between NO-mediated and NO-independent K(Ca) channel contributions to resting vasomotor tone and impairment of both mechanisms of endothelium-dependent vasodilatation.     

Vasoactivity of the gasotransmitters hydrogen sulfide and carbon monoxide in the chicken ductus arteriosus

Besides nitric oxide (NO) and carbon monoxide (CO), hydrogen sulfide (H(2)S) is a third gaseous messenger that may play a role in controlling vascular tone and has been proposed to serve as an O(2) sensor. However, whether H(2)S is vasoactive in the ductus arteriosus (DA) has not yet been studied. We investigated, using wire myography, the mechanical responses induced by Na(2)S (1 μM-1 mM), which forms H(2)S and HS(-) in solution, and by authentic CO (0.1 μM-0.1 mM) in DA rings from 19-day chicken embryos. Na(2)S elicited a 100% relaxation (pD(2) 4.02) of 21% O(2)-contracted and a 50.3% relaxation of 62.5 mM KCl-contracted DA rings. Na(2)S-induced relaxation was not affected by presence of the NO synthase inhibitor l-NAME, the soluble guanylate cyclase (sGC) inhibitor ODQ, or the K(+) channel inhibitors tetraethylammonium (TEA; nonselective), 4-aminopyridine (4-AP, K(V)), glibenclamide (K(ATP)), iberiotoxin (BK(Ca)), TRAM-34 (IK(Ca)), and apamin (SK(Ca)). CO also relaxed O(2)-contracted (60.8% relaxation) and KCl-contracted (18.6% relaxation) DA rings. CO-induced relaxation was impaired by ODQ, TEA, and 4-AP (but not by L-NAME, glibenclamide, iberiotoxin, TRAM-34 or apamin), suggesting the involvement of sGC and K(V) channel stimulation. The presence of inhibitors of H(2)S or CO synthesis as well as the H(2)S precursor L-cysteine or the CO precursor hemin did not significantly affect the response of the DA to changes in O(2) tension. Endothelium-dependent and -independent relaxations were also unaffected. In conclusion, our results indicate that the gasotransmitters H(2)S and CO are vasoactive in the chicken DA but they do not suggest an important role for endogenous H(2)S or CO in the control of chicken ductal reactivity.     

An amino acid outside the pore region influences apamin sensitivity in small conductance Ca2+-activated K+ channels

Small conductance calcium-activated potassium channels (SK, K(Ca)) are a family of voltage-independent K+ channels with a distinct physiology and pharmacology. The bee venom toxin apamin inhibits exclusively the three cloned SK channel subtypes (SK1, SK2, and SK3) with different affinity, highest for SK2, lowest for SK1, and intermediate for SK3 channels. The high selectivity of apamin made it a valuable tool to study the molecular makeup and function of native SK channels. Three amino acids located in the outer vestibule of the pore are of particular importance for the different apamin sensitivities of SK channels. Chimeric SK1 channels, enabling the homomeric expression of the rat SK1 (rSK1) subunit and containing the core domain (S1-S6) of rSK1, are apamin-insensitive. By contrast, channels formed by the human orthologue human SK1 (hSK1) are sensitive to apamin. This finding hinted at the involvement of regions beyond the pore as determinants of apamin sensitivity, because hSK1 and rSK1 have an identical amino acid sequence in the pore region. Here we investigated which parts of the channels outside the pore region are important for apamin sensitivity by constructing chimeras between apamin-insensitive and -sensitive SK channel subunits and by introducing point mutations. We demonstrate that a single amino acid situated in the extracellular loop between the transmembrane segments S3 and S4 has a major impact on apamin sensitivity. Our findings enabled us to convert the hSK1 channel into a channel that was as sensitive for apamin as SK2, the SK channel with the highest sensitivity.     

SK but not IK channels regulate human detrusor smooth muscle spontaneous and nerve-evoked contractions

Animal studies suggest that the small (SK) and intermediate (IK) conductance Ca(2+)-activated K(+) channels may contribute to detrusor smooth muscle (DSM) excitability and contractility. However, the ability of SK and IK channels to control DSM spontaneous phasic and nerve-evoked contractions in human DSM remains unclear. We first investigated SK and IK channels molecular expression in native human DSM and further assessed their functional role using isometric DSM tension recordings and SK/IK channel-selective inhibitors. Quantitative PCR experiments revealed that SK3 channel mRNA expression in isolated DSM single cells was ～12- to 44-fold higher than SK1, SK2, and IK channels. RT-PCR studies at the single-cell level detected mRNA messages for SK3 channels but not SK1, SK2, and IK channels. Western blot and immunohistochemistry analysis further confirmed protein expression for the SK3 channel and lack of detectable protein expression for IK channel in whole DSM tissue. Apamin (1 μM), a selective SK channel inhibitor, significantly increased the spontaneous phasic contraction amplitude, muscle force integral, phasic contraction duration, and muscle tone of human DSM isolated strips. Apamin (1 μM) also increased the amplitude of human DSM electrical field stimulation (EFS)-induced contractions. However, TRAM-34 (1 μM), a selective IK channel inhibitor, had no effect on the spontaneous phasic and EFS-induced DSM contractions suggesting a lack of IK channel functional role in human DSM. In summary, our molecular and functional studies revealed that the SK, particularly the SK3 subtype, but not IK channels are expressed and regulate the spontaneous and nerve-evoked contractions in human DSM.     

Ca(2+)-activated K+ channels: molecular determinants and function of the SK family

Ca(2+)-activated K(+) (K(Ca)) channels of small (SK) and intermediate (IK) conductance are present in a wide range of excitable and non-excitable cells. On activation by low concentrations of Ca(2+), they open, which results in hyperpolarization of the membrane potential and changes in cellular excitability. K(Ca)-channel activation also counteracts further increases in intracellular Ca(2+), thereby regulating the concentration of this ubiquitous intracellular messenger in space and time. K(Ca) channels have various functions, including the regulation of neuronal firing properties, blood flow and cell proliferation. The cloning of SK and IK channels has prompted investigations into their gating, pharmacology and organization into calcium-signalling domains, and has provided a framework that can be used to correlate molecularly identified K(Ca) channels with their native currents.     

Apical Ca2+-activated potassium channels in mouse parotid acinar cells

Ca(2+) activation of Cl and K channels is a key event underlying stimulated fluid secretion from parotid salivary glands. Cl channels are exclusively present on the apical plasma membrane (PM), whereas the localization of K channels has not been established. Mathematical models have suggested that localization of some K channels to the apical PM is optimum for fluid secretion. A combination of whole cell electrophysiology and temporally resolved digital imaging with local manipulation of intracellular [Ca(2+)] was used to investigate if Ca(2+)-activated K channels are present in the apical PM of parotid acinar cells. Initial experiments established Ca(2+)-buffering conditions that produced brief, localized increases in [Ca(2+)] after focal laser photolysis of caged Ca(2+). Conditions were used to isolate K(+) and Cl(-) conductances. Photolysis at the apical PM resulted in a robust increase in K(+) and Cl(-) currents. A localized reduction in [Ca(2+)] at the apical PM after photolysis of Diazo-2, a caged Ca(2+) chelator, resulted in a decrease in both K(+) and Cl(-) currents. The K(+) currents evoked by apical photolysis were partially blocked by both paxilline and TRAM-34, specific blockers of large-conductance "maxi-K" (BK) and intermediate K (IK), respectively, and almost abolished by incubation with both antagonists. Apical TRAM-34-sensitive K(+) currents were also observed in BK-null parotid acini. In contrast, when the [Ca(2+)] was increased at the basal or lateral PM, no increase in either K(+) or Cl(-) currents was evoked. These data provide strong evidence that K and Cl channels are similarly distributed in the apical PM. Furthermore, both IK and BK channels are present in this domain, and the density of these channels appears higher in the apical versus basolateral PM. Collectively, this study provides support for a model in which fluid secretion is optimized after expression of K channels specifically in the apical PM.     

Cloning and functional expression of a liver isoform of the small conductance Ca2+-activated K+ channel SK3

Small conductance Ca2+-activated K+ (SK) channels have been cloned from mammalian brain, but little is known about the molecular characteristics of SK channels in nonexcitable tissues. Here, we report the isolation from rat liver of an isoform of SK3. The sequence of the rat liver isoform differs from rat brain SK3 in five amino acid residues in the NH3 terminus, where it more closely resembles human brain SK3. SK3 immunoreactivity was detectable in hepatocytes in rat liver and in HTC rat hepatoma cells. Human embryonic kidney (HEK-293) cells transfected with liver SK3 expressed 10 pS K+ channels that were Ca2+ dependent (EC(50) 630 nM) and were blocked by the SK channel inhibitor apamin (IC(50) 0.6 nM); whole cell SK3 currents inactivated at membrane potentials more positive than -40 mV. Notably, the Ca2+ dependence, apamin sensitivity, and voltage-dependent inactivation of SK3 are strikingly similar to the properties of hepatocellular and biliary epithelial SK channels evoked by metabolic stress. These observations raise the possibility that SK3 channels influence membrane K+ permeability in hepatobiliary cells during liver injury.     

ACh-induced relaxations of rabbit small mesenteric arteries: role of arachidonic acid metabolites and K+

ACh-induced endothelium-dependent relaxation in rabbit small mesenteric arteries is resistant to N-nitro-L-arginine (L-NA) and indomethacin but sensitive to high K+, indicating the relaxations are mediated by endothelium-derived hyperpolarizing factors (EDHFs). The identity of the EDHFs in this vascular bed remains undefined. Small mesenteric arteries pretreated with L-NA and indomethacin were contracted with phenylephrine. ACh (10(-10) to 10(-6) M) caused concentration-dependent relaxations that were shifted to the right by lipoxygenase inhibition and the Ca(2+)-activated K+ channel inhibitors apamin (100 nM) or charybdotoxin (100 nM) and eliminated by the combination of apamin plus charybdotoxin. Relaxations to ACh were also blocked by a combination of barium (200 microM) and apamin but not barium plus charybdotoxin. Addition of K+ (10.9 mM final concentration) to the preconstricted arteries elicited small relaxations. K+ addition before ACh restored the charybdotoxin-sensitive component of relaxations to ACh. K+ (10.9 mM) also relaxed endothelium-denuded arteries, and the relaxations were inhibited by barium but not by charybdotoxin and apamin. With the use of whole cell patch-clamp analysis, ACh (10(-7) M) stimulated voltage-dependent outward K+ current from endothelial cells, which was inhibited by charybdotoxin, indicating K+ efflux. Arachidonic acid (10(-7) to 10(-4) M) induced concentration-related relaxations that were inhibited by apamin but not by charybdotoxin and barium. Addition of arachidonic acid after K+ (10.9 mM) resulted in more potent relaxations to arachidonic acid compared with control without K+ (5.9 mM). These findings suggest that, in rabbit mesenteric arteries, ACh-induced, L-NA- and indomethacin-resistant relaxation is mediated by endothelial cell K+ efflux and arachidonic acid metabolites, and a synergism exists between these two separate mechanisms.     

Modulation of endothelial SK3 channel activity by Ca²⁺-dependent caveolar trafficking

Small- and intermediate-conductance Ca(2+)-activated K(+) channels (SK3/Kcnn3 and IK1/Kcnn4) are expressed in vascular endothelium. Their activities play important roles in regulating vascular tone through their modulation of intracellular concentration ([Ca(2+)](i)) required for the production of endothelium-derived vasoactive agents. Activation of endothelial IK1 or SK3 channels hyperpolarizes endothelial cell membrane potential, increases Ca(2+) influx, and leads to the release of vasoactive factors, thereby impacting blood pressure. To examine the distinct roles of IK1 and SK3 channels, we used electrophysiological recordings to investigate IK1 and SK3 channel trafficking in acutely dissociated endothelial cells from mouse aorta. The results show that SK3 channels undergo Ca(2+)-dependent cycling between the plasma membrane and intracellular organelles; disrupting Ca(2+)-dependent endothelial caveolae cycling abolishes SK3 channel trafficking. Moreover, transmitter-induced changes in SK3 channel activity and surface expression modulate endothelial membrane potential. In contrast, IK1 channels do not undergo rapid trafficking and their activity remains unchanged when either exo- or endocytosis is block. Thus modulation of SK3 surface expression may play an important role in regulating endothelial membrane potential in a Ca(2+)-dependent manner.     

Dietary obesity increases NO and inhibits BKCa-mediated, endothelium-dependent dilation in rat cremaster muscle artery: association with caveolins and caveolae

Obesity is a risk factor for hypertension and other vascular disease. The aim of this study was to examine the effect of diet-induced obesity on endothelium-dependent dilation of rat cremaster muscle arterioles. Male Sprague-Dawley rats (213 ± 1 g) were fed a cafeteria-style high-fat or control diet for 16-20 wk. Control rats weighed 558 ± 7 g compared with obese rats 762 ± 12 g (n = 52-56; P < 0.05). Diet-induced obesity had no effect on acetylcholine (ACh)-induced dilation of isolated, pressurized (70 mmHg) arterioles, but sodium nitroprusside (SNP)-induced vasodilation was enhanced. ACh-induced dilation of arterioles from control rats was abolished by a combination of the K(Ca) blockers apamin, 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34), and iberiotoxin (IBTX; all 0.1 μmol/l), with no apparent role for nitric oxide (NO). In arterioles from obese rats, however, IBTX had no effect on responses to ACh while the NO synthase (NOS)/guanylate cyclase inhibitors N(ω)-nitro-L-arginine methyl ester (L-NAME; 100 μmol/l)/1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10 μmol/l) partially inhibited ACh-induced dilation. Furthermore, NOS activity (but not endothelial NOS expression) was increased in arteries from obese rats. L-NAME/ODQ alone or removal of the endothelium constricted arterioles from obese but not control rats. Expression of caveolin-1 and -2 oligomers (but not monomers or caveolin-3) was increased in arterioles from obese rats. The number of caveolae was reduced in the endothelium of arteries, and caveolae density was increased at the ends of smooth muscle cells from obese rats. Diet-induced obesity abolished the contribution of large-conductance Ca(2+)-activated K(+) channel to ACh-mediated endothelium-dependent dilation of rat cremaster muscle arterioles, while increasing NOS activity and inducing an NO-dependent component.     

Tranilast Increases Vasodilator Response to Acetylcholine in Rat Mesenteric Resistance Arteries through Increased EDHF Participation

Background and Purpose

Tranilast, in addition to its capacity to inhibit mast cell degranulation, has other biological effects, including inhibition of reactive oxygen species, cytokines, leukotrienes and prostaglandin release. In the current study, we analyzed whether tranilast could alter endothelial function in rat mesenteric resistance arteries (MRA).

Experimental Approach

Acetylcholine-induced relaxation was analyzed in MRA (untreated and 1-hour tranilast treatment) from 6 month-old Wistar rats. To assess the possible participation of endothelial nitric oxide or prostanoids, acetylcholine-induced relaxation was analyzed in the presence of L-NAME or indomethacin. The participation of endothelium-derived hyperpolarizing factor (EDHF) in acetylcholine-induced response was analyzed by preincubation with TRAM-34 plus apamin or by precontraction with a high K⁺ solution. Nitric oxide (NO) and superoxide anion levels were measured, as well as vasomotor responses to NO donor DEA-NO and to large conductance calcium-activated potassium channel opener NS1619.

Key Results

Acetylcholine-induced relaxation was greater in tranilast-incubated MRA. Acetylcholine-induced vasodilation was decreased by L-NAME in a similar manner in both experimental groups. Indomethacin did not modify vasodilation. Preincubation with a high K⁺ solution or TRAM-34 plus apamin reduced the vasodilation to ACh more markedly in tranilast-incubated segments. NO and superoxide anion production, and vasodilator responses to DEA-NO or NS1619 remained unmodified in the presence of tranilast.

Conclusions and Implications

Tranilast increased the endothelium-dependent relaxation to acetylcholine in rat MRA. This effect is independent of the nitric oxide and cyclooxygenase pathways but involves EDHF, and is mediated by an increased role of small conductance calcium-activated K+ channels.     

Interaction of SK(Ca) channels and L-type Ca(2+) channels in catecholamine secretion in the rat adrenal gland

We elucidated the interaction of small-conductance Ca(2+)-activated K(+) (SK(Ca)) channels and L-type Ca(2+) channels in muscarinic receptor-mediated control of catecholamine secretion in the isolated perfused rat adrenal gland. The muscarinic agonist methacholine (10-300 microM) produced concentration-dependent increases in adrenal output of epinephrine and norepinephrine. The SK(Ca) channel blocker apamin (1 microM) enhanced the methacholine-induced catecholamine responses. The facilitatory effect of apamin on the methacholine-induced catecholamine responses was not observed during treatment with the L-type Ca(2+) channel blocker nifedipine (3 microM) or Ca(2+)-free solution. Nifedipine did not affect the methacholine-induced catecholamine responses, but it inhibited the responses during treatment with apamin. The L-type Ca(2+) channel activator Bay k 8644 (1 microM) enhanced the methacholine-induced catecholamine responses, whereas the enhancement of the methacholine-induced epinephrine and norepinephrine responses were prevented and attenuated by apamin, respectively. These results suggest that SK(Ca) channels are activated by muscarinic receptor stimulation, which inhibits the opening of L-type Ca(2+) channels and thereby attenuates adrenal catecholamine secretion.     

Ca2+-activated IK1 channels associate with lipid rafts upon cell swelling and mediate volume recovery

Restoration of cell volume in the continued presence of osmotic stimuli is essential, particularly in hepatocytes, which swell upon nutrient uptake. Responses to swelling involve the Ca2+-dependent activation of K+ channels, which promote fluid efflux to drive volume recovery; however, the channels involved in hepatocellular volume regulation have not been identified. We found that hypotonic exposure of HTC hepatoma cells evoked the opening of 50 pS K+-permeable channels, consistent with intermediate conductance (IK) channels. We isolated from rat liver and HTC cells a cDNA with sequence identity to the coding region of IK1. Swelling-activated currents were inhibited by transfection with a dominant interfering IK1 mutant. The IK channel blockers clotrimazole and TRAM-34 inhibited whole cell swelling-activated K+ currents and volume recovery. To determine whether IK1 underwent volume-sensitive localization, we expressed a green fluorescent protein fusion of IK1 in HTC cells. The localization of IK1 was suggestive of distribution in lipid rafts. Consistent with this, there was a time-dependent increase in colocalization between IK1 and the lipid raft ganglioside GM1 on the plasma membrane, which subsequently decreased with volume recovery. Pharmacological disruption of lipid rafts altered the plasma membrane distribution of IK1 and inhibited volume recovery after hypotonic exposure. Collectively, these findings support the hypothesis that IK1 regulates compensatory responses to hepatocellular swelling and suggest that regulation of cell volume involves coordination of signaling from lipid rafts with IK1 function.