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The effects of caffeine on ATP-sensitive K(+) channels in smooth muscle cells from pig urethra.

ABSTRACT: The effects of caffeine on both levcromakalim-induced macroscopic and unitary currents in pig proximal urethra were investigated by the use of patch-clamp techniques (conventional whole-cell configuration and cell-attached configuration). The effects of caffeine were also examined on currents in inside-out patches of COS7 cells expressing carboxy terminus truncated inwardly rectifying K(+) channel (Kir6.2) subunits (i.e. Kir6.2DeltaC36) which form ATP-sensitive K(+) channels (K(ATP) channels). In conventional whole-cell configuration, the levcromakalim (100 microM)-induced inward current (symmetrical 140 mM K(+) conditions) was inhibited by caffeine (> or =1 mM) at a holding potential of -50 mV. In contrast, ryanodine (10 microM) caused no significant inhibitory effect on the gradual decay of the levcromakalim-induced current at -50 mV. The amplitude of the 30 microM levcromakalim-induced current was enhanced by 3-isobutyl-1-methylxanthine (IBMX, 100 microM). In cell-attached configuration, the levcromakalim-induced K(+) channel openings were inhibited by subsequent application of 10 mM caffeine, decreasing the channel open probability at -50 mV. Reverse transcriptase-polymerase chain reaction (RT - PCR) analysis revealed the presence of Kir6.2 transcript in pig urethra. Caffeine (> or =3 mM) inhibited the channel activity of Kir6.2DeltaC36 expressed in COS7 cells (3 mM caffeine, 65+/-6%, n=4; 10 mM caffeine, 29+/-2%, n=4). These results suggest that caffeine can inhibit the activity of K(ATP) channels through a direct blocking effect on the pore-forming Kir subunit.

SUBMITTER: Teramoto N

PROVIDER: S-EPMC1572345 | biostudies-literature |

REPOSITORIES: biostudies-literature

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The involvement of L-type Ca(2+) channels in the relaxant effects of the ATP-sensitive K(+) channel opener ZD6169 on pig urethral smooth muscle.

Project description:1. The effects of ZD6169, a novel ATP-sensitive K(+) channel (K(ATP) channel) opener, were investigated on membrane currents in isolated myocytes using patch-clamp techniques. Tension measurement was also performed to study the effects of ZD6169 on the resting tone of pig urethral smooth muscle. 2. Levcromakalim was more potent than ZD6169 in lowering the resting urethral tone. Relaxation induced by low concentrations of ZD6169 (< or =3 microM) was completely suppressed by additional application of glibenclamide (1 microM). In contrast, glibenclamide (1-10 microM) only partially inhibited the relaxation induced by higher concentrations of ZD6169 (> or = microM). 3. Bay K8644 (1 microM) reduced the maximum relaxation produced by ZD6169 (> or =10 microM). 4. In whole-cell configuration, ZD6169 suppressed the peak amplitude of voltage-dependent Ba(2+) currents in a concentration- and voltage-dependent manner, and at 100 microM, shifted the steady-state inactivation curve of the voltage-dependent Ba(2+) currents to the left at a holding potential of -90 mV. 5. In cell-attached configuration, open probability of unitary voltage-dependent Ba(2+) channels (27 pS, 90 mM Ba(2+)) was inhibited by 100 microM ZD6169 and by 10 microM nifedipine. 6. Reverse transcriptase-polymerase chain reaction (RT - PCR) analysis revealed the presence of the transcript of the alpha(1C) subunit of L-type Ca(2+) channels in pig urethra. 7. These results demonstrate that ZD6169 causes urethral relaxation through two distinct mechanisms, activation of K(ATP) channels at lower concentrations and inhibition of voltage-dependent Ca(2+) channels at higher concentrations (about 10 microM).

| S-EPMC1573091 | biostudies-literature

Contribution of Kv2.1 channels to the delayed rectifier current in freshly dispersed smooth muscle cells from rabbit urethra.

Project description:We have characterized the native voltage-dependent K(+) (K(v)) current in rabbit urethral smooth muscle cells (RUSMC) and compared its pharmacological and biophysical properties with K(v)2.1 and K(v)2.2 channels cloned from the rabbit urethra and stably expressed in human embryonic kidney (HEK)-293 cells (HEK(Kv2.1) and HEK(Kv2.2)). RUSMC were perfused with Hanks' solution at 37°C and studied using the patch-clamp technique with K(+)-rich pipette solutions. Cells were bathed in 100 nM Penitrem A (Pen A) to block large-conductance Ca(2+)-activated K(+) (BK) currents and depolarized to +40 mV for 500 ms to evoke K(v) currents. These were unaffected by margatoxin, ?-dendrotoxin, or ?-dendrotoxin (100 nM, n = 3-5) but were blocked by stromatoxin-1 (ScTx, IC(50) ?130 nM), consistent with the idea that the currents were carried through K(v)2 channels. RNA was detected for K(v)2.1, K(v)2.2, and the silent subunit K(v)9.3 in urethral smooth muscle. Immunocytochemistry showed membrane staining for both K(v)2 subtypes and K(v)9.3 in isolated RUSMC. HEK(Kv2.1) and HEK(Kv2.2) currents were blocked in a concentration-dependent manner by ScTx, with estimated IC(50) values of ?150 nM (K(v)2.1, n = 5) and 70 nM (K(v)2.2, n = 6). The mean half-maximal voltage (V(1/2)) of inactivation of the USMC K(v) current was -56 ± 3 mV (n = 9). This was similar to the HEK(Kv2.1) current (-55 ± 3 mV, n = 13) but significantly different from the HEK(Kv2.2) currents (-30 ± 3 mV, n = 11). Action potentials (AP) evoked from RUSMC studied under current-clamp mode were unaffected by ScTx. However, when ScTx was applied in the presence of Pen A, the AP duration was significantly prolonged. Similarly, ScTx increased the amplitude of spontaneous contractions threefold, but only after Pen A application. These data suggest that K(v)2.1 channels contribute significantly to the K(v) current in RUSMC.

| S-EPMC3213918 | biostudies-literature

The effects of flecainide on ATP-sensitive K(+) channels in pig urethral myocytes.

Project description:The effects of the antiarrhythmic drug flecainide on levcromakalim-induced hyperpolarization, macroscopic and unitary K(+) currents in pig urethra were investigated using patch-clamp techniques. The effects of flecainide were also examined on currents in inside-out patches of COS7 cells expressing carboxy terminus truncated inwardly rectifying K(+) channel (Kir6.2) subunits (i.e. Kir6.2DeltaC36) which form ATP-sensitive K(+) channels (K(ATP) channels). In current-clamp mode, application of flecainide (> or =100 microM) caused a significant depolarization after the membrane potential had been hyperpolarized by levcromakalim. In voltage-clamp experiments, the levcromakalim-induced outward current was suppressed by 300 microM flecainide in quasi-physiological K(+) conditions (K(i)=51 microM). In contrast, approximately 20% of the levcromakalim-induced inward current still remained even after application of 300 microM flecainide in symmetrical 140 mM K(+) conditions (K(i)=51 microM). In contrast, approximately 20% of the levcromakalim-induced inwar=126 microM). In cell-attached configuration, the channel activity of the levcromakalim-induced K(ATP) channels was reversibly inhibited by flecainide (> or =30 microM) at -50 mV. Their activity was also suppressed by either disopyramide or cibenzoline. Flecainide reversibly inhibited the channel activity of Kir6.2DeltaC36 expressed in COS7 cells using inside-out configuration. Inhibitory effects of flecainide on the levcromakalim-induced currents became more potent when the value of external pH increased, although this slightly reduced the proportion of drug molecules carrying a positive charge. These results suggest that flecainide inhibits channel activity through blocking the pore site of the K(ATP) channel in pig urethra.

| S-EPMC1572826 | biostudies-other

Glimepiride block of cloned beta-cell, cardiac and smooth muscle K(ATP) channels.

Project description:1. We examined the effect of the sulphonylurea glimepiride on three types of recombinant ATP-sensitive potassium (K(ATP)) channels. 2. K(ATP) channels share a common pore-forming subunit, Kir6.2, which associates with different sulphonylurea receptor isoforms (SUR1 in beta-cells, SUR2A in heart and SUR2B in smooth muscle). 3. Kir6.2 was coexpressed with SUR1, SUR2A or SUR2B in Xenopus oocytes and macroscopic K(ATP) currents were recorded from giant inside-out membrane patches. Glimepiride was added to the intracellular membrane surface. 4. Glimepiride inhibited Kir6.2/SUR currents by interaction with two sites: a low-affinity site on Kir6.2 (IC(50)= approximately 400 microM) and a high-affinity site on SUR (IC(50)=3.0 nM for SUR1, 5.4 nM for SUR2A and 7.3 nM for SUR2B). The potency of glimepiride at the high-affinity site is close to that observed for glibenclamide (4 nM for SUR1, 27 nM for SUR2A), which has a similar structure. 5. Glimepiride inhibition of Kir6.2/SUR2A and Kir6.2/SUR2B currents, but not Kir6.2/SUR1 currents, reversed rapidly. 6. Our results indicate that glimepiride is a high-affinity sulphonylurea that does not select between the beta-cell, cardiac and smooth muscle types of recombinant K(ATP) channel, when measured in inside-out patches. High-affinity inhibition is mediated by interaction of the drug with the sulphonylurea receptor subunit of the channel.

| S-EPMC1572773 | biostudies-literature

Synthesis and characterization of a quinolinonic compound activating ATP-sensitive K(+) channels in endocrine and smooth muscle tissues.

Project description:Original quinolinone derivatives structurally related to diazoxide were synthesized and their effects on insulin secretion from rat pancreatic islets and the contractile activity of rat aortic rings determined. A concentration-dependent decrease of insulin release was induced by 6-chloro-2-methylquinolin-4(1H)-one (HEI 713). The average IC(50) values were 16.9+/-0.8 microM for HEI 713 and 18.4+/-2.2 microM for diazoxide. HEI 713 increased the rate of (86)Rb outflow from perifused pancreatic islets. This effect persisted in the absence of external Ca(2+) but was inhibited by glibenclamide, a K(ATP) channel blocker. Inside-out patch-clamp experiments revealed that HEI 713 increased K(ATP) channel openings. HEI 713 decreased (45)Ca outflow, insulin output and cytosolic free Ca(2+) concentration in pancreatic islets and islet cells incubated in the presence of 16.7 or 20 mM glucose and extracellular Ca(2+). The drug did not affect the K(+)(50 mM)-induced increase in (45)Ca outflow. In aortic rings, the vasorelaxant effects of HEI 713, less potent than diazoxide, were sensitive to glibenclamide and to the extracellular K(+) concentration. The drug elicited a glibenclamide-sensitive increase in (86)Rb outflow from perifused rat aortic rings. Our data describe an original compound which inhibits insulin release with a similar potency to diazoxide but which has fewer vasorelaxant effects. Our results suggest that, in both aortic rings and islet tissue, the biological effects of HEI 713 mainly result from activation of K(ATP) channels ultimately leading to a decrease in Ca(2+) inflow.

| S-EPMC1572960 | biostudies-other

Functional coupling between the caffeine/ryanodine-sensitive Ca2+ store and mitochondria in rat aortic smooth muscle cells.

Project description:We investigated the role of mitochondria in the agonist-induced and/or caffeine-induced Ca2+ transients in rat aortic smooth muscle cells. We explored the possibility that proliferation modulates the coupling between mitochondria and endoplasmic reticulum. Ca2+ transients induced by either ATP or caffeine were measured in presence or absence of drugs interfering with mitochondrial activity in freshly dissociated cells (day 1) and in subconfluent primary culture (day 12). We found that the mitochondrial inhibitors, rotenone or carbonyl cyanide m-chlorophenylhydrazone, as well as the permeability transition pore inhibitor, cyclosporin A, had no effect on the ATP-induced Ca2+ transient at either day 1 or day 12, but prevented caffeine-induced cytosolic Ca2+ increase at day 12 but not at day 1. Close connections between ryanodine receptors and mitochondria were observed at both day 1 and 12. Thapsigargin (TG) prevented ATP- and caffeine-induced Ca2+ transients at day 1. At day 12, where only 50% of the cells were sensitive to caffeine, TG did not prevent the caffeine-induced Ca2+ transient, and prevented ATP-induced Ca2+ transient in only half of the cells. Together, these data demonstrate that rat aortic smooth muscle cells at day 1 have an ATP- and caffeine-sensitive pool, which is functionally independent but physically closely linked to mitochondria and totally inhibited by TG. At day 12, we propose the existence of two cell populations: half contains IP3 receptors and TG-sensitive Ca2+ pumps only; the other half contains, in addition to the IP3-sensitive pool independent from mitochondria, a caffeine-sensitive pool. This latter pool is linked to mitochondria through the permeability transition pore and is refilled by both TG-sensitive and insensitive mechanisms.

| S-EPMC1221962 | biostudies-other

The Selective Rat Toxicant Norbormide Blocks K<sub>ATP</sub> Channels in Smooth Muscle Cells But Not in Insulin-Secreting Cells.

Project description:Norbormide is a toxicant selective for rats to which it induces a widespread vasoconstriction. In a recent paper, we hypothesized a role of ATP-sensitive potassium (KATP) channels in norbormide-induced vasoconstriction. The current study was undertaken to verify this hypothesis by comparing the effects of norbormide with those of glibenclamide, a known KATP channel blocker. The whole-cell patch-clamp method was used to record KATP currents in myocytes freshly isolated from the rat and mouse caudal artery and from the rat gastric fundus, as well as in insulin-secreting pancreatic beta cells (INS-1 cells). Smooth muscle contractile function was assessed on either rat caudal artery rings or gastric fundus strips. Molecular modeling and docking simulation to KATP channel proteins were investigated in silico. Both norbormide (a racemic mixture of endo and exo isomers) and glibenclamide inhibited KATP currents in rat and mouse caudal artery myocytes, as well as in gastric fundus smooth muscle cells. In rat INS-1 cells, only glibenclamide blocked KATP channels, whereas norbormide was ineffective. The inhibitory effect of norbormide in rat caudal artery myocytes was not stereo-specific as both the endo isomers (active as vasoconstrictor) and the exo isomers (inactive as vasoconstrictor) had similar inhibitory activity. In rat caudal artery rings, norbormide-induced contraction was partially reverted by the KATP channel opener pinacidil. Computational approaches indicated the SUR subunit of KATP channels as the binding site for norbormide. KATP channel inhibition may play a role in norbormide-induced vasoconstriction, but does not explain the species selectivity, tissue selectivity, and stereoselectivity of its constricting activity. The lack of effect in INS-1 cells suggests a potential selectivity of norbormide for smooth muscle KATP channels.

| S-EPMC6540933 | biostudies-literature

ATP induces guinea pig gallbladder smooth muscle excitability via the P2Y4 receptor and COX-1 activity.

Project description:The purpose of this study was to elucidate the mechanisms by which ATP increases guinea pig gallbladder smooth muscle (GBSM) excitability. We evaluated changes in membrane potential and action potential (AP) frequency in GBSM by use of intracellular recording. Application of ATP (100 microM) caused membrane depolarization and a significant increase in AP frequency that were not sensitive to block by tetrodotoxin (0.5 microM). The nonselective P2 antagonist, suramin (100 microM), blocked the excitatory response, resulting in decreased AP frequency in the presence of ATP. The excitatory response to ATP was not altered by pyridoxal-phosphate-6-azophenyl-2,4-disulfonic acid (30 microM), a nonselective P2X antagonist. UTP also caused membrane depolarization and increased AP frequency, with a similar dose-response relationship as ATP. RT-PCR demonstrated that the P2Y(4), but not P2Y(2), receptor subtype is expressed in guinea pig gallbladder muscularis. ATP induced excitation was blocked by indomethacin (10 microM) and the cyclooxygenase (COX)-1 inhibitor SC-560 (300 nM), but not the COX-2 inhibitor nimesulide (500 nM). These data suggest that ATP stimulates P2Y(4) receptors within the gallbladder muscularis and, in turn, stimulate prostanoid production via COX-1 leading to increased excitability of GBSM.

| S-EPMC2921626 | biostudies-literature

TRPML1 channels initiate Ca2+ sparks in vascular smooth muscle cells.

Project description:TRPML1 (transient receptor potential mucolipin 1) is a Ca2+-permeable, nonselective cation channel localized to the membranes of endosomes and lysosomes and is not present or functional on the plasma membrane. Ca2+ released from endosomes and lysosomes into the cytosol through TRPML1 channels is vital for trafficking, acidification, and other basic functions of these organelles. Here, we investigated the function of TRPML1 channels in fully differentiated contractile vascular smooth muscle cells (SMCs). In live-cell confocal imaging studies, we found that most endosomes and lysosomes in freshly isolated SMCs from cerebral arteries were essentially immobile. Using nanoscale super-resolution microscopy, we found that TRPML1 channels present in late endosomes and lysosomes formed stable complexes with type 2 ryanodine receptors (RyR2) on the sarcoplasmic reticulum (SR). Spontaneous Ca2+ signals resulting from the release of SR Ca2+ through RyR2s ("Ca2+ sparks") and corresponding Ca2+-activated K+ channel activity are critically important for balancing vasoconstriction. We found that these signals were essentially absent in SMCs from TRPML1-knockout (Mcoln1-/- ) mice. Using ex vivo pressure myography, we found that loss of this critical signaling cascade exaggerated the vasoconstrictor responses of cerebral and mesenteric resistance arteries. In vivo radiotelemetry studies showed that Mcoln1-/- mice were spontaneously hypertensive. We conclude that TRPML1 is crucial for the initiation of Ca2+ sparks in SMCs and the regulation of vascular contractility and blood pressure.

| S-EPMC7397860 | biostudies-literature

Modulation by simvastatin of iberiotoxin-sensitive, Ca2+-activated K+ channels of porcine coronary artery smooth muscle cells.

Project description:Statins (3-hydroxy-3-methyl-glutaryl coenzyme A (HMG CoA) reductase inhibitors) have been demonstrated to reduce cardiovascular mortality. It is unclear how the expression level of HMG CoA reductase in cardiovascular tissues compares with that in cells derived from the liver. We hypothesized that this enzyme exists in different cardiovascular tissues, and simvastatin modulates the vascular iberiotoxin-sensitive Ca2+-activated K(+) (BK(Ca)) channels.Expression of HMG CoA reductase in different cardiovascular preparations was measured. Effects of simvastatin on BK(Ca) channel gatings of porcine coronary artery smooth muscle cells were evaluated.Western immunoblots revealed the biochemical existence of HMG CoA reductase in human cardiovascular tissues and porcine coronary artery. In porcine coronary artery smooth muscle cells, extracellular simvastatin (1, 3 and 10 microM) (hydrophobic), but not simvastatin Na+ (hydrophilic), inhibited the BK(Ca) channels with a minimal recovery upon washout. Isopimaric acid (10 microM)-mediated enhancement of the BK(Ca) amplitude was reversed by external simvastatin. Simvastatin Na+ (10 microM, applied internally), markedly attenuated isopimaric acid (10 microM)-induced enhancement of the BK(Ca) amplitude. Reduced glutathione (5 mM; in the pipette solution) abolished simvastatin -elicited inhibition. Mevalonolactone (500 microM) and geranylgeranyl pyrophosphate (20 microM) only prevented simvastatin (1 and 3 microM)-induced responses. simvastatin (10 microM ) caused a rottlerin (1 microM)-sensitive (cycloheximide (10 microM)-insensitive) increase of PKC-delta protein expression.Our results demonstrated the biochemical presence of HMG CoA reductase in different cardiovascular tissues, and that simvastatin inhibited the BK(Ca) channels of the arterial smooth muscle cells through multiple intracellular pathways.

| S-EPMC2042927 | biostudies-other

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