Tetraethylammonium-evoked oscillatory contractions of rat tail artery: a K-K model

Spontaneously rhythmic contraction of peripheral blood vessels actively modulates the peripheral circulation and blood pressure. However, the underlying mechanisms for the complex rhythmic contraction patterns of various vascular tissues are not yet fully understood. In the present study, the tetraethylammonium (TEA)-induced spontaneously oscillatory contractions of isolated rat tail artery tissues were examined. It was found that TEA evoked arterial oscillatory contractions in a concentration-dependent, but endothelium-independent manner. The voltage-dependent K+ (Kv) channel specific blocker, 4-aminopyridine (4-AP), induced a sustained, but not oscillated, vascular contraction. The presence of 4-AP had no effect on the TEA-induced oscillatory contractions. The blockade of KCa channels with charybdotoxin or apamin did not affect the basal force of vascular tissues. Neither the TEA-induced oscillatory contraction was affected by these blockers. The opening of KATP channels by levcromakalim or their blockade by glybenclamide ceased or increased, respectively, the oscillation of TEA-induced contractions. The absence of Ca2+ or the presence of nifedipine in the bath solution completely abolished the effects of TEA. The inhibition of Ca2+-ATPase in the sarcoplasmic reticulum with micromolar concentrations of thapsigargin or cyclopiazonic acid either abolished or enhanced, respectively, the TEA-induced oscillatory contractions. Ryanodine did not affect the TEA-induced oscillatory contraction. In conclusion, the TEA-induced oscillatory contraction may be initiated by the blockade of the TEA-sensitive delayed rectifier K+ channels and maintained by the TEA-insensitive but ATP-sensitive K+ channels. This K-K model presents a novel mechanism for the depolarization-induced rhythmic contractions of small arteries.     

Opposing roles of K(+) and Cl(-) channels in maintenance of opossum lower esophageal sphincter tone

The ionic basis underlying the maintenance of myogenic tone of lower esophageal sphincter circular muscle (LES) was investigated in opossum with the use of standard isometric tension and conventional intracellular microelectrode recordings in vitro. In tension recording studies, nifedipine (1 microM) reduced basal tone to 27.7 +/- 3.8% of control. The K(+) channel blockers tetraethylammonium (TEA, 2 mM), charybdotoxin (100 nM), and 4-aminopyridine (4-AP, 2 mM) enhanced resting tone, whereas apamin and glibenclamide were without affect. Cl(-) channel blockers DIDS (500 microM) and 5-nitro-2-(3-phenylpropylamino)-benzoic acid (500 microM), as well as niflumic acid (0.1-300 microM), decreased basal tone, but tamoxifen was without effect. Intracellular microelectrode recordings revealed ongoing, spontaneous, spike-like action potentials (APs). Nifedipine abolished APs and depolarized resting membrane potential (RMP). Both TEA and 4-AP significantly depolarized RMP and augmented APs, whereas niflumic acid dose-dependently hyperpolarized RMP and abolished APs. These data suggest that, in the opossum, basal tone is associated with continuous APs and that K(+) and Ca(2+)-activated Cl(-) channels have important opposing roles in the genesis of LES tone.     

Quinine inhibits Ca2+-independent K+ channels whereas tetraethylammonium inhibits Ca2+-activated K+ channels in insulin-secreting cells

The effects of quinine and tetraethylammonium (TEA) on single-channel K+ currents recorded from excised membrane patches of the insulin-secreting cell line RINm5F were investigated. When 100 microM quinine was applied to the external membrane surface K+ current flow through inward rectifier channels was abolished, while a separate voltage-activated high-conductance K+ channel was not significantly affected. On the other hand, 2 mM TEA abolished current flow through voltage-activated high-conductance K+ channels without influencing the inward rectifier K+ channel. Quinine is therefore not a specific inhibitor of Ca2+-activated K+ channels, but instead a good blocker of the Ca2+-independent K+ inward rectifier channel whereas TEA specifically inhibits the high-conductance voltage-activated K+ channel which is also Ca2+-activated.     

L-type Ca2+ channels and K+ channels specifically modulate the frequency and amplitude of spontaneous Ca2+ oscillations and have distinct roles in prolactin release in GH3 cells

GH3 cells showed spontaneous rhythmic oscillations in intracellular calcium concentration ([Ca2+]i) and spontaneous prolactin release. The L-type Ca2+ channel inhibitor nimodipine reduced the frequency of Ca2+ oscillations at lower concentrations (100nM-1 microM), whereas at higher concentrations (10 microM), it completely abolished them. Ca2+ oscillations persisted following exposure to thapsigargin, indicating that inositol 1,4,5-trisphosphate-sensitive intracellular Ca2+ stores were not required for spontaneous activity. The K+ channel inhibitors Ba2+, Cs+, and tetraethylammonium (TEA) had distinct effects on different K+ currents, as well as on Ca2+ oscillations and prolactin release. Cs+ inhibited the inward rectifier K+ current (KIR) and increased the frequency of Ca2+ oscillations. TEA inhibited outward K+ currents activated at voltages above -40 mV (grouped within the category of Ca2+ and voltage-activated currents, KCa,V) and increased the amplitude of Ca2+ oscillations. Ba2+ inhibited both KIR and KCa,V and increased both the amplitude and the frequency of Ca2+ oscillations. Prolactin release was increased by Ba2+ and Cs+ but not by TEA. These results indicate that L-type Ca2+ channels and KIR channels modulate the frequency of Ca2+ oscillations and prolactin release, whereas TEA-sensitive KCa,V channels modulate the amplitude of Ca2+ oscillations without altering prolactin release. Differential regulation of these channels can produce frequency or amplitude modulation of calcium signaling that stimulates specific pituitary cell functions.     

Regulation of urinary bladder smooth muscle contractions by ryanodine receptors and BK and SK channels

This study examines the roles of voltage-dependent Ca(2+) channels (VDCC), ryanodine receptors (RyRs), large-conductance Ca(2+)-activated K(+) (BK) channels, and small-conductance Ca(2+)-activated K(+) (SK) channels in the regulation of phasic contractions of guinea pig urinary bladder smooth muscle (UBSM). Nisoldipine (100 nM), a dihydropyridine inhibitor of VDCC, abolished spontaneous UBSM contractions. Ryanodine (10 microM) increased contraction frequency and thereby integrated force and, in the presence of the SK blocker apamin, had a greater effect on integrated force than ryanodine alone. Blocking BK (iberiotoxin, 100 nM) or SK (apamin, 100 nM) channels increased contraction amplitude and duration but decreased frequency. The contractile response to iberiotoxin was more pronounced than to apamin. The increases in contraction amplitude and duration to apamin were substantially augmented with ryanodine pretreatment. These results indicate that BK and SK channels have prominent roles as negative feedback elements to limit UBSM contraction amplitude and duration. RyRs also appear to play a significant role as a negative feedback regulator of contraction frequency and duration, and this role is influenced by the activity of SK channels.     

Diversity of K+ channels in circular smooth muscle of opossum lower esophageal sphincter

We previously demonstrated that a balance of K+ and Ca2+-activated Cl- channel activity maintained the basal tone of circular smooth muscle of opossum lower esophageal sphincter (LES). In the current studies, the contribution of major K+ channels to the LES basal tone was investigated in circular smooth muscle of opossum LES in vitro. K+ channel activity was recorded in dispersed single cells at room temperature using patch-clamp recordings. Whole-cell patch-clamp recordings displayed an outward current beginning to activate at -60 mV by step test pulses lasting 400 ms (-120 mV to +100 mV) with increments of 20 mV from holding potential of -80 mV ([K+]I = 150 mM, [K+]o = 2.5 mM). However, no inward rectification was observed. The outward current peaked within 50 ms and showed little or no inactivation. It was significantly decreased by bath application of nifedipine, tetraethylammonium (TEA), 4-aminopyridine (4-AP), and iberiotoxin (IBTN). Further combination of TEA with 4-AP, nifedipine with 4-AP, and IBTN with TEA, or vice versa, blocked more than 90% of the outward current. Ca2+-sensitive single channels were recorded at asymetrical K+ gradients in cell-attached patch-clamp configurations (100.8+/-3.2 pS, n = 8). Open probability of the single channels recorded in inside-out patch-clamp configurations were greatly decreased by bath application of IBTN (100 nM) (Vh = -14.4+/-4.8 mV in control vs. 27.3+/-0.1 mV, n = 3, P < 0.05). These data suggest that large conductance Ca2+-activated K+ and delayed rectifier K+ channels contribute to the membrane potential, and thereby regulate the basal tone of opossum LES circular smooth muscle.     

Calcium-sensitive potassium channel inhibitors antagonize genistein- and daidzein-induced arterial relaxation in vitro

Estradiol-17beta relaxes rabbit coronary artery rings via large conductance Ca2+-activated K+-channels (K(Ca)). Genistein and daidzein are plant-derived estrogen-like compounds. The aim of the present study was to investigate whether potassium channels participate in the genistein- and daidzein-induced arterial relaxation like they do in the case of estradiol-17beta. Endothelium-denuded superior mesenteric arterial rings from non-pregnant Wistar female rats were used. At a concentration of 10 microM, estradiol-17beta, genistein and daidzein relaxed noradrenaline precontracted arterial rings, (58 +/- 4%, 45 +/- 5% and 31 +/- 3%, respectively; (n=6-8)). Genistein- and daidzein-induced relaxations were inhibited both by iberiotoxin (1-10 nM) and charybdotoxin (30 nM), the antagonists of large conductance Ca2+-activated K+-channels (K(Ca)). Estradiol-17beta-induced relaxation was reduced by iberiotoxin (30 nM). Estradiol-17beta- and daidzein-induced relaxations were also decreased by apamin (0.1-0.3 microM), an antagonist of small conductance Ca2+-activated K+-channels. The antagonists of voltage-dependent K+-channels (K(V)) (4-aminopyridine), ATP-sensitive K+-channels (K(ATP)) (glibenclamide), or inward rectifier K+-channels (KIR) (barium) had no effect on the relaxation responses of any of the compounds studied. Estrogen receptor antagonist tamoxifen did not inhibit the relaxations. In conclusion, in the noradrenaline precontracted rat mesenteric arteries, the relaxations caused by estradiol-17beta, genistein and daidzein were antagonized by large and small conductance K(Ca)-channel inhibitors, suggesting the role of these channels as one of the relaxation mechanisms.     

Voltage-gated potassium channels in brown fat cells

We studied the membrane currents of isolated cultured brown fat cells from neonatal rats using whole-cell and single-channel voltage-clamp recording. All brown fat cells that were recorded from had voltage-gated K currents as their predominant membrane current. No inward currents were seen in these experiments. The K currents of brown fat cells resemble the delayed rectifier currents of nerve and muscle cells. The channels were highly selective for K+, showing a 58-mV change in reversal potential for a 10-fold change in the external [K+]. Their selectivity was typical for K channels, with relative permeabilities of K+ greater than Rb+ greater than NH+4 much greater than Cs+, Na+. The K currents in brown adipocytes activated with a sigmoidal delay after depolarizations to membrane potentials positive to -50 mV. Activation was half maximal at a potential of -28 mV and did not require the presence of significant concentrations of internal calcium. Maximal voltage-activated K conductance averaged 20 nS in high external K+ solutions. The K currents inactivated slowly with sustained depolarization with time constants for the inactivation process on the order of hundreds of milliseconds to tens of seconds. The K channels had an average single-channel conductance of 9 pS and a channel density of approximately 1,000 channels/cell. The K current was blocked by tetraethylammonium or 4-aminopyridine with half maximal block occurring at concentrations of 1-2 mM for either blocker. K currents were unaffected by two blockers of Ca2+-activated K channels, charybdotoxin and apamin. Bath-applied norepinephrine did not affect the K currents or other membrane currents under our experimental conditions. These properties of the K channels indicate that they could produce an increase in the K+ permeability of the brown fat cell membrane during the depolarization that accompanies norepinephrine-stimulated thermogenesis, but that they do not contribute directly to the norepinephrine-induced depolarization.     

Characterization of outward K(+) currents in isolated smooth muscle cells from sheep urethra

The perforated-patch techniquewas used to measure membrane currents in smooth muscle cells from sheepurethra. Depolarizing pulses evoked large transient outward currentsand several components of sustained current. The transient current anda component of sustained current were blocked by iberiotoxin, penitremA, and nifedipine but were unaffected by apamin or 4-aminopyridine,suggesting that they were mediated by large-conductanceCa²⁺-activated K⁺ (BK) channels. When the BKcurrent was blocked by exposure to penitrem A (100 nM) andCa²⁺-free bath solution, there remained a voltage-sensitiveK⁺ current that was moderately sensitive to blockade withtetraethylammonium (TEA; half-maximal effective dose = 3.0 ± 0.8 mM) but not 4-aminopyridine. Penitrem A (100 nM) increasedthe spike amplitude and plateau potential in slow waves evoked insingle cells, whereas addition of TEA (10 mM) further increased theplateau potential and duration. In conclusion, bothCa²⁺-activated and voltage-dependent K⁺currents were found in urethral myocytes. Both of these currents arecapable of contributing to the slow wave in these cells, suggesting that they are likely to influence urethral tone under certain conditions.

     

Ca2+-activated K+ channel blockers induce PKC modulated oscillatory contractions in guinea pig trachea

Mechanisms underlying the Ca2+-activated K+ channel (K(Ca)) blockers-induced oscillatory contractions were investigated in guinea pig tracheal smooth muscle. The mean oscillatory frequencies induced by charybdotoxin (ChTX; 100 nM) and iberiotoxin (IbTX; 100 nM) were 9.8+/-0.8 (counts/h) and 8.0+/-1.3 (counts/h), respectively. Apamin (1 microM ), a blocker of SK(Ca), induced no contraction in guinea pig trachea and did not affect ChTX-induced oscillatory contractions. In Ca2+ free solution, no ChTX-induced contraction was observed. Nifedipine (100 nM), a blocker of voltage-dependent Ca2+ channels, and SK&F 96365 (10 microM), a blocker of capacitative Ca2+ entry, completely abolished ChTX-induced oscillatory contractions. Ryanodine (1 microM) decreased the amplitude, but increased the frequency of the oscillatory contractions. Thapsigargin (1 microM) changed contractions from the oscillatory type to the sustained type. Moreover, the protein kinase C (PKC) inhibitor, bisindolylamaleimide I (1 microM), decreased the amplitude and frequency, but PKC activator, phorbol 12-myristate 13-acetate (1 microM), increased the frequency of oscillatory contractions. These results suggest that K(Ca) inhibitors-induced oscillatory contractions are initiated by Ca2+ influx through L-type voltage-dependent Ca2+ channels. The ryanodine-sensitive calcium release channels in the sarcoplasmic reticulum may play an important role in maintaining the oscillatory contractions. Moreover, PKC activity modulates these oscillatory contractions.     

The role of Ca2+ in the contractility of rabbit small intestine in vitro.

This study evaluated the role of Ca2+ in spontaneous and ACh- and KCl-induced contractions in longitudinal and circular smooth muscle from rabbit small intestine in vitro. In the first experiment, the amplitude, frequency and tone of spontaneous contractions in longitudinal and circular smooth muscle of small intestine were determined and, in the second experiment, the ACh- and KCl-induced responses of longitudinal and circular smooth muscle were measured. Atropine and guanethidine reduced the amplitude and tone of contractions in longitudinal and circular muscle, but reduced the frequency of contractions in circular muscle, only. TTX attenuated the amplitude of contractions and decreased the tone of contractions in longitudinal muscle, but increased the tone in circular muscle. Ca2+-free solutions, verapamil, nifedipine and caffeine diminished the three parameters of spontaneous contractions. Thapsigargin and cyclopiazonic acid increased the amplitude and tone of contractions in ileum longitudinal muscle, only, and cyclopiazonic acid increased the amplitude of contractions in circular muscle. Ca2+-free solutions, verapamil, nifedipine, thapsigargin, cyclopiazonic acid, and caffeine diminished ACh- and KCl-induced contractions. Those results suggest that extracellular Ca2+ plays a role in spontaneous contractions, and extracellular and intracellular Ca2+ participate in the ACh- and KCl-induced contractions of rabbit small intestine.     

Evidence that a Ca2+ sparks/STOCs coupling mechanism is responsible for the inhibitory effect of caffeine on electro-mechanical coupling in guinea pig ureteric smooth muscle

Recent studies have highlighted the role of the sarcoplasmic reticulum (SR) in controlling excitability, Ca2+ signalling and contractility in smooth muscle. Caffeine, an agonist of ryanodine receptors (RyRs) on the SR has been previously shown to effect Ca2+ signalling but its effects on excitability and contractility are not so clear. We have studied the effects of low concentration of caffeine (1 mM) on Ca2+ signalling, action potential and contractility of guinea pig ureteric smooth muscle. Caffeine produced reversible inhibition of the action potentials, Ca2+ transients and phasic contractions evoked by electrical stimulation. It had no effect on the inward Ca2+ current or Ca2+ transient but increased the amplitude and the frequency of spontaneous transient outward currents (STOCs) in voltage clamped ureteric myocytes, suggesting Ca2+-activated K+ channels (BK) are affected by it. In isolated cells and cells in situ caffeine produced an increase in the frequency and the amplitude of Ca2+ sparks as well the number of spark discharging sites per cell. Inhibition of Ca2+ sparks by ryanodine (50 microM) or SR Ca2+-ATPase (SERCA) cyclopiazonic acid (CPA, 20 microM) or BKCa channels by iberiotoxin (200 nM) or TEA (1 mM), fully reversed the inhibitory effect of caffeine on Ca2+ transients and force evoked by electrical field stimulation (EFS). These data suggest that the inhibitory effect of caffeine on the action potential, Ca2+ transients and force in ureteric smooth muscle is caused by activation of Ca2+ sparks/STOCs coupling mechanism.     

Blockers of potassium current and resting membrane potential in rat muscle fibers.

Rat diaphragm fibers were equilibrated for several hours in 150 mM KCl; when they were returned to 5 mM KCl the resting potential went back to its original level with a half time of 17 min. This repolarization was blocked by 5 mM BaCl2, a blocker of the inward rectifier K channel. On the other hand, 0.1 mM apamin and 0.02 mM glibenclamide which block the Ca-dependent and ATP sensitive K channels, respectively, and 0.1 mM 9-AC a blocker of the Cl- channel did not affect the repolarization. 5 mM barium decreased the K conductance measured under current-clamp conditions in diaphragm muscle fibers. The possible role of the inward rectifier system in the repolarization following return to normal [K]o is discussed.     

Membrane responses to norepinephrine in cultured brown fat cells

We used the "perforated-patch" technique (Horn, R., and A. Marty, 1988. Journal of General Physiology. 92:145-159) to examine the effects of adrenergic agonists on the membrane potentials and membrane currents in isolated cultured brown fat cells from neonatal rats. In contrast to our previous results using traditional whole-cell patch clamp, 1-23-d cultured brown fat cells clamped with the perforated patch consistently showed vigorous membrane responses to both alpha- and beta-adrenergic agonists, suggesting that cytoplasmic components essential for the thermogenic response are lost in whole-cell experiments. The membrane responses to adrenergic stimulation varied from cell to cell but were consistent for a given cell. Responses to bath-applied norepinephrine in voltage-clamped cells had three possible components: (a) a fast transient inward current, (b) a slower outward current carried by K+ that often oscillated in amplitude, and (c) a sustained inward current largely by Na+. The fast inward and outward currents were activated by alpha-adrenergic agonists while the slow inward current was mediated by beta-adrenergic agonists. Oscillating outward currents were the most frequently seen response to norepinephrine stimulation. Activation of this current, termed IK,NE, was independent of voltage and seemed to be carried by Ca2(+)-activated K channels since the current oscillated in amplitude at constant membrane potential and gradually decreased when the cells were bathed with calcium-free external solution. IK,NE had a novel pharmacology in that it could be blocked by 4-aminopyridine, tetraethylammonium, apamin, and charybdotoxin. Both IK,NE and the voltage-gated K channels also present in brown fat (Lucero, M. T., and P. A. Pappone, 1989a. Journal of General Physiology. 93:451-472) may play a role in maintaining cellular homeostasis in the face of the high metabolic activity involved in thermogenesis.     

A whole-cell and single-channel study of the voltage-dependent outward potassium current in avian hepatocytes

Voltage-dependent membrane currents were studied in dissociated hepatocytes from chick, using the patch-clamp technique. All cells had voltage-dependent outward K+ currents; in 10% of the cells, a fast, transient, tetrodotoxin-sensitive Na+ current was identified. None of the cells had voltage-dependent inward Ca2+ currents. The K+ current activated at a membrane potential of about -10 mV, had a sigmoidal time course, and did not inactivate in 500 ms. The maximum outward conductance was 6.6 +/- 2.4 nS in 18 cells. The reversal potential, estimated from tail current measurements, shifted by 50 mV per 10-fold increase in the external K+ concentration. The current traces were fitted by n2 kinetics with voltage-dependent time constants. Omitting Ca2+ from the external bath or buffering the internal Ca2+ with EGTA did not alter the outward current, which shows that Ca2+-activated K+ currents were not present. 1-5 mM 4-aminopyridine, 0.5-2 mM BaCl2, and 0.1-1 mM CdCl2 reversibly inhibited the current. The block caused by Ba was voltage dependent. Single-channel currents were recorded in cell-attached and outside-out patches. The mean unitary conductance was 7 pS, and the channels displayed bursting kinetics. Thus, avian hepatocytes have a single type of K+ channel belonging to the delayed rectifier class of K+ channels.     

The role of ether-a-go-go-related gene K(+) channels in glucocorticoid inhibition of adrenocorticotropin release by rat pituitary cells

The present study investigated the role of K(+) channels in the inhibitory effect of glucocorticoid on adrenocorticotropin (ACTH) release induced by corticotropin-releasing hormone (CRH) using cultured rat anterior pituitary cells. Apamin and charybdotoxin (CTX) did not have a significant effect on ACTH release induced by CRH (1 nM). Tetraethylammonium (TEA), a broad spectrum K(+) channel blocker, increased the ACTH response to CRH only at the highest concentration (10 mM). The exposure to 100 nM corticosterone for 60 min inhibited the CRH-induced ACTH release. Neither TEA, apamin, nor CTX affected the inhibitory effect of corticosterone. In contrast, astemizole (Ast) and E-4031, ether-a-go-go-related gene (erg) K(+) channel blockers, abolished the inhibitory effect of corticosterone on CRH-induced ACTH release (1.25+/-0.10 vs. 1.45+/-0.11 ng/well at 10 microM Ast, p>0.05, 1.71+/-0.16 vs. 1.91+/-0.32 ng/well at 10 microM E-4031, p>0.05, vehicle vs. corticosterone). RT-PCR demonstrated all three subtypes of rat-erg mRNAs in the pituitary and corticosterone increased only erg1 mRNA up to 2.47+/-0.54 fold. In conclusion, erg K(+) channels were up-regulated by glucocorticoid, and have indispensable roles in delayed glucocorticoid inhibition of CRH-induced ACTH release by rat pituitary cells.     

Ca(2+)-activated K+ channels in human leukemic T cells

Using the patch-clamp technique, we have identified two types of Ca(2+)-activated K+ (K(Ca)) channels in the human leukemic T cell line. Jurkat. Substances that elevate the intracellular Ca2+ concentration ([Ca2+]i), such as ionomycin or the mitogenic lectin phytohemagglutinin (PHA), as well as whole-cell dialysis with pipette solutions containing elevated [Ca2+]i, activate a voltage-independent K+ conductance. Unlike the voltage-gated (type n) K+ channels in these cells, the majority of K(Ca) channels are insensitive to block by charybdotoxin (CTX) or 4-aminopyridine (4-AP), but are highly sensitive to block by apamin (Kd less than 1 nM). Channel activity is strongly dependent on [Ca2+]i, suggesting that multiple Ca2+ binding sites may be involved in channel opening. The Ca2+ concentration at which half of the channels are activated is 400 nM. These channels show little voltage dependence over a potential range of -100 to 0 mV and have a unitary conductance of 4-7 pS in symmetrical 170 mM K+. In the presence of 10 nM apamin, a less prevalent type of K(Ca) channel with a unitary conductance of 40-60 pS can be observed. These larger-conductance channels are sensitive to block by CTX. Pharmacological blockade of K(Ca) channels and voltage-gated type n channels inhibits oscillatory Ca2+ signaling triggered by PHA. These results suggest that K(Ca) channels play a supporting role during T cell activation by sustaining dynamic patterns of Ca2+ signaling.     

Potassium channels regulate tone in rat pulmonary veins

Intrapulmonary veins (PVs) contribute to pulmonary vascular resistance, but the mechanisms controlling PV tone are poorly understood. Although smooth muscle cell (SMC) K(+) channels regulate tone in most vascular beds, their role in PV tone is unknown. We show that voltage-gated (K(V)) and inward rectifier (K(ir)) K(+) channels control resting PV tone in the rat. PVs have a coaxial structure, with layers of cardiomyocytes (CMs) arrayed externally around a subendothelial layer of typical SMCs, thus forming spinchterlike structures. PVCMs have both an inward current, inhibited by low-dose Ba(2+), and an outward current, inhibited by 4-aminopyridine. In contrast, PVSMCs lack inward currents, and their outward current is inhibited by tetraethylammonium (5 mM) and 4-aminopyridine. Several K(V), K(ir), and large-conductance Ca(2+)-sensitive K(+) channels are present in PVs. Immunohistochemistry showed that K(ir) channels are present in PVCMs and PV endothelial cells but not in PVSMCs. We conclude that K(+) channels are present and functionally important in rat PVs. PVCMs form sphincters rich in K(ir) channels, which may modulate venous return both physiologically and in disease states including pulmonary edema.     

K(+) channel inhibition, calcium signaling, and vasomotor tone in canine pulmonary artery smooth muscle

We investigated the role of K(+) channels in the regulation of baseline intracellular free Ca(2+) concentration ([Ca(2+)](i)), alpha-adrenoreceptor-mediated Ca(2+) signaling, and capacitative Ca(2+) entry in pulmonary artery smooth muscle cells (PASMCs). Inhibition of voltage-gated K(+) channels with 4-aminopyridine (4-AP) increased the membrane potential and the resting [Ca(2+)](i) but attenuated the amplitude and frequency of the [Ca(2+)](i) oscillations induced by the alpha-agonist phenylephrine (PE). Inhibition of Ca(2+)-activated K(+) channels (with charybdotoxin) and inhibition (with glibenclamide) or activation of ATP-sensitive K(+) channels (with lemakalim) had no effect on resting [Ca(2+)](i) or PE-induced [Ca(2+)](i) oscillations. Thapsigargin was used to deplete sarcoplasmic reticulum Ca(2+) stores in the absence of extracellular Ca(2+). Under these conditions, 4-AP attenuated the peak and sustained components of capacitative Ca(2+) entry, which was observed when extracellular Ca(2+) was restored. Capacitative Ca(2+) entry was unaffected by charybdotoxin, glibenclamide, or lemakalim. In isolated pulmonary arterial rings, 4-AP increased resting tension and caused a leftward shift in the KCl dose-response curve. In contrast, 4-AP decreased PE-induced contraction, causing a rightward shift in the PE dose-response curve. These results indicate that voltage-gated K(+) channel inhibition increases resting [Ca(2+)](i) and tone in PASMCs but attenuates the response to PE, likely via inhibition of capacitative Ca(2+) entry.     

A family of calcium-dependent potassium channels from rat brain

By incorporating rat brain plasma membrane vesicles into planar lipid bilayers, we have found and characterized four types of Ca2(+)-activated K+ channels. The unitary conductances of these channels are 242 +/- 14 pS, 236 +/- 16 pS, 135 +/- 10 pS, and 76 +/- 6 pS in symmetrical 150 mM KCI buffers. These channels share a number of properties. They are all activated by depolarizing voltages, activated by micromolar concentrations of internal Ca2+ with a Hill coefficient for Ca2+ activation of between 2 and 3, noninactivating under our assay conditions, blocked by low millimolar concentrations of TEA from the outside, apamin-insensitive, and very selective for K+ over Na+ and Cl-. Three of the four channels are also blocked by nanomolar concentrations of charybdotoxin. One of the high conductance Ca2(+)-activated K+ channels is novel in that it is not blocked by charybdotoxin and exhibits gating kinetics highlighted by long closed times and long open times. This family of closely related Ca2(+)-activated K+ channels may share structural domains underlying particular functions.