Biomedical subjects

A D Bonev

Publications and source records attributed to A D Bonev.

At least 19 recordsLinked to original sources

PubMed2000

Vasoregulation by the beta1 subunit of the calcium-activated potassium channel.

Small arteries exhibit tone, a partially contracted state that is an important determinant of blood pressure. In arterial smooth muscle cells, intracellular calcium paradoxically controls both contraction and relaxation. The mechanisms by which calcium can differentially regulate diverse physiological responses within a single cell remain unresolved. Calcium-dependent relaxation is mediated by local calcium release from the sarcoplasmic reticulum. These 'calcium sparks' activate calcium-dependent potassium (BK) channels comprised of alpha and beta1 subunits. Here we show that targeted deletion of the gene for the beta1 subunit leads to a decrease in the calcium sensitivity of BK channels, a reduction in functional coupling of calcium sparks to BK channel activation, and increases in arterial tone and blood pressure. The beta1 subunit of the BK channel, by tuning the channel's calcium sensitivity, is a key molecular component in translating calcium signals to the central physiological function of vasoregulation.

Animals↗

PubMed2000

Intracellular calcium events activated by ATP in murine colonic myocytes.

ATP is a candidate enteric inhibitory neurotransmitter in visceral smooth muscles. ATP hyperpolarizes visceral muscles via activation of small-conductance, Ca(2+)-activated K(+) (SK) channels. Coupling between ATP stimulation and SK channels may be mediated by localized Ca(2+) release. Isolated myocytes of the murine colon produced spontaneous, localized Ca(2+) release events. These events corresponded to spontaneous transient outward currents (STOCs) consisting of charybdotoxin (ChTX)-sensitive and -insensitive events. ChTX-insensitive STOCs were inhibited by apamin. Localized Ca(2+) transients were not blocked by ryanodine, but these events were reduced in magnitude and frequency by xestospongin C (Xe-C), a blocker of inositol 1,4,5-trisphosphate receptors. Thus we have termed the localized Ca(2+) events in colonic myocytes "Ca(2+) puffs. " The P(2Y) receptor agonist 2-methylthio-ATP (2-MeS-ATP) increased the intensity and frequency of Ca(2+) puffs. 2-MeS-ATP also increased STOCs in association with the increase in Ca(2+) puffs. Pyridoxal-phospate-6-azophenyl-2',4'-disculfonic acid tetrasodium, a P(2) receptor inhibitor, blocked responses to 2-MeS-ATP. Spontaneous Ca(2+) transients and the effects of 2-MeS-ATP on Ca(2+) puffs and STOCs were blocked by U-73122, an inhibitor of phospholipase C. Xe-C and ryanodine also blocked responses to 2-MeS-ATP, suggesting that, in addition to release from IP(3) receptor-operated stores, ryanodine receptors may be recruited during agonist stimulation to amplify release of Ca(2+). These data suggest that localized Ca(2+) release modulates Ca(2+)-dependent ionic conductances in the plasma membrane. Localized Ca(2+) release may contribute to the electrical responses resulting from purinergic stimulation.

Adenosine Triphosphate↗

PubMed1999

Kir2.1 encodes the inward rectifier potassium channel in rat arterial smooth muscle cells.

1. The molecular nature of the strong inward rectifier K+ channel in vascular smooth muscle was explored by using isolated cell RT-PCR, cDNA cloning and expression techniques. 2. RT-PCR of RNA from single smooth muscle cells of rat cerebral (basilar), coronary and mesenteric arteries revealed transcripts for Kir2.1. Transcripts for Kir2.2 and Kir2.3 were not found. 3. Quantitative PCR analysis revealed significant differences in transcript levels of Kir2.1 between the different vascular preparations (n = 3; P < 0.05). A two-fold difference was detected between Kir2.1 mRNA and beta-actin mRNA in coronary arteries when compared with relative levels measured in mesenteric and basilar preparations. 4. Kir2.1 was cloned from rat mesenteric vascular smooth muscle cells and expressed in Xenopus oocytes. Currents were strongly inwardly rectifying and selective for K+. 5. The effect of extracellular Ba2+, Ca2+, Mg2+ and Cs2+ ions on cloned Kir2.1 channels expressed in Xenopus oocytes was examined. Ba2+ and Cs+ block were steeply voltage dependent, whereas block by external Ca2+ and Mg2+ exhibited little voltage dependence. The apparent half-block constants and voltage dependences for Ba2+, Cs+, Ca2+ and Mg2+ were very similar for inward rectifier K+ currents from native cells and cloned Kir2.1 channels expressed in oocytes. 6. Molecular studies demonstrate that Kir2.1 is the only member of the Kir2 channel subfamily present in vascular arterial smooth muscle cells. Expression of cloned Kir2.1 in Xenopus oocytes resulted in inward rectifier K+ currents that strongly resemble those that are observed in native vascular arterial smooth muscle cells. We conclude that Kir2.1 encodes for inward rectifier K+ channels in arterial smooth muscle.

Animals↗

PubMed1999

Functional coupling of ryanodine receptors to KCa channels in smooth muscle cells from rat cerebral arteries.

The relationship between Ca2+ release ("Ca2+ sparks") through ryanodine-sensitive Ca2+ release channels in the sarcoplasmic reticulum and KCa channels was examined in smooth muscle cells from rat cerebral arteries. Whole cell potassium currents at physiological membrane potentials (-40 mV) and intracellular Ca2+ were measured simultaneously, using the perforated patch clamp technique and a laser two-dimensional (x-y) scanning confocal microscope and the fluorescent Ca2+ indicator, fluo-3. Virtually all (96%) detectable Ca2+ sparks were associated with the activation of a spontaneous transient outward current (STOC) through KCa channels. A small number of sparks (5 of 128) were associated with currents smaller than 6 pA (mean amplitude, 4.7 pA, at -40 mV). Approximately 41% of STOCs occurred without a detectable Ca2+ spark. The amplitudes of the Ca2+ sparks correlated with the amplitudes of the STOCs (regression coefficient 0.8; P < 0.05). The half time of decay of Ca2+ sparks (56 ms) was longer than the associated STOCs (9 ms). The mean amplitude of the STOCs, which were associated with Ca2+ sparks, was 33 pA at -40 mV. The mean amplitude of the "sparkless" STOCs was smaller, 16 pA. The very significant increase in KCa channel open probability (>10(4)-fold) during a Ca2+ spark is consistent with local Ca2+ during a spark being in the order of 1-100 microM. Therefore, the increase in fractional fluorescence (F/Fo) measured during a Ca2+ spark (mean 2.04 F/Fo or approximately 310 nM Ca2+) appears to significantly underestimate the local Ca2+ that activates KCa channels. These results indicate that the majority of ryanodine receptors that cause Ca2+ sparks are functionally coupled to KCa channels in the surface membrane, providing direct support for the idea that Ca2+ sparks cause STOCs.

Animals↗

PubMed1998

Ontogeny of local sarcoplasmic reticulum Ca2+ signals in cerebral arteries: Ca2+ sparks as elementary physiological events.

Ca2+ release through ryanodine receptors (RyRs) in the sarcoplasmic reticulum is a key element of excitation-contraction coupling in muscle. In arterial smooth muscle, Ca2+ release through RyRs activates Ca2+-sensitive K+ (KCa) channels to oppose vasoconstriction. Local Ca2+ transients ("Ca2+ sparks"), apparently caused by opening of clustered RyRs, have been observed in smooth and striated muscle. We explored the fundamental issue of whether RyRs generate Ca2+ sparks to regulate arterial smooth muscle tone by examining the function of RyRs during ontogeny of arteries in the brain. In the present study, Ca2+ sparks were measured using the fluorescent Ca2+ indicator fluo-3 combined with laser scanning confocal microscopy. Diameter and arterial wall [Ca2+] measurements obtained from isolated pressurized arteries were also used in this study to provide functional insights. Neonatal arteries (<1 day postnatal), although still proliferative, have the molecular components for excitation-contraction coupling, including functional voltage-dependent Ca2+ channels, RyRs, and KCa channels and also constrict to elevations in intravascular pressure. Despite having functional RyRs, Ca2+ spark frequency in intact neonatal arteries was approximately 1/100 of adult arteries. In marked contrast to adult arteries, neonatal arteries did not respond to inhibitors of RyRs and KCa channels. These results support the hypothesis that RyRs organize during postnatal development to cause Ca2+ sparks, and RyRs must generate Ca2+ sparks to regulate the function of the intact tissue.

Animals↗

PubMed1998

Ca2+ channels, ryanodine receptors and Ca(2+)-activated K+ channels: a functional unit for regulating arterial tone.

Local calcium transients ('Ca2+ sparks') are thought to be elementary Ca2+ signals in heart, skeletal and smooth muscle cells. Ca2+ sparks result from the opening of a single, or the coordinated opening of many, tightly clustered ryanodine receptor (RyR) channels in the sarcoplasmic reticulum (SR). In arterial smooth muscle, Ca2+ sparks appear to be involved in opposing the tonic contraction of the blood vessel. Intravascular pressure causes a graded membrane potential depolarization to approximately -40 mV, an elevation of arterial wall [Ca2+]i and contraction ('myogenic tone') of arteries. Ca2+ sparks activate calcium-sensitive K+ (KCa) channels in the sarcolemmal membrane to cause membrane hyperpolarization, which opposes the pressure induced depolarization. Thus, inhibition of Ca2+ sparks by ryanodine, or of KCa channels by iberiotoxin, leads to membrane depolarization, activation of L-type voltage-gated Ca2+ channels, and vasoconstriction. Conversely, activation of Ca2+ sparks can lead to vasodilation through activation of KCa channels. Our recent work is aimed at studying the properties and roles of Ca2+ sparks in the regulation of arterial smooth muscle function. The modulation of Ca2+ spark frequency and amplitude by membrane potential, cyclic nucleotides and protein kinase C will be explored. The role of local Ca2+ entry through voltage-dependent Ca2+ channels in the regulation of Ca2+ spark properties will also be examined. Finally, using functional evidence from cardiac myocytes, and histological evidence from smooth muscle, we shall explore whether Ca2+ channels, RyR channels, and KCa channels function as a coupled unit, through Ca2+ and voltage, to regulate arterial smooth muscle membrane potential and vascular tone.

Animals↗

PubMed1998

Frequency modulation of Ca2+ sparks is involved in regulation of arterial diameter by cyclic nucleotides.

Forskolin, which elevates cAMP levels, and sodium nitroprusside (SNP) and nicorandil, which elevate cGMP levels, increased, by two- to threefold, the frequency of subcellular Ca2+ release ("Ca2+ sparks") through ryanodine-sensitive Ca2+ release (RyR) channels in the sarcoplasmic reticulum (SR) of myocytes isolated from cerebral and coronary arteries of rats. Forskolin, SNP, nicorandil, dibutyryl-cAMP, and adenosine increased the frequency of Ca(2+)-sensitive K+ (KCa) currents ["spontaneous transient outward currents" (STOCs)] by two- to threefold, consistent with Ca2+ sparks activating STOCs. These agents also increased the mean amplitude of STOCs by 1.3-fold, an effect that could be explained by activation of KCa channels, independent of effects on Ca2+ sparks. To test the hypothesis that cAMP could act to dilate arteries through activation of the Ca2+ spark-->KCa channel pathway, the effects of blockers of KCa channels (iberiotoxin) and of Ca2+ sparks (ryanodine) on forskolin-induced dilations of pressurized cerebral arteries were examined. Forskolin-induced dilations were partially inhibited by iberiotoxin and ryanodine (with no additive effects) and were entirely prevented by elevating external K+. Forskolin lowered average Ca2+ in pressurized arteries while increasing ryanodine-sensitive, caffeine-induced Ca2+ transients. These experiments suggest a new mechanism for cyclic nucleotide-mediated dilations through an increase in Ca2+ spark frequency, caused by effects on SR Ca2+ load and possibly on the RyR channel, which leads to increased STOC frequency, membrane potential hyperpolarization, closure of voltage-dependent Ca2+ channels, decrease in arterial wall Ca2+, and, ultimately, vasodilation.

Animals↗

PubMed1997

Ca(2+)-activated K+ channels regulate action potential repolarization in urinary bladder smooth muscle.

The goal of this study was to examine the role of large conductance Ca(2+)-activated K+ channels in the regulation of cell excitability in urinary bladder smooth muscle from the guinea pig. Ca(2+)-activated K+ channels were studied with single-channel recording techniques and found to be intracellular Ca2+ and voltage dependent and sensitive to external tetraethylammonium and blocked by nanomolar concentrations of iberiotoxin (apparent dissociation constant of 4 nM). Spontaneous action potentials recorded from intact tissue strips depended on external Ca2+ and were inhibited by Ca2+ channel blockers. Iberiotoxin (100 nM) significantly altered the configuration of the action potential by increasing the duration and peak amplitude of the action potential and decreasing the rate of decay. Iberiotoxin also increased the action potential frequency from 0.11 to 0.29 Hz. This study suggests that Ca(2+)-activated K+ channels play a significant role in the repolarization of the action potential and in the maintenance of the resting membrane potential of the urinary bladder smooth muscle.

Action Potentials↗

PubMed1997

Activators of protein kinase C decrease Ca2+ spark frequency in smooth muscle cells from cerebral arteries.

Local Ca2+ transients ("Ca2+ sparks") caused by the opening of one or the coordinated opening of a number of tightly clustered ryanodine-sensitive Ca(2+)-release (RyR) channels in the sarcoplasmic reticulum (SR) activate nearby Ca(2+)-dependent K+ (KCa) channels to cause an outward current [referred to as a "spontaneous transient outward current" (STOC)]. These KCa currents cause membrane potential hyperpolarization of arterial myocytes, which would lead to vasodilation through decreasing Ca2+ entry through voltage-dependent Ca2+ channels. Therefore, modulation of Ca2+ spark frequency should be a means to regulation of KCa channel currents and hence membrane potential. We examined the frequency modulation of Ca2+ sparks and STOCs by activation of protein kinase C (PKC). The PKC activators, phorbol 12-myristate 13-acetate (PMA; 10 nM) and 1,2-dioctanoyl-sn-glycerol (1 microM), decreased Ca2+ spark frequency by 72% and 60%, respectively, and PMA reduced STOC frequency by 83%. PMA also decreased STOC amplitude by 22%, which could be explained by an observed reduction (29%) in KCa channel open probability in the absence of Ca2+ sparks. The reduction in STOC frequency occurred in the presence of an inorganic blocker (Cd2+) of voltage-dependent Ca2+ channels. The reduction in Ca2+ spark frequency did not result from SR Ca2+ depletion, since caffeine-induced Ca2+ transients did not decrease in the presence of PMA. These results suggest that activators of PKC can modulate the frequency of Ca2+ sparks, through an effect on the RyR channel, which would decrease STOC frequency (i.e., KCa channel activity).

Animals↗

PubMed1996

Vasoconstrictors inhibit ATP-sensitive K+ channels in arterial smooth muscle through protein kinase C.

The effects of vasoconstrictor-receptor (neuropeptide Y, alpha-adrenergic, serotonergic, histaminergic) stimulation on currents through ATP-sensitive potassium (KATP) channels in arterial smooth muscle cells were examined. Whole-cell KATP currents, activated by the synthetic KATP channel opener pinacidil or by the endogenous vasodilator, calcitonin gene-related peptide, which acts through protein kinase A, were measured in smooth muscle cells isolated from mesenteric arteries of rabbit. Stimulation of NPY-, alpha 1-, serotonin (5-HT2)-, and histamine (H1)-receptors inhibited KATP currents by 40-56%. The signal transduction pathway that links these receptors to KATP channels was investigated. An inhibitor of phospholipase C (D609) and of protein kinase C (GF 109203X) reduced the inhibitory effect of these vasoconstrictors on KATP currents from 40-56% to 11-23%. Activators of protein kinase C, a diacylglycerol analogue and phorbol 12-myristate 13-acetate (PMA), inhibited KATP currents by 87.3 and 84.2%, respectively. KATP currents, activated by calcitonin gene-related peptide, were also inhibited (47-87%) by serotonin, phenylephrine, and PMA. We propose that KATP channels in these arterial myocytes are subject to dual modulation by protein kinase C (inhibition) and protein kinase A (activation).

Adenosine Triphosphate↗

PubMed1996

Inward rectifier K+ currents in smooth muscle cells from rat coronary arteries: block by Mg2+, Ca2+, and Ba2+.

Inward rectifier K+ channels have been implicated in the control of membrane potential and external K(+)-induced dilations of small coronary arteries. To identify and characterize inward rectifier K+ currents in coronary artery smooth muscle, whole cell K+ currents in smooth muscle cells enzymatically isolated from rat coronary (septal) arteries (diameters, 100-150 microns) were measured in the conventional and perforated configurations of the patch-clamp technique. Ba(2+)-sensitive, whole cell K+ current-voltage relationships exhibited inward rectification. Blockers of Ca(2+)-activated K+ channels (1 mM tetraethylammonium ion), ATP-sensitive K+ channels (10 microM glibenclamide), and voltage-dependent K+ channels (1 mM 4-aminopyridine) in smooth muscle did not affect inward rectifier K+ currents. The nonselective K+ channel inhibitor phencyclidine (100 microM) reduced inward rectifier K+ currents by approximately 50%. External Ba2+ reduced inward currents, with membrane potential hyperpolarization increasing inhibition. The half-inhibition constant for Ba2+ was 2.1 microM at -60 mV, decreasing e-fold for a 25-mV hyperpolarization. External Cs+ also blocked inward rectifier K+ currents, with the half-inhibition constant for Cs+ of 2.9 mM at -60 mV. External Ca2+ and Mg2+ reduced inward rectifier K+ currents. At -60 mV, Ca2+ and Mg2+ (1 mM) reduced inward currents by 33 and 21%, respectively. Inward rectification was not affected by dialysis of the cell's interior with a nominally Ca(2+)- and Mg(2+)-free solution. These findings indicate that inward rectifier K+ channels exist in coronary artery smooth muscle and that Ba2+ may be a useful probe for the functional role of inward rectifier K+ channels in coronary arteries.

Animals↗

PubMed1996

Gender differences in coronary artery diameter involve estrogen, nitric oxide, and Ca(2+)-dependent K+ channels.

During their reproductive years, women have a much lower incidence of coronary heart disease compared with men of similar age. Estrogen appears to be largely responsible for this decrease in cardiovascular mortality in women. In the present study, isolated pressurized coronary arteries from rats were used to assess the role of gender and circulating estrogen on coronary vascular function. Pressure-induced constrictions ("myogenic tone") were greater (approximately 2-fold) in isolated coronary arteries from estrogen-deficient male or ovariectomized (OVX) rats compared with similar arteries obtained from female rats or OVX rats receiving physiological levels of estrogen replacement (OVX+E group). These differences in coronary artery diameter were abolished by removal of the vascular endothelium or chemical inhibition of NO synthase. The anti-estrogen, tamoxifen, increased pressure-induced constrictions of coronary arteries from female and OVX+E rats. Dilations of pressurized coronary arteries from female and OVX animals to sodium nitroprusside, a nitrovasodilator that generates NO, were reduced by > 50% by iberiotoxin (IBTX), an inhibitor of Ca(2+)-dependent K+ (KCa) channels. Sodium nitroprusside (10 mumol/L) hyperpolarized coronary arteries by 13 +/- 2 mV, an effect that was greatly diminished (approximately 80%) by IBTX. Coronary arteries isolated from female rats produced greater constrictions in response to IBTX and KT 5823, an inhibitor of cGMP-dependent protein kinase, compared with coronary arteries from OVX rats. cGMP-dependent protein kinase increased the activity of KCa channels 16.5 +/- 5-fold in excised membrane patches from smooth muscle cells enzymatically isolated from these small coronary arteries. We propose that physiological levels of circulating 17 beta-estradiol elevate basal NO release from the endothelial cells, which increases the diameter of pressurized coronary arteries. Further, our results suggest that part of the effect of this NO is through activation of KCa channels in the smooth muscle cells of the coronary arteries.

Animals↗

PubMed1996

Studies of the K(ATP) channel opening activity of the new dihydropyridine compound 9-(3-cyanophenyl)-3,4,6,7,9,10-hexahydro-1,8-(2H,5H)-acridined ione in bladder detrusor in vitro.

The potassium (K+) channel opening activity of ZM244085 (9-(3-cyanophenyl)-3,4,6,7,9,10-hexahydro-1,8-(2H,5H)-acridined ione, CAS 149398-59-4), a novel dihydropyridine (DHP), was ascertained. In a set of functional assays, its mechanoinhibitory effect on myogenic activity of guinea pig bladder detrusor muscles, either mildly or highly depolarized with 15 or 80 mmol/l KCl, was measured. ZM244085 had negligible effect on the tone of the detrusor contracted with 80 mmol/l KCl but reduced the myogenic activity induced with 15 mmol/l KCl (IC50=4.2 +/- 0.4 mumol/l). Glibenclamide, an ATP-sensitive K+ (KATP) channel blocker, competitively antagonized this action of ZM244085 with a pA2 value of 7.6. This functional profile of ZM244085 is similar to that of the prototypic K+ channel opener cromakalim but stands in contrast to that of typical DHP Ca2+ channel blockers such as nifedipine and nimodipine. The membrane potential of the guinea pig detrusor, recorded with intracellular microelectrodes, was hyperpolarized 6.8 +/- 3.1 mV by ZM244085 (10 mumol/l). This hyperpolarization was completely blocked by glibenclamide but not affected by apamin (10 mumol/l), a toxin blocking specifically small conductance and Ca2+ dependent K+ (SKCa) channels. ZM244085 (10 mumol/l) increased the whole cell KATP current in isolated guinea pig detrusor cells by 8.8 +/- 2.5 pA, but failed to activate large conductance and Ca2+ dependent K+ (BKCa) channels in excised inside-out membrane patches from those cells. The results from these studies showed that ZM244085 is a K+ channel opener which activates predominantly KATP channels in vitro to relax bladder detrusors.

Acridines↗

PubMed1995

Relaxation of arterial smooth muscle by calcium sparks.

Local increases in intracellular calcium ion concentration ([Ca2+]i) resulting from activation of the ryanodine-sensitive calcium-release channel in the sarcoplasmic reticulum (SR) of smooth muscle cause arterial dilation. Ryanodine-sensitive, spontaneous local increases in [Ca2+]i (Ca2+ sparks) from the SR were observed just under the surface membrane of single smooth muscle cells from myogenic cerebral arteries. Ryanodine and thapsigargin inhibited Ca2+ sparks and Ca(2+)-dependent potassium (KCa) currents, suggesting that Ca2+ sparks activate KCa channels. Furthermore, KCa channels activated by Ca2+ sparks appeared to hyperpolarize and dilate pressurized myogenic arteries because ryanodine and thapsigargin depolarized and constricted these arteries to an extent similar to that produced by blockers of KCa channels. Ca2+ sparks indirectly cause vasodilation through activation of KCa channels, but have little direct effect on spatially averaged [Ca2+]i, which regulates contraction.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed1995

Pharmacology of ATP-sensitive K+ currents in smooth muscle cells from rabbit mesenteric artery.

The inference that ATP-sensitive K+ (KATP) channels are involved in arterial responses to the synthetic K+ channel openers, hypoxia, adenosine, and calcitonin gene-related peptide, has relied on the sensitivity of these responses to the sulfonylureas glibenclamide and tolbutamide and to tetraethylammonium (TEA+). The inhibition of KATP currents by glibenclamide, tolbutamide, and TEA+ was investigated in single smooth muscle cells from rabbit mesenteric artery by use of the whole cell patch-clamp technique. The synthetic K+ channel openers pinacidil (half-activation 0.6 microM), cromakalim (half-activation 1.9 microM), and diazoxide (half-activation 37.1 microM) activated K(+)-selective currents that were blocked by glibenclamide. Elevation of pipette (intracellular) ATP concentration decreased K+ currents induced by pinacidil. Half-inhibition of KATP currents by glibenclamide and tolbutamide occurred at 101 nM and 351 microM, respectively. KATP currents were also inhibited by external TEA+, with half-inhibition at 6.2 mM. The results indicate that glibenclamide is an effective inhibitor of KATP channels in arterial smooth muscle and that tolbutamide and TEA+ are much less effective. Furthermore, these results support numerous functional studies that have demonstrated that the vasorelaxations to K+ channel openers are inhibited by < 10 microM glibenclamide but not by < 1 mM TEA+.

Adenosine Triphosphate↗

PubMed1995

Zeneca ZD6169 and its analogs from a novel series of anilide tertiary carbinols: in vitro KATP channel opening activity in bladder detrusor.

The potassium (K+) channel opening activity of Zeneca ZD6169 and one of its pyridylsulfonyl analogs from the anilide tertiary carbinol series was ascertained. Their mechanoinhibitory effects on the myogenic activity of the guinea pig bladder detrusor muscle were measured in a set of functional assays. Elevating the K+ concentration in the tissue bath from 15 to 80 mmol/l increased the IC50 value of ZD6169 from 1.61 +/- 0.22 223 +/- 37 mumol/l. This result suggests that ZD6169 may act as a K+ channel opener. Similar to the prototypic ATP-sensitive K+ (KATP) channel opener cromakalim, the K+ channel openers from the anilide tertiary carbinol series displayed stereoselective mechanoinhibitory activity only in the test protocol in which the detrusor was stimulated with 15 mmol/l KCl. Being the active enantiomer, ZD6169 has an activity more than 30-fold higher than the less active enantiomer. ZD6169 at 10 mumol/l hyperpolarized the guinea pig detrusor membrane potential by 6.1 +/- 1.2 mV and increased the whole cell KATP current in isolated guinea pig smooth muscle cells by 34.9 +/- 7.9 pA. This is comparable to the increase of 26.8 +/- 5.0 pA obtained with 10 mumol/l of lemakalim, the active enantiomer of cromakalim. The K+ channel opening activity of ZD6169 and the pyridylsulfonyl analog was competitively antagonized by the KATP channel blocker glibenclamide in the guinea pig detrusor with a pA2 value of 7.2. This activity, however, was unaffected by blockers of small and large conductance Ca-dependent K+ channels, such as apamin and charybdotoxin, respectively. The present study showed that Zeneca ZD6169 and its analog from the anilide tertiary carbinol series are K+ channel openers that activate KATP channels in vitro to relax bladder detrusors.

Adenosine Triphosphate↗

PubMed1994

Activation of ATP-sensitive potassium currents in guinea-pig gall-bladder smooth muscle by the neuropeptide CGRP.

1. The actions of a neuroactive peptide, calcitonin gene-related peptide (CGRP), and the ATP-sensitive potassium (K+ATP) channel activator lemakalim on guinea-pig gall-bladder smooth muscle cells were investigated using intracellular recording from intact preparations and whole-cell patch clamp recording from acutely dissociated myocytes. 2. CGRP and lemakalim caused a glibenclamide-sensitive hyperpolarization of the plasma membrane of intact cells with an associated suppression of spontaneous action potentials. 3. In isolated smooth muscle cells, CGRP (10 nM) and lemakalim (10 microM) activated currents that were glibenclamide sensitive, voltage independent and potassium selective. 4. External TEA+ at 1.0 and 10.0 mM inhibited glibenclamide-sensitive, CGRP-activated currents by 3.8 and 66.5%, respectively. 5. Increases in the intracellular ATP concentration from 0.1 to 5.0 mM reduced the glibenclamide-sensitive potassium current in the presence of CGRP (10 nM) or lemakalim (10 microM) by > 60%. The increase in the intracellular ATP also reduced the steady-state glibenclamide-sensitive current by > 80%. 6. These findings indicate that CGRP activates K+ATP channels to hyperpolarize the membrane of gall-bladder smooth muscle cells. This hyperpolarization may be an important mechanism underlying the relaxant effects of CGRP on the gall-bladder.

Adenosine Triphosphate↗

PubMed1994

Calcitonin gene-related peptide activated ATP-sensitive K+ currents in rabbit arterial smooth muscle via protein kinase A.

1. Whole-cell K+ currents activated by calcitonin gene-related peptide (CGRP) in smooth muscle cells enzymatically isolated from rabbit mesenteric arteries were measured in the conventional and perforated configurations of the patch clamp technique. The signal transduction pathway from CGRP receptors to activation of potassium currents was investigated. 2. CGRP (10 nM) activated a whole-cell current that was blocked by glibenclamide (10 microM), an inhibitor of ATP-sensitive K+ channels. Elevating intracellular ATP reduced glibenclamide-sensitive currents. CGRP increased the glibenclamide-sensitive currents by 3- to 6-fold in cells dialysed with 0.1 mM ATP, 3.0 mM ATP or in intact cells. The reversal potential of the glibenclamide-sensitive current in the presence of CGRP shifted with the potassium equilibrium potential, while its current-voltage relationship exhibited little voltage dependence. 3. Forskolin (10 microM), an adenylyl cyclase activator, Sp-cAMPS (500 microM) and the catalytic subunit of protein kinase A increased glibenclamide-sensitive K+ currents 2.1-, 3.3- and 8.2-fold, respectively. 4. Nitric oxide and nitroprusside did not activate glibenclamide-sensitive K+ currents. 5. Dialysis of the cell's interior with inhibitors of protein kinase A (synthetic peptide inhibitor, 4.6 microM or H-8, 100 microM) completely blocked activation of K+ currents by CGRP. 6. Our results suggest the following signal transduction scheme for activation of K+ currents by CGRP in arterial smooth muscle: (1) CGRP stimulates adenylyl cyclase, which leads to an elevation of cAMP; (2) cAMP activates protein kinase A, which opens ATP-sensitive K+ channels.

Adenosine Triphosphate↗