Mesenteric Artery Smooth Muscle Cells Research Articles

Effects of aging on Ca 2+ signaling in murine mesenteric arterial smooth muscle cells

Pathophysiological changes in arterial smooth muscle structure and function occur with aging and there are a number of reports illustrating reductions in vascular responsiveness with aging. While much is known about arterial remodeling and functional adaptations with aging, very little is known about the biophysical adaptations in individual arterial myocytes. Cytosolic Ca 2+ signaling, involving activation of L-type Ca 2+ channels on the plasma membrane as well as InsP 3 and ryanodine receptors on the sarcoplasmic reticulum, is integral to vascular tone and reactivity. Thus, we tested the hypothesis that aging results in reductions in the functional expression of L-type channels and temporal aspects of ryanodine receptor and InsP 3 receptor Ca 2+ signaling, in mesenteric arterial smooth muscle cells isolated from 6 and 30 months old C57Bl/6 mice. Comparisons of L-type current activity were made using dialyzed, whole-cell voltage-clamp techniques and Ba 2+ as charge carrier. Ca 2+ signaling was measured using fura-2 fluorescence microscopy techniques. Cell morphological changes were also investigated using electrophysiological and immunocytochemical approaches. The amplitudes of L-type Ca 2+ currents were increased in older mice, but this was associated with membrane surface area increases of ∼50%, due to increases in cell length not cell width. Consequently, L-type Ca 2+ current densities were preserved with age, indicating functional channel expression was unchanged. In contrast, aging was associated with decrements in Ca 2+ signaling in response to either ryanodine receptor stimulation by caffeine or InsP 3 receptor activation with phenylephrine. These changes with aging may be related to the previously reported depression in myogenic reactivity.

Pharmacological and Electrophysiological Characterization of Store-Operated Currents and Capacitative Ca2+Entry in Vascular Smooth Muscle Cells

Capacitative Ca(2+) entry (CCE) in vascular smooth muscle cells contributes to vasoconstrictor and mitogenic effects of vasoactive hormones. In A7r5 rat aortic smooth muscle cells, measurements of cytosolic free Ca(2+) concentration ([Ca(2+)](i)) have demonstrated that depletion of intracellular Ca(2+) stores activates CCE. However, there is disagreement in published studies regarding the regulation of this mechanism by the vasoconstrictor hormone [Arg(8)]-vasopressin (AVP). We have employed electrophysiological methods to characterize the membrane currents activated by store depletion [store-operated current (I(SOC))]. Because of different recording conditions, it has not been previously determined whether I(SOC) corresponds to CCE measured using fura-2; nor has the channel protein responsible for CCE been identified. In the present study, the pharmacological characteristics of I(SOC), including its sensitivity to blockade by 2-aminoethoxydiphenylborane, diethylstilbestrol, or micromolar Gd(3+), were found to parallel the effects of these drugs on thapsigargin- or AVP-activated CCE measured under identical external ionic conditions using fura-2. Thapsigargin-stimulated I(SOC) was also measured in freshly isolated rat mesenteric artery smooth muscle cells (MASMC). Members of the transient receptor potential (TRP) family of nonselective cation channels, TRPC1, TRPC4, and TRPC6, were detected by reverse transcription-polymerase chain reaction and Western blot in both A7r5 cells and MASMC. TRPC1 expression was reduced in a stable A7r5 cell line expressing a small interfering RNA (siRNA) or by infection of A7r5 cells with an adenovirus expressing a TRPC1 antisense nucleotide sequence. Thapsigargin-stimulated I(SOC) was reduced in both the TRPC1 siRNA- and TRPC1 antisense-expressing cells, suggesting that the TRPC1 channel contributes to the I(SOC)/CCE pathway.

Urocortin II Inhibits the Apoptosis of Mesenteric Arterial Smooth Muscle Cells Via L-type Calcium Channels in Spontaneously Hypertensive Rats

Urocortin (UCN) II, a newly isolated corticotropinreleasing-factor (CRF) related peptide, has beenfound to have potent cardiovascular protective effects.To investigate the mechanisms of its vascularprotective effects, we exposed mesenteric arterialsmooth muscle cells (MASMC) from spontaneouslyhypertensive rats (SHR) to UCN II to observe thechange in cell apoptosis using TUNEL assay andmeasured intracellular calcium concentration ([Ca2+]i)using confocal laser scanning microscope. In addition,effects of UCN II on L-type calcium currents (ICa,L) werealso measured using whole-cell patch clamp. Ourresults showed that UCN II concentration-dependently,but time-independently inhibited cell apoptosis.Astressin 2B, a special CRF 2 receptor antagonist,had no influence on this inhibition. Hypoxia or BayK8644, the L-type calcium channel activator, inducedthe apoptosis of MASMC from SHR. Pretreatment ofthe cells with UCN II diminished the effects of hypoxiaor Bay K8644. UCN II was also observed to reduce[Ca2+]i increase induced by KCl or Bay K8644. UCN IIconcentration-dependently inhibited ICa,L, which wasnot affected by astressin 2B. It did not affect theactivation of ICa,L, but markedly shifted the inactivationcurve to the left. In conclusion, UCN II inhibits theapoptosis of MASMC from SHR via inhibiting L-typecalcium channels.

TTX-sensitive voltage-gated Na+ channels are expressed in mesenteric artery smooth muscle cells

The presence and properties of voltage-gated Na+ channels in mesenteric artery smooth muscle cells (SMCs) were studied using whole cell patch-clamp recording. SMCs from mouse and rat mesenteric arteries were enzymatically dissociated using two dissociation protocols with different enzyme combinations. Na+ and Ca2+ channel currents were present in myocytes isolated with collagenase and elastase. In contrast, Na+ currents were not detected, but Ca2+ currents were present in cells isolated with papain and collagenase. Ca2+ currents were blocked by nifedipine. The Na+ current was insensitive to nifedipine, sensitive to changes in the extracellular Na+ concentration, and blocked by tetrodotoxin with an IC50 at 4.3 nM. The Na+ conductance was half maximally activated at -16 mV, and steady-state inactivation was half-maximal at -53 mV. These values are similar to those reported in various SMC types. In the presence of 1 microM batrachotoxin, the Na+ conductance-voltage relationship was shifted by 27 mV in the hyperpolarizing direction, inactivation was almost completely eliminated, and the deactivation rate was decreased. The present study indicates that TTX-sensitive, voltage-gated Na+ channels are present in SMCs from the rat and mouse mesenteric artery. The presence of these channels in freshly isolated SMC depends critically on the enzymatic dissociation conditions. This could resolve controversy about the presence of Na+ channels in arterial smooth muscle.

Alteration of arterial smooth muscle potassium channel composition and BK Ca current modulation in hypertension

We investigated K + currents and their regulation by the sarcoplasmic reticulum in mesenteric arterial smooth muscle cells of the spontaneously hypertensive rat (SHR). Using perforated patch-clamp technique, we found the overall K + current density was significantly lower in adult SHR compared to adult Wistar–Kyoto rats (WKY). The K + currents were almost exclusively of large-conductance Ca 2+-dependent (BK Ca) variety in SHR, but largely of voltage-gated (Kv) variety in WKY. Western blot assay showed parallel findings. These differences were not observed in pre-hypertensive rats. Depleting the intracellular Ca 2+ store inhibited the K + currents in adult SHR. Ryanodine augmented the K + current at 1 μM, but suppressed it at 10 μM; 2-aminoethoxydiphenyl borate demonstrated concentration-dependent inhibition. We conclude that an alteration of membrane K + channel composition has resulted in lower overall K + current density. The changes in K + current type may indicate an underlying defect in Ca 2+-handling that predisposes smooth muscle cells to the hypertensive phenotype.

The pro-oxidant role of methylglyoxal in mesenteric artery smooth muscle cells

Methylglyoxal (MG), a highly reactive metabolite of glucose, causes non-enzymatic glycation of proteins to form irreversible advanced glycation endproducts (AGEs). The present study investigated whether methylglyoxal induced oxidative stress and activated nuclear factor kappa B (NF-kappaB) in freshly isolated and cultured smooth muscle cells (SMCs) from rat mesenteric artery. The treatment of cells with MG (50 or 100 micromol/L) induced a significant increase in AGE formation and oxidation of DCF. MG-enhanced generation of AGEs and the oxidation of DCF was markedly inhibited by antioxidant n-acetylcysteine (NAC, 600 micromol/L). MG at a concentration of 100 micromol/L increased the heme-oxygenase-1 expression in these cells. Moreover, MG activated NF-kappaB p65, indicated by an increased immuno cytochemistry stain for NF-kappaB p65 located in the nucleus after the treatment of mesenteric artery SMCs with MG. MG-induced activation of NF-kappaB p65 was inhibited by NAC. In summary, MG significantly increases oxidative stress and activates NF-kappaB p65 in mesenteric artery SMCs. The pro-oxidant role of methylglyoxal may contribute to various pathological changes of SMCs from resistance arteries.

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.

Vasorelaxation and hyperpolarisation of rat small mesenteric artery by the quaternary anion tetraphenylboron

This study characterises the vasorelaxation and hyperpolarisation effects of the negatively charged quaternary compound tetraphenylboron (TPB) in the rat small mesenteric artery. Segments of rat small mesenteric artery were mounted in a myograph and vessel tone and membrane potential were measured simultaneously. In vessels pre-contracted with vasopressin (0.3-0.6 nM), U46619 (30-90 nM) or methoxamine (0.3-3 microM), TPB (0.1-100 microM) produced a marked endothelium-independent relaxation. However, vasorelaxation responses to TPB were abolished in tissues pre-contracted with K(+) (50 mM), and significantly inhibited by glibenclamide (glib, 10 microM). In the absence of tone, TPB (1-30 microM) caused a concentration-dependent membrane hyperpolarisation of rat mesenteric artery smooth muscle cells, which was not dependent on the endothelium, but sensitive to glibenclamide (10 microM). In methoxamine (0.3-3 microM) pre-contracted vessels, the relaxation response was associated with a marked hyperpolarisation, which was also sensitive to glibenclamide (10 microM), further inhibited by a combination of K(+) channel blockers (glib [10 microM], charybdotoxin [100 nM], apamin [100 nM], 4-aminopyridine [1 mM] and Ba(2+) [30 microM]) and abolished by 50 mM K(+). The results of this study show that TPB causes a vasorelaxation and hyperpolarisation response in the rat small mesenteric artery through a direct action on the vascular smooth muscle. TPB exerts its effects partially via the activation of K(ATP) channels, but also by another mechanism involving K(+)-dependent hyperpolarisation.

Single cGMP-activated Ca(+)-dependent Cl(-) channels in rat mesenteric artery smooth muscle cells.

The present study describes the single channel properties of a novel cGMP-activated Ca(2+)-dependent Cl(-) channel in rat mesenteric artery smooth muscle cells. Single channel currents were recorded in cell-attached patches in the presence of 8 Br cGMP in response to the addition of caffeine or noradrenaline and in both outside-out and inside-out patches when the internal patch surface was bathed in cGMP and Ca(2+). The channels were permeable to Cl(-) ions with an anion permeability sequence of SCN(-) (1.7) > Cl(-) (1.0) > I(-) (0.6). Single channel mean open probability (NP(o)) was independent of voltage and the channels displayed three conductance levels of 15, 35 and 55 pS. cGMP was required for channel activation and the single channel NP(o) increased sharply with raised [Ca(2+)](i), maximal activation occurring at a [Ca(2+)](i) of about 100 nM. The relationship between NP(o) and cGMP concentration was voltage independent and could be fitted by the Hill equation giving a K(d) of about 3 microM and a Hill coefficient (n(H)) of 3. cGMP- and Ca(2+)-dependent channel currents were inhibited by 10 microM ZnCl(2) but niflumic acid, an inhibitor of Ca(2+)-activated Cl(-) channels, had no effect. Inhibition of cGMP-dependent protein kinase activity by the cGMP-dependent protein kinase inhibitor KT5823 or replacement of ATP by AMP-PNP reduced NP(o), while activation of cGMP-dependent protein kinase by guanosine 3', 5'-cyclic monophosphate, beta-phenyl-1, N(2)-etheno-8-bromo-sodium salt (8 Br PET cGMP) produced a significant increase in single channel NP(o). It is likely that these single channel currents underlie the noradrenaline-activated inward current important for vasomotion in these resistance arteries.

Direct block by bisindolylmaleimide of the voltage-dependent K + currents of rat mesenteric arterial smooth muscle

We investigated the effect of bisindolylmaleimide (I), a widely used protein kinase C (PKC) inhibitor, on the voltage-dependent K + (Kv) currents of rat mesenteric arterial smooth muscle cells using the whole-cell patch-clamp technique. Bisindolylmaleimide (I) reversibly and dose-dependently inhibited the Kv currents with an apparent K d value of 0.23±0.001 μM. The blockade was apparently through the acceleration of the decay rate of the Kv currents. The apparent rate constants of association and dissociation for bisindolylmaleimide (I) were 17.9±1.6 μM −1 s −1 and 4.1±1.5 s −1, respectively. The inhibition of Kv current by bisindolylmaleimide (I) was steeply voltage-dependent between −30 and 0 mV (voltage range of channel activation). Bisindolylmaleimide (I) had no effect on the steady-state activation and inactivation of the Kv currents. Applications of trains of pulses at 1 or 2 Hz lead to a progressive increase in the bisindolylmaleimide (I)-blockade, and the recovery from bisindolylmaleimide (I)-block at −80 mV exhibited a time constant of 577.2±52.7 ms. Bisindolylmaleimide (V), an inactive analogue of bisindolylmaleimide (I), similarly inhibited the Kv currents with an apparent K d value of 1.48±0.004 μM, but other PKC inhibitor chelerythrine little affected the Kv currents. These results suggest that bisindolylmaleimide (I) directly inhibits the Kv currents of rat mesenteric arterial smooth muscle cells independently of PKC inhibition, in a state-, voltage-, time- and use-dependent manner.

Opioid receptor antagonists modulate Ca2+-activated K+ channels in mesenteric arterial smooth muscle cells of rats in hemorrhagic shock.

Previous study has indicated a significant enhancement of activity of large-conductance Ca2+-activated K+ channel (BKCa) in mesenteric arterial vascular smooth muscle cells isolated from rats in vascular hyporesponsive stage of hemorrhagic shock. In the present study, the effect of opioid receptor antagonism on BKCa activity in the vascular smooth muscle cells of rats in the hyporesponse stage of hemorrhagic shock was investigated by using inside-out configuration of the patch-clamp technique. The results showed that naloxone (10 microM) down-regulated the activity of BKCa by reducing open probability (Po) and open frequency of the channels. The reduction of Po resulted from a decrease of mean open time and an increase of the slow closed time constant. Naltrindole and nor-binaltorphimine (100 nM) had the similar effects to that of naloxone, but no significant effect of beta-funaltrexamine (100 nM) on the activity of the channels could be found. These results suggest that delta- and kappa-opioid receptors, but not mu-receptors, may be involved in the regulation of BKCa in vascular hyporesponse stage, and that inhibition of BKCa may be one of the mechanisms of the opioid receptor antagonists improving the response of resistance arteries to vasoactive stimulants during the decompensatory stage of hemorrhagic shock.

Bell-shaped curves for prostaglandin-induced modulation of adenylate cyclase: two mutually opposing effects

Each of the natural prostanoid is at least one order of magnitude more potent for its specific receptor (DP, EP, FP, IP and TP) than any of the other prostanoids. However, they are able to interact also with one or more of the other classes of prostanoid receptors. The concentration–response curves for modulation of adenylate cyclase activity in rabbit mesenteric artery smooth muscle cells by different prostaglandins are not always monotonic, i.e. simple sigmoidal curves in logarithmic scale, but they are often biphasic. Prostacyclin, iloprost and prostaglandin E 1 showed a convex bell-shaped curve, i.e. adenylate cyclase activity is stimulated at lower concentrations and inhibited at higher concentrations, while the curve of prostaglandin E 2 showed a concave bell-shaped curve, i.e. adenylate cyclase is inhibited at lower concentrations and stimulated at higher concentrations. By selectively inhibiting one of the transduction mechanisms present in mesenteric smooth muscle cells, we have demonstrated that the observed responses to these prostanoids are likely due to two mutually opposing effects. Thus, the data previously published by our laboratory on a prostacyclin analog, 5( Z)-carbacyclin, might be reinterpreted more correctly in the light of this new possibility.

Modulation of Ca2+ channels by opioid receptor antagonists in mesenteric arterial smooth muscle cells of rats in hemorrhagic shock.

The effects of hemorrhagic shock on Ba currents ( ) via Ca channels and the regulation of the channels in the vascular hyporesponse stage of hemorrhagic shock by opioid receptor antagonists were examined by using the whole-cell recording of patch-clamp technique in mesenteric arterial smooth muscle cells of rats. The results showed that hemorrhagic shock induced an inhibition of Ca channels in the cells; 10 micro M of naloxone and 100 n of naltrindole, nor-binaltorphimine, and beta-funaltrexamine increased the in the cells of rats in shock. After inhibition of protein kinase C by using 1-(5-isoquindinesulfonyl)-2-methylpiperazine via electrodes, the enhancement of by the antagonists was not observed. These results suggested that the inhibition of Ca channel induced by hemorrhagic shock was mediated by delta-, kappa-, and mu -opioid receptors in the cells and may be partly responsible for vascular hyporesponse. The enhancement of was mediated by activation of protein kinase C and may be responsible for the antagonist-caused improvement in the response of resistance arteries to vasoactive stimulants at the decompensatory stage of hemorrhagic shock.

Decreased endothelin binding and [Ca2+]i signaling in microvessels of DOCA-salt hypertensive rats.

The deoxycorticosterone acetate (DOCA)-salt model of hypertension is characterized by elevated vascular endothelin-1 (ET-1) and by reduced contraction to ET-1 in isolated mesenteric small arteries. The decreased contraction to ET-1 may be a compensatory mechanism caused by elevations in ET-1 and arterial pressure. The present study was designed to determine whether down-regulation of endothelin receptors or altered Ca2+ signaling contribute to the decreased contraction to ET-1. Contraction to ET-1 (10 to 10 mol/l) was significantly reduced in isolated mesenteric small arteries (87-286 microm intraluminal diameter) from DOCA-salt rats compared with placebo rats. Membrane protein was obtained for measurement of [125I]ET-1 receptor binding and ET receptor expression. Maximum binding was significantly reduced in vascular membranes from DOCA-salt rats (670 +/- 71 fmol/mg protein) compared with placebo rats (1165 +/- 75 fmol/mg protein), but binding affinity was unchanged. Conversely, ETA receptor protein was increased in DOCA-salt rat vessels. To assess Ca2+ signaling, freshly dissociated mesenteric small artery smooth muscle cells were loaded with fura-2 for measurement of the average myoplasmic free Ca2+ concentration ([Ca2+ ] ). The ET-1 (10 mol/l) induced increase in [Ca2+ ] was significantly less in cells from DOCA-salt rats compared with from placebo rats. This effect was not due to a loss of L-type Ca2+ channels since expression was increased in membrane protein from DOCA-salt rats compared with placebo rats, as measured by Western blot analysis. These findings indicate that decreases in receptor binding and Ca2+ signaling contribute to the impaired contraction to ET-1 in DOCA-salt hypertensive rats. However, these changes are not due to reduced expression of ETA receptors or L-type Ca2+ channels.

Contributions of Kv1.2, Kv1.5 and Kv2.1 subunits to the native delayed rectifier K + current in rat mesenteric artery smooth muscle cells

A large array of voltage-gated K + channel (Kv) genes has been identified in vascular smooth muscle tissues. This molecular diversity underlies the vast repertoire of native Kv channels that regulate the excitability of vascular smooth muscle tissues. The contributions of different Kv subunit gene products to the native Kv currents are poorly understood in vascular smooth muscle cells (SMCs). In the present study, Kv subunit-specific antibodies were applied intracellularly to selectively block various Kv channel subunits and the whole-cell outward Kv currents were recorded using the patch-clamp technique in rat mesenteric artery SMCs. Anti-Kv1.2 antibody (8 μg/ml) inhibited the Kv currents by 29.2 ± 5.9% (n = 6, P < 0.05), and anti-Kv1.5 antibody (6 μg/ml) by 24.5 ± 2.6% (n = 7, P < 0.05). Anti-Kv2.1 antibody inhibited the Kv currents in a concentration-dependent fashion (4–20 μg/ml). Co-application of antibodies against Kv1.2 and Kv2.1 (8 μg/ml each) induced an additive inhibition of Kv currents by 42.3 ± 3.1% (n = 7, P < 0.05). In contrast, anti-Kv1.3 antibody (6 μg/ml) did not have any effect on the native Kv current (n = 6, P > 0.05). A control antibody (anti-GIRK1) also had no effect on the native Kv currents. This study demonstrates that Kv1.2, Kv1.5, and Kv2.1 subunit genes all contribute to the formation of the native Kv channels in rat mesenteric artery SMCs.

Bradykinin B1 receptor blocks PDGF-induced mitogenesis by prolonging ERK activation and increasing p27Kip1.

The mechanism by which the bradykinin B1 receptor (B1R) inhibits platelet-derived growth factor (PDGF)-stimulated proliferation was investigated in cultured rat mesenteric arterial smooth muscle cells. The B1R agonist des-Arg9-bradykinin (DABK) was found to inhibit PDGF-mediated activation of the cyclin E-cyclin-dependent kinase 2 (Cdk2) complex and to prevent hyperphosphorylation of retinoblastoma protein. DABK did not inhibit upregulation of cyclin E expression but increased expression of the Cdk2 inhibitor p27Kip1 and the association of p27Kip1 with the cyclin E-Cdk2 complex. In addition, DABK inhibited the PDGF-stimulated expression of cyclin D that would otherwise siphon p27Kip1 away from inhibition of cyclin E-Cdk2. The signaling mechanism by which DABK regulated p27Kip1 was explored. DABK was found to stimulate the activity of mitogen-activated protein kinase kinase (MEK) and extracellular signal-regulated kinase (ERK) and to prolong activation of MEK and ERK by PDGF. Inhibition of ERK activation with the MEK inhibitors PD-98059 and U-0126 as well as the Src family kinase inhibitor PP2 completely blocked the effect of DABK to increase p27Kip1 and partially reversed the DABK-mediated inhibition of PDGF-stimulated proliferation. These studies demonstrate that the B1R inhibits PDGF-stimulated mitogenesis in part by prolonged activation of ERK leading to increased expression of p27Kip1.

Natural Bile Acids and Synthetic Analogues Modulate Large Conductance Ca2+-activated K+ (BKCa) Channel Activity in Smooth Muscle Cells

Bile acids have been reported to produce relaxation of smooth muscle both in vitro and in vivo. The cellular mechanisms underlying bile acid–induced relaxation are largely unknown. Here we demonstrate, using patch-clamp techniques, that natural bile acids and synthetic analogues reversibly increase BKCa channel activity in rabbit mesenteric artery smooth muscle cells. In excised inside-out patches bile acid–induced increases in channel activity are characterized by a parallel leftward shift in the activity-voltage relationship. This increase in BKCa channel activity is not due to Ca2+-dependent mechanism(s) or changes in freely diffusible messengers, but to a direct action of the bile acid on the channel protein itself or some closely associated component in the cell membrane. For naturally occurring bile acids, the magnitude of bile acid–induced increase in BKCa channel activity is inversely related to the number of hydroxyl groups in the bile acid molecule. By using synthetic analogues, we demonstrate that such increase in activity is not affected by several chemical modifications in the lateral chain of the molecule, but is markedly favored by polar groups in the side of the steroid rings opposite to the side where the methyl groups are located, which stresses the importance of the planar polarity of the molecule. Bile acid–induced increases in BKCa channel activity are also observed in smooth muscle cells freshly dissociated from rabbit main pulmonary artery and gallbladder, raising the possibility that a direct activation of BKCa channels by these planar steroids is a widespread phenomenon in many smooth muscle cell types. Bile acid concentrations that increase BKCa channel activity in mesenteric artery smooth muscle cells are found in the systemic circulation under a variety of human pathophysiological conditions, and their ability to enhance BKCa channel activity may explain their relaxing effect on smooth muscle.

Natural bile acids and synthetic analogues modulate large conductance Ca2+-activated K+ (BKCa) channel activity in smooth muscle cells.

Bile acids have been reported to produce relaxation of smooth muscle both in vitro and in vivo. The cellular mechanisms underlying bile acid–induced relaxation are largely unknown. Here we demonstrate, using patch-clamp techniques, that natural bile acids and synthetic analogues reversibly increase BKCa channel activity in rabbit mesenteric artery smooth muscle cells. In excised inside-out patches bile acid–induced increases in channel activity are characterized by a parallel leftward shift in the activity-voltage relationship. This increase in BKCa channel activity is not due to Ca2+-dependent mechanism(s) or changes in freely diffusible messengers, but to a direct action of the bile acid on the channel protein itself or some closely associated component in the cell membrane. For naturally occurring bile acids, the magnitude of bile acid–induced increase in BKCa channel activity is inversely related to the number of hydroxyl groups in the bile acid molecule. By using synthetic analogues, we demonstrate that such increase in activity is not affected by several chemical modifications in the lateral chain of the molecule, but is markedly favored by polar groups in the side of the steroid rings opposite to the side where the methyl groups are located, which stresses the importance of the planar polarity of the molecule. Bile acid–induced increases in BKCa channel activity are also observed in smooth muscle cells freshly dissociated from rabbit main pulmonary artery and gallbladder, raising the possibility that a direct activation of BKCa channels by these planar steroids is a widespread phenomenon in many smooth muscle cell types. Bile acid concentrations that increase BKCa channel activity in mesenteric artery smooth muscle cells are found in the systemic circulation under a variety of human pathophysiological conditions, and their ability to enhance BKCa channel activity may explain their relaxing effect on smooth muscle.

Anorexic effect of K+ channel blockade in mesenteric arterial smooth muscle and intestinal epithelial cells.

Activity of voltage-gated K+ (Kv) channels controls membrane potential (E(m)). Membrane depolarization due to blockade of K+ channels in mesenteric artery smooth muscle cells (MASMC) should increase cytoplasmic free Ca2+ concentration ([Ca2+]cyt) and cause vasoconstriction, which may subsequently reduce the mesenteric blood flow and inhibit the transportation of absorbed nutrients to the liver and adipose tissue. In this study, we characterized and compared the electrophysiological properties and molecular identities of Kv channels and examined the role of Kv channel function in regulating E(m) in MASMC and intestinal epithelial cells (IEC). MASMC and IEC functionally expressed multiple Kv channel alpha- and beta-subunits (Kv1.1, Kv1.2, Kv1.3, Kv1.4, Kv1.5, Kv2.1, Kv4.3, and Kv9.3, as well as Kvbeta1.1, Kvbeta2.1, and Kvbeta3), but only MASMC expressed voltage-dependent Ca2+ channels. The current density and the activation and inactivation kinetics of whole cell Kv currents were similar in MASMC and IEC. Extracellular application of 4-aminopyridine (4-AP), a Kv-channel blocker, reduced whole cell Kv currents and caused E(m) depolarization in both MASMC and IEC. The 4-AP-induced E(m) depolarization increased [Ca2+]cyt in MASMC and caused mesenteric vasoconstriction. Furthermore, ingestion of 4-AP significantly reduced the weight gain in rats. These results suggest that MASMC and IEC express multiple Kv channel alpha- and beta-subunits. The function of these Kv channels plays an important role in controlling E(m). The membrane depolarization-mediated increase in [Ca2+]cyt in MASMC and mesenteric vasoconstriction may inhibit transportation of absorbed nutrients via mesenteric circulation and limit weight gain.

Evidence for involvement of A-kinase anchoring protein in activation of rat arterial K(ATP) channels by protein kinase A.

1. We have investigated the possible role of A-kinase anchoring proteins (AKAPs) in protein kinase A (PKA) signalling to ATP-sensitive K+ (K(ATP)) channels of rat isolated mesenteric arterial smooth muscle cells using whole-cell patch clamp and peptides that inhibit PKA-AKAP binding. 2. Intracellular Ht31 peptide (20 microM), which inhibits the PKA-AKAP interaction, blocked K(ATP) current activation by either dibutyryl cAMP or calcitonin gene-related peptide. Ht31-proline (20 microM), which does not inhibit PKA binding to AKAP, did not block K(ATP) current activation. 3. Ht31 reduced K(ATP) current activated by pinacidil and also prevented its inhibition by Rp-cAMPS, effects consistent with Ht31 blocking steady-state K(ATP) channel activation by PKA. However, Ht31 did not prevent K(ATP) current activation by the catalytic subunit of PKA. 4. An antibody to the RII subunit of PKA showed localization of PKA near to the cell membrane. Our results provide evidence that both steady-state and receptor-driven activation of K(ATP) channels by PKA involve the localization of PKA by an AKAP.