Inhibition of the cardiac L-type calcium channel current by the TRPM8 agonist, (-)-menthol.

(-)-Menthol and icilin are agonists of the thermoreceptor non-selective cation channel, TRPM8, and are commonly used to investigate TRPM8 function without a full appreciation of their non-specific effects. To investigate the hypothesis that (-)-menthol and icilin inhibit cardiovascular-type L-type Ca(2+) channel currents (I(Ca,L)), the actions of the TRPM8 agonists on rabbit ventricular myocyte I(Ca,L) were examined at near-physiological temperature (≈35°C) using whole-cell recording. Icilin (3-100 µM) did not significantly inhibit I(Ca,L). (3) in contrast, (-)-menthol concentration-dependently inhibited peak I(Ca,L) (IC(50)=74.6 µM; log(10)IC(50)(M)=-4.13±0.14). (-)-Menthol blocked the late I(Ca,L) remaining at the end of depolarising pulses with greater efficacy (96.1±2.4% block at 1 mM) than peak I(Ca,L) (68.9±5.7% block at 1 mM, P<0.01), although there was no difference in potency of block of peak and late currents. Block by (-)-menthol showed no voltage-dependence. The actions of (-)-menthol were compared with those of nimodipine. Nimodipine was a more efficacious (97.3±1.5 % block at 30 µM, P<0.01) and potent (IC(50)=0.74 µM; log(10)IC(50)(M)=-6.13±0.08, P<0.0001) blocker of peak I(Ca,L) than (-)-menthol. In contrast to (-)-menthol, nimodipine showed greater potency (IC(50)=0.056 µM; log(10)IC(50)(M)=-7.25±0.17, P<0.0001), but not greater efficacy, in block of late compared with peak I(Ca,L). In summary, these data demonstrate that, at near-physiological temperature, (-) -menthol blocks cardiac I(Ca,L) at concentrations similar to those reportedly effective in TRPM8-agonism. The data suggest that the mechanism of L-type Ca(2+) channel block by (-)-menthol differs from that of nimodipine.

Measurement of chloride flux associated with the myogenic response in rat cerebral arteries.

1. Self-referencing ion-selective (SERIS) electrodes were used to measure the temperature and pressure dependence of Cl(-) efflux, during myogenic contraction of pressurized rat cerebral resistance arteries. 2. At room temperature (18-21 degrees C), a small, pressure-independent Cl(-) efflux was measured. On warming to 37 degrees C, arteries developed pressure-dependent myogenic tone, and this was associated with a pressure-dependent increase in Cl(-) efflux (n = 5). 3. Both myogenic tone and the pressure- and temperature-dependent Cl(-) efflux were abolished on application of 10 microM tamoxifen, a Cl(-) channel blocker (IC(50) 3.75 +/- 0.2 microM). Tamoxifen (10 microM) also prevented contraction to 60 mM K(+), suggesting non-specific effects of tamoxifen (n = 5). 4. Myogenic tone was abolished by 2 microM nimodipine, but Cl(-) efflux was unaffected. In the presence of nimodipine, 10 microM tamoxifen still abolished pressure- and temperature-dependent Cl(-) efflux (n = 3). 5. In summary, a Cl(-) efflux can be measured from rat cerebral arteries, with a temperature dependence that is closely correlated with myogenic contraction. We conclude that Cl(-) efflux through Cl(-) channels contributes to the depolarization associated with myogenic contraction.

Blockade of chloride channels reveals relaxations of rat small mesenteric arteries to raised potassium.

1. Raised extracellular K(+) relaxes some arteries, and has been proposed as Endothelium-Derived Hyperpolarizing Factor (EDHF). However, relaxation of rat small mesenteric arteries to K(+) is highly variable. We have investigated the mechanism of K(+)-induced dilatation and relaxation of pressurized arteries and arteries mounted for measurement of isometric force. 2. Raising [K(+)](o) from 5.88 - 10.58 mM did not dilate or relax pressurized or isometric arteries. Relaxation to raised [K(+)](o) was revealed in the presence of 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB); this effect of NPPB was concentration-dependent (IC(50): 1.16 microM). 3. Relaxations to raised [K(+)](o) in the presence of NPPB, were abolished by 30 microM Ba(2+) or endothelial-denudation. Acetycholine (10 microM) relaxed endothelium-intact arteries in presence of raised [K(+)](o) NPPB and Ba(2+). 4. Relaxations to raised [K(+)](o) were revealed in hyperosmotic superfusate (+60 mM sucrose). These relaxations were abolished by 30 microM Ba(2+). In the presence of raised [K(+)](o), 60 mM sucrose and 30 microM Ba(2+), 10 microM acetycholine still relaxed all arteries. 5. Fifty microM 18 alpha-glycyrrhetinic acid (18 alpha-GA), a gap junction inhibitor, depressed relaxations to both 10 microM acetylcholine and raised [K(+)](o), in the presence of 10 microM NPPB. 6. In summary, blockade of a volume-sensitive Cl(-) conductance in small rat mesenteric arteries, using NPPB or hyperosmotic superfusion, reveals a endothelium-dependent, Ba(2+) sensitive dilatation or relaxation of rat mesenteric arteries to raised [K(+)](o). We conclude that inwardly rectifying potassium channels on the endothelium underlie relaxations to raised [K(+)](o) in rat small mesenteric arteries.

Potassium does not mimic EDHF in rat mesenteric arteries.

1. K(+) has been proposed to be EDHF in small arteries. We compared ACh-stimulated, EDHF-mediated dilatation/relaxation with raised [K(+)](o) in rat mesenteric arteries. 2. In pressurized arteries, ACh (10 microM) dilated all arteries. Raising [K(+)](o) from 5.88 to 10. 58 mM only dilated 30% of arteries. Ba(2+) (30 microM) did not affect dilatation to ACh, but abolished 40% of dilatations to raised [K(+)](o). 3. If [K(+)](o) was lowered to 1.18 mM, restoring [K(+)](o) to 5.88 mM produced dilatation which was depressed by Ba(2+) or ouabain (1 mM). Combined application of Ba(2+) and ouabain abolished dilatation. In 1.18 mM K(+), dilatation to ACh was depressed by ouabain, but not by Ba(2+). Combined application of Ba(2+) and ouabain depressed dilatation further. Gap junction inhibitors (Gap-27; 300 microM and 18-alpha-glycyrrhetinic acid; 100 microM) also depressed dilatation to ACh. 4. In arteries mounted isometrically, ACh (1 microM) relaxed endothelium intact (+E), but not endothelium denuded (-E) arteries. Raising [K(+)](o) from 5.9 - 10.9 mM failed to relax all arteries. When [K(+)](o) was lowered to 1 mM, raising [K(+)](o) to 6 mM produced relaxation. In -E arteries, relaxation was unaffected by Ba(2+) but abolished by ouabain. In +E arteries, Ba(2+) depressed and ouabain abolished relaxation. In +E arteries, with 1 mM K(+), ACh relaxation was depressed by ouabain but not Ba(2+). The combined application of Ba(2+) and ouabain further depressed relaxation. 5. In summary, both EDHF and raised [K(+)](o) dilate/relax rat mesenteric arteries, though sensitivities to barium and ouabain differ. K(+) may be a relaxing factor in this tissue, but its characteristics differ from EDHF. Gap junction inhibitors depress EDHF, implying an important role for myo-endothelial gap junctions.

Charybdotoxin and apamin block EDHF in rat mesenteric artery if selectively applied to the endothelium.

In rat mesenteric artery, endothelium-derived hyperpolarizing factor (EDHF) is blocked by a combination of apamin and charybdotoxin (ChTX). The site of action of these toxins has not been established. We compared the effects of ChTX and apamin applied selectively to the endothelium and to the smooth muscle. In isometrically mounted arteries, ACh (0.01-10 micrometers), in the presence of indomethacin (2.8 microM) and Nomega-nitro-L-arginine methyl ester (L-NAME) (100 microM), concentration dependently relaxed phenylephrine (PE)-stimulated tone (EC50 50 nM; n = 10). Apamin (50 nM) and ChTX (50 nM) abolished this relaxation (n = 5). In pressurized arteries, ACh (10 microM), applied intraluminally in the presence of indomethacin (2.8 microM) and L-NAME (100 microM), dilated both PE-stimulated (0.3-0.5 microM; n = 5) and myogenic tone (n = 3). Apamin (50 nM ) and ChTX (50 nM) applied intraluminally abolished ACh-induced dilatations. Bath superperfusion of apamin and ChTX did not affect ACh-induced dilatations of either PE-stimulated (n = 5) or myogenic tone (n = 3). This is the first demonstration that ChTX and apamin act selectively on the endothelium to block EDHF-mediated relaxation.

Streptomycin inhibition of myogenic tone, K+-induced force and block of L-type calcium current in rat cerebral arteries.

1. Streptomycin has been demonstrated to inhibit mechanosensitive conductances in a wide variety of cell types, including muscle. The action of streptomycin on rat cerebral arteries that exhibit pressure-induced myogenic response was investigated. 2. Pressure-induced tone, measured using isobaric myography, in isolated pressurized cerebral arteries was reversibly and concentration-dependently inhibited by streptomycin with an IC50 of 2.6 mM. 3. Isometric K+-induced force, measured using isometric myography, is supported by voltage-gated Ca2+ entry. Streptomycin reversibly and concentration-dependently inhibited isometric force with an IC50 of 1.71 mM. 4. Voltage-gated macroscopic inward Ca2+ channel currents were recorded from freshly isolated rat basilar myocytes. These were reversibly and concentration-dependently inhibited by streptomycin with an IC50 of 1.79 and 0.47 mM when 10 and 1.8 mM CaCl2, respectively, was used as the charge carrier. 5. These data suggest that streptomycin inhibits myogenic tone and K+-induced isometric force largely by blockade of L-type, dihydropyridine-sensitive Ca2+ channels. In conclusion, streptomycin is not useful in the investigation of stretch-activated channels which may underlie the myogenic response of rat small cerebral arteries.

A transient dilatation of pressurised rat cerebral arteries during rapid pressure increases is mediated by nitric oxide.

In pressurised resistance arteries in vitro, rapid pressure increases cause a transient "peak" dilatation, followed by a myogenic constriction. The mechanism of transient dilatation was investigated in isolated rat cerebral arteries in vitro using pressure myography. The peak increased with the amplitude of the pressure step. A near-maximal dilatation to 118+/-1.6% (SEM, n=20) of the diameter at 30 mmHg was produced by pressure steps from 30 to 75 mmHg. Nomega-nitro-l-arginine methyl ester (L-NAME, 20 microM) depressed the peak at the onset of a 30 to 75 mmHg pressure step to 49.8+/-14% of the control (n=6; P=0.04). D-NAME (20 microM) had no significant effect (82.1+/-13%; n=4; P=0.13). L-Arginine (400 microM) enhanced the peak (164+/-17% of control; n=8; P=0.01). Oxadiazolol (4,3-a) quinoxalin-1-one (ODQ, 2 microM) depressed the peak to 33.2+/-12% of control (n=5; P=0.012). 6-Anilino-5, 8-quinolinedone (LY 83583, 10 microM) depressed the peak to 18.8+/-2. 9% of control (n=3; P=0.01). Removing the endothelium decreased the peak to 15.3+/-11% of control (n=3; P=0.04). In conclusion, in rat cerebral arteries, the initial dilatation at the onset of a rapid step increase in pressure is an active dilatation involving endothelial NO release.

Non-specificity of chloride channel blockers in rat cerebral arteries: block of the L-type calcium channel.

1. The effects of chloride channel blockers on pressure-induced constriction, K(+)-induced force, and whole-cell calcium channel currents were tested in rat cerebral arteries using isobaric and isometric myography, and patch clamp. 2. Under isobaric conditions at 75 mmHg, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), a chloride channel blocker, reversibly depressed the myogenic constriction with an IC50 of 32.8 +/- 0.52 microM (mean +/- S.E.M., n = 5). Blockers of Ca(2+)-activated chloride channels, flufenamic acid (100 microM) and 9-anthracene chloride (9-AC; 1 mM), and the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel blocker, glibenclamide (100 microM), were without effect in this tissue (n = 3). 3. Under isobaric conditions at 20 mmHg, 37 degrees C, raising [K+]o to 45 mM induced a constriction which was unaffected by 100 microM NPPB (n = 4). In contrast, at 75 mmHg and 18-21 degrees C, 100 microM NPPB completely and reversibly blocked a 45 mM K(+)-induced constriction (n = 3). 4. Under isometric conditions, NPPB reversibly depressed a 45 mM K(+)-induced force with an IC50 of 10.0 +/- 0.76 microM (mean +/- S.E.M., n = 5). Indanyloxyacetic acid 94 (IAA-94), another chloride channel blocker, depressed the K(+)-induced force with an IC50 of 17.0 +/- 1.2 microM (mean +/- S.E.M., n = 4). 5. Using whole-cell patch clamp, 100 microM NPPB or 200 microM IAA-94 blocked calcium channel currents carried by 10 mM Ba2+ by 79.1 +/- 1.7 and 39.8 +/- 7.0%, respectively (mean +/- S.E.M., n = 6). 6. In summary, chloride channel blockers depress calcium channel currents in rat cerebral arteries, which could contribute to a reduction in myogenic contraction.

Myogenic contraction by modulation of voltage-dependent calcium currents in isolated rat cerebral arteries.

1. Tissue blood flow and blood pressure are regulated by the spontaneous, myogenic, contraction developed by resistance arteries. However, the cellular mechanisms underlying myogenic contraction are not understood. In this study, the mechanisms of myogenic contraction in cerebral resistance arteries were investigated. 2. The vasoconstriction observed in response to increased pressure in cerebral resistance arteries (myogenic reactivity) was dependent on Ca2+ entry through voltage-dependent Ca2+ channels, since it was abolished by Ca2+ removal and by dihydropyridine antagonists of voltage-dependent Ca2+ channels. 3. Myogenic reactivity persisted in a high-K+ saline, with reduced Ca2+, where membrane potential is presumed to be clamped. Therefore, membrane depolarization alone does not fully account for the increased voltage-dependent Ca2+ channel opening. 4. Voltage-dependent Ca2+ currents in single smooth muscle cells isolated from the resistance artery were substantially increased by applying positive pressure to the patch electrode evoking membrane stretch. 5. Myogenic reactivity remained unaffected by ryanodine and therefore was independent of internal ryanodine-sensitive Ca2+ stores. 6. The myofilament Ca2+ sensitivity was not increased by elevated pressure in alpha-toxin-permeabilized arteries. However, pharmacological activation of protein kinase C or G proteins did increase the myofilament Ca2+ sensitivity. 7. Myogenic contraction over the pressure range 30-70 mmHg could be accounted for by an increase in [Ca2+]i from 100 to 200 nM. 8. It is concluded that modest increases in [Ca2+]i within the range 100-200 nM can account for that myogenic contraction, and that stretch-evoked modulation of Ca2+ currents may contribute to the myogenic response.

Neomycin inhibits K+-induced force and Ca2+ channel current in rat arterial smooth muscle.

The techniques of small vessel isometric myography and patch clamp were used to investigate the action of neomycin on K+-induced isometric force and voltage-gated Ca2+ channel currents in rat arterial smooth muscle. Neomycin and the dihydropyridine (DHP) Ca2+ channel antagonist (-)202-791 concentration-dependently and reversibly inhibited 40 mM K+-induced isometric force in rings of rat mesenteric and basilar arteries (IC50 values of 70 microM and 1. 2 nM, respectively, n = 10 and 4). Elevation of [Ca2+]o by a factor of 2 significantly reduced the IC50 values for inhibition of K+-induced force for both neomycin and (-)202-791 (192 microM and 3. 7 nM, respectively, n = 6 and 4), but did not affect the Hill coefficient of the concentration/effect relationships. In patch-clamp experiments using freshly isolated basilar arterial myocytes, the voltage-gated inward current carried by Ba2+ was reversibly and concentration-dependently inhibited by neomycin (IC50 32 microM, n = 3). The concentration/effect curve for inhibition of the inward Ba2+ current by neomycin was significantly shifted to the right when [Ba2+]o was raised from 1.8 mM to 10 mM (IC50 144 microM, n = 8). Our findings suggest that neomycin relaxes high-K+-induced force in rat isolated mesenteric and basilar arteries largely by inhibition of voltage-dependent and DHP-sensitive Ca2+ channels.

Effects of the BKCa channel activator, NS1619, on rat cerebral artery smooth muscle.

1. We have investigated the actions of NS1619, a putative activator of large conductance calcium-activated potassium channels (BKCa) by use of the patch-clamp technique on smooth muscle cells enzymatically isolated from the rat basilar artery. 2. Using whole cell current-clamp to measure membrane potential, addition of 30 microM NS1619 produced cellular hyperpolarization, moving the membrane potential towards the calculated equilibrium potential for potassium. This hyperpolarization was rapidly reversed by IbTX (100 nM), a selective inhibitor of BKCa. 3. In whole cell recordings made from cells voltage-clamped at 0 mV using the perforated-patch technique, addition of NS1619 (10-30 microM) activated an outward current, which reversed following washout of NS1619. 4. This outward current was unaffected by application of either glibenclamide (5 microM), an inhibitor of ATP-sensitive potassium channels, or apamin (100 nM), an inhibitor of small-conductance calcium-activated potassium channels. However, this current was almost completely abolished by iberiotoxin (IbTX; 50-100nM). 5. Depolarizing voltage steps activated small outward currents from cells held at -15 mV. Application of NS1619 (10-30 microM) increased the size of these currents, producing a shift to the left of the current-voltage (I-V) relationship. These currents were largely inhibited by IbTX (100 nM). 6. Measurements of the unitary amplitude of the single channels activated by NS1619 which could be resolved in whole cell recordings yielded a value of 5.6 +/- 0.14 pA at 0 mV. 7. NS1619 (10-30 microM) directly activated single channels contained in excised inside-out and outside-out membrane patches. In both configurations NS1619 (10-30 microM) rapidly increased the open probability of a large conductance calcium-dependent channel. The activation produced by NS1619 was calcium-dependent and inhibited by external IbTX (100 nM). The unitary current amplitude was unaffected by NS1619. 8. By use of conventional whole cell recording methods and conditions that suppressed BKCa openings, outward potassium currents were activated by depolarizing potentials positive to -35 mV from a holding potential of -65 mV. NS1619 (10-30 microM) inhibited this current in a concentration-dependent manner. This inhibition was reversed following washout of NS1619, recovering to 60-90% of control values within 2 min. 9. Ba2+ currents, measured by conventional whole cell recording, were activated by depolarizing voltage steps from negative holding potentials. NS1619 (1-30 microM) inhibited the evoked current in a concentration-dependent manner, yielding an IC50 value of 7 microM with a Hill coefficient approaching unity. This inhibition was reversible, with the currents recovering to 65-100% of control values after washout of NS1619 for 2 min. 10. NS1619 (0.3-100 microM) induced concentration-dependent relaxation of basilar artery segments contracted with histamine/5-HT (IC50 = 12.5 +/- 2.0 microM; n = 4). This relaxation curve was shifted to the right, but not abolished, when the tissue was treated with a blocker of BKCa channels (IbTX; 100nM). Additionally, NS1619 produced concentration-dependent relaxation of basilar artery contracted with a depolarizing, isotonic salt solution containing 80 mM K+. 11. Thus NS1619 produces hyperpolarization of basilar artery myocytes through direct activation of BKCa and also directly inhibits Ca2+ currents and voltage-activated K+ channels. We discuss the implications of these results for its vasorelaxant actions.

Properties of the ATP-sensitive K+ current activated by levcromakalim in isolated pulmonary arterial myocytes.

Tension and patch clamp recording techniques were used to investigate the relaxation of rabbit pulmonary artery and the properties of the K+ current activated by levcromakalim in isolated myocytes. Under whole-cell voltage clamp, holding at -60 mV in symmetrical 139 mM K+, levcromakalim (10 microM) induced a noisy inward current of -116 +/- 19 pA (n = 13) which developed over 1 to 2 min. This current could be blocked by either glibenclamide (10 microM) or phencyclidine (5-50 microM) and was unaffected when extracellular Ca2+ was removed. Both these drugs inhibited the levcromakalim-induced relaxation of muscle strips precontracted with 20 mM [K+]o. Application of voltage ramps in symmetrical 139 mM K+ confirmed that the levcromakalim-induced current was carried by K+ ions and was weakly voltage dependent over the potential range from -100 to +40 mV. The unitary current amplitude and density of the channels underlying the levcromakalim-activated whole-cell K+ current was estimated from the noise in the current record. We estimate that levcromakalim caused activation of around 300 channels per cell, with a single channel current of 1.1 pA, corresponding to a slope conductance of about 19 pS. Furthermore, cells dialyzed with an ATP-free pipette solution developed a large noisy inward current at -60 mV, which could subsequently be blocked by flash photolysis of caged ATP. Analysis of the noise associated with this current indicated that the single channel amplitude underlying the ATP-blocked current was 1.4 pA, a value similar to that estimated for the levcromakalim-induced current.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium channel currents recorded from isolated myocytes of rat basilar artery are stretch sensitive.

1. Conventional and perforated patch whole-cell methods were used to measure Ca2+ channel currents from isolated rat basilar arterial myocytes in response to voltage steps. Peak currents measured in response to repeated steps to +2 mV increased over the first 3 min after gaining whole-cell access. The increase followed an exponential time course, was not due to decrease in underlying outward current and was independent of the presence of ATP and GTP in the pipette filling solution. 2. Application of positive and negative pressure to the pipette resulted in significant increase and decrease in peak inward current, respectively. Membrane stretch, associated with changes in cell volume, rather than applied pressure per se appeared to determine changes in peak current. 3. Using amphotericin B perforated patch recording, changes in cell volume on application of 80% hypo- or 120% hyperosmotic superfusing solutions were found to effect similarly peak inward current. Hyposmotically induced cell swelling increased and hyperosmotic cell shrinkage decreased peak inward current at all test potentials studied. 4. The increase in inward current in response to hyposmotic superfusate was reversible and appears to reflect an increase in voltage-dependent Ca2+ channel current since it was not due to a change in non-voltage-sensitive conductance(s) and it was as sensitive as the control current to the dihydropyridine (DHP) antagonist (-)202-791.

Calcium currents elicited by voltage steps and steady voltages in myocytes isolated from the rat basilar artery.

1. Whole-cell patch clamp methods were used to record Ca2+ channel currents from isolated rat basilar arterial myocytes either in response to voltage steps or at steady holding potentials. Inward currents were rapidly and reversibly blocked by 2 mM Co2+, and this solution was routinely used to subtract leakage currents. 2. Peak currents measured in response to voltage steps were -85 +/- 23 pA (n = 7) in physiological Ca2+ (1.8 mM) and -256 +/- 39 pA (n = 19) in 10 mM Ba2+. The time course of activation and inactivation were unaffected by changing the holding potential from -88 to -48 mV, and thus we found no evidence for a component of current flowing through transient Ca2+ channels. 3. Activation curves were constructed by dividing the macroscopic current by the single Ca2+ channel current. The product of the number of functional channels and the open-state probability (NPo) measured in this way rose to around 2000. Its dependence on voltage was fitted by a Boltzmann function with half-activation at -8.6 mV in physiological Ca2+ concentrations and a steepness factor in the range 6-8 mV. 4. In physiological solution, block by external Mg2+ reduced peak current through Ca2+ channels by 17%. 5. Steady-state currents were measured by holding the cell at a fixed voltage and rapidly applying Co2+ to block the current through Ca2+ channels. Steady-state currents could be detected at voltages as negative as -58 mV, and persisted for more than 15 min, suggesting that Ca2+ channels in this artery may provide a steady-state Ca2+ influx that contributes to resting contractile tone.

Block of calcium-activated potassium channels in mammalian arterial myocytes by tetraethylammonium ions.

The effects of tetraethylammonium ions (TEA+) and tetrapentylammonium ions (TPeA+) on Ca2(+) -activated K+ (KCa) channels were studied in membrane patches from mesenteric arterial myocytes. External TEA+ produced a flickery block. The concentration dependence for reduction in mean unitary current was consistent with 1:1 binding, with dissociation constants (Kd) in rat and rabbit of 196 and 159 microM at 0 mV, and the block was weakly voltage dependent. Rate constants for blocking and unblocking were 380 mM-1.ms-1 and 73 ms-1, respectively. In patches containing several channels TEA+ reduced average current to the same extent as mean unitary current, implying that TEA+ block is independent of the channel state. Block was unaffected by raising external K+ to 120 mM. External TPeA+ blocked with slower kinetics and lower affinity than TEA+ (Kd, 1.49 mM). The sulfonylurea glibenclamide (10-100 microM), the hyperpolarizing vasodilator cromakalim (5 microM), and internal ATP (1 mM) were without effect on channel activity. We conclude that TEA+ is a relatively effective blocker of single KCa channels of arterial smooth muscle and should block macroscopic currents equally well, whereas external TPeA+ is about eight times less effective.

Participation of Ca currents in colonic electrical activity.

Canine colonic myocytes were studied with the whole cell patch-clamp technique. In 1.8 mM Ca2+, inward currents were evoked by depolarization. Currents activated positive to -50 mV, peaked at approximately 0 mV, and reversed at approximately +50 mV. Inward current was potentiated by high external Ca2+ concentration and BAY K8644 and was decreased by low external Ca2+, nifedipine, and Mn2+, indicating that the current was carried by Ca2+. Overlap of the activation-inactivation properties indicated a "window current" range (-40 to -20 mV) in which inward current might be sustained for long durations at potentials achieved during electrical slow waves. Voltage-clamp protocols simulating physiological depolarizations elicited sustained inward currents. Maximum changes in intracellular Ca2+ resulting from sustained inward currents were calculated, which suggested that depolarizations at the level of slow waves may increase cell Ca2+ sufficiently to cause contraction. The data suggest that electrical slow waves in colonic myocytes are due in part to inward Ca current. This current appears to be sufficient to explain the relationship between slow waves and contractions and provides an explanation for the mechanical threshold in colonic muscles.

Voltage and time dependency of calcium mediated phasic and tonic responses in rat vas deferens smooth muscle--the effect of some calcium agonist and antagonist agents.

1. Phasic and tonic components of the K+-induced contracture response were found to be expressed to different degrees in the prostatic and epididymal portions of the rat vas deferens. 2. With elevation of external potassium greater than 25 mM, the mechanisms underlying the phasic component operate only transiently before inactivation and replacement with tonic tension. 3. Both phasic and tonic components of the vas deferens response to potassium were markedly dependent upon external calcium ions. 4. Nifedipine and verapamil equally inhibited the phasic and tonic components of the K+ response in the prostatic vas deferens. However, inhibition by these agents was far more pronounced in the phasic components than in the tonic component of the epididymal vas deferens. 5. BAY K 8644 potentiated, in a dose-dependent manner, the phasic components of the K+ response, particularly in the prostatic vas deferens. 6. Abscisic acid also potentiated, in a dose-dependent manner the K+ response of rat vas deferens, but this action was far more pronounced in the tonic component of the epididymal portion. 7. Papaverine abolished the BAY K 8644 potentiated epididymal K+ response but did not affect the BAY K 8644 potentiated prostatic K+ response. 8. It is concluded that abscisic acid potentiated responses associated with the activation of voltage-dependent, non-inactivating slow calcium channels. 9. Papaverine, nifedipine and verapamil appear to be less selective than abscisic acid in that they equally inhibit phasic and tonic responses in prostatic vas deferens, but these may be interdependent, yet they more strongly inhibit phasic responses of epididymal vas deferens.

The involvement of fast calcium channel activity in the selective activation of phasic contractions by partial depolarization in rat vas deferens smooth muscle.

In the prostatic portion of the rat vas deferens, 65% of the preparations studied developed pronounced rapid twitch activity in response to slight depolarization by 15 mM K+ salines. The mechanism underlying this response was studied using treatments designed to inhibit the influence of endogenous transmitters and using recognized calcium antagonist drugs. Although the action of phentolamine was inconclusive, experiments employing guanethidine, reserpine, 6-hydroxydopamine, atropine and alpha,beta-methylene ATP suggest that endogenous transmitter release was not responsible for the observed twitch activity. Twitch activity was strongly dependent upon [Ca]0. The 15 mM K+ twitch activity was inhibited by verapamil (5 X 10(-5) M) but was resistant to 10(-3) M lanthanum. Twitch activity was, however, abolished by 10(-3) M Mn2+ ions and was markedly potentiated by 2 X 10(-3) M TEA. The rapid twitch activity exhibited a strong voltage-dependency, being abolished by [K]0 elevations of 25 mM and above. It is concluded that this phasic activity of the vas deferens smooth muscle may depend upon fast calcium channel activity which, in contrast to voltage-dependent slow calcium channel activity, shows ready voltage-inactivation on substantial depolarization.

Inhibition by papaverine of calcium movements and tension in the smooth muscles of rat vas deferens and urinary bladder.

Papaverine, up to 150 micron, was without effect on resting tension of vas deferens and urinary bladder smooth muscle strips, but caused a dose-dependent inhibition of the phasic component of the 100 mM-K contracture. At 150 micron, papaverine caused an 80 and 60% inhibition of phasic tension in vas deferens and urinary bladder muscle respectively. Papaverine caused a marked dose-dependent reduction in the tonic component of 100 mM-K contractures in both preparations. At 150 micron, papaverine often relaxed preparations below initial starting tension. Addition of 150 micron-papaverine to incubation media caused a fall of 25-30% in 45Ca uptake by both preparations. Papaverine at 150 micron caused a small fall in resting 45Ca efflux from vas deferens and a larger fall in efflux from bladder muscle. However, this papaverine concentration caused a large inhibition of the 45Ca uptake by and 45Ca efflux from both preparations when stimulated by high-K depolarization. Papaverine at 150 micron was without significant effect on Ca binding by either microsomes or mitochondria isolated from both vas deferens and urinary bladder smooth muscle. The main site of papaverine action appears to be on the Ca-influx mechanism responsible for regulating Ca entry during prolonged K depolarization.