Nanoscale imaging of rat atrial myocytes by scanning ion conductance microscopy reveals heterogeneity of T-tubule openings and ultrastructure of the cell membrane

In contrast to ventricular myocytes, the structural and functional importance of atrial transverse tubules (T-tubules) is not fully understood. Therefore, we investigated the ultrastructure of T-tubules of living rat atrial myocytes in comparison with ventricular myocytes. Nanoscale cell surface imaging by scanning ion conductance microscopy (SICM) was accompanied by confocal imaging of intracellular T-tubule network, and the effect of removal of T-tubules on atrial excitation-contraction coupling (EC-coupling) was observed. By SICM imaging, we classified atrial cell surface into 4 subtypes. About 38% of atrial myocytes had smooth cell surface with no clear T-tubule openings and intracellular T-tubules (smooth-type). In 33% of cells, we found a novel membrane nanostructure running in the direction of cell length and named it 'longitudinal fissures' (LFs-type). Interestingly, T-tubule openings were often found inside the LFs. About 17% of atrial cells resembled ventricular myocytes, but they had smaller T-tubule openings and a lower Z-groove ratio than the ventricle (ventricular-type). The remaining 12% of cells showed a mixed structure of each subtype (mixed-type). The LFs-, ventricular-, and mixed-type had an appreciable amount of reticular form of intracellular T-tubules. Formamide-induced detubulation effectively removed atrial T-tubules, which was confirmed by both confocal images and decreased cell capacitance. However, the LFs remained intact after detubulation. Detubulation reduced action potential duration and L-type Ca2+ channel (LTCC) density, and prolonged relaxation time of the myocytes. Taken together, we observed heterogeneity of rat atrial T-tubules and membranous ultrastructure, and the alteration of atrial EC-coupling by disruption of T-tubules.

Nanoscale imaging of rat atrial myocytes by scanning ion conductance microscopy reveals heterogeneity of T-tubule openings and ultrastructure of the cell membrane

In contrast to ventricular myocytes, the structural and functional importance of atrial transverse tubules (T-tubules) is not fully understood. Therefore, we investigated the ultrastructure of T-tubules of living rat atrial myocytes in comparison with ventricular myocytes. Nanoscale cell surface imaging by scanning ion conductance microscopy (SICM) was accompanied by confocal imaging of intracellular T-tubule network, and the effect of removal of T-tubules on atrial excitation-contraction coupling (EC-coupling) was observed. By SICM imaging, we classified atrial cell surface into 4 subtypes. About 38% of atrial myocytes had smooth cell surface with no clear T-tubule openings and intracellular T-tubules (smooth-type). In 33% of cells, we found a novel membrane nanostructure running in the direction of cell length and named it 'longitudinal fissures' (LFs-type). Interestingly, T-tubule openings were often found inside the LFs. About 17% of atrial cells resembled ventricular myocytes, but they had smaller T-tubule openings and a lower Z-groove ratio than the ventricle (ventricular-type). The remaining 12% of cells showed a mixed structure of each subtype (mixed-type). The LFs-, ventricular-, and mixed-type had an appreciable amount of reticular form of intracellular T-tubules. Formamide-induced detubulation effectively removed atrial T-tubules, which was confirmed by both confocal images and decreased cell capacitance. However, the LFs remained intact after detubulation. Detubulation reduced action potential duration and L-type Ca2+ channel (LTCC) density, and prolonged relaxation time of the myocytes. Taken together, we observed heterogeneity of rat atrial T-tubules and membranous ultrastructure, and the alteration of atrial EC-coupling by disruption of T-tubules.

Deficiency of Anoctamin 5/TMEM16E causes nuclear positioning defect and impairs Ca²⁺ signaling of differentiated C2C12 myotubes

Anoctamin 5 (ANO5)/TMEM16E belongs to a member of the ANO/TMEM16 family member of anion channels. However, it is a matter of debate whether ANO5 functions as a genuine plasma membrane chloride channel. It has been recognized that mutations in the ANO5 gene cause many skeletal muscle diseases such as limb girdle muscular dystrophy type 2L (LGMD2L) and Miyoshi muscular dystrophy type 3 (MMD3) in human. However, the molecular mechanisms of the skeletal myopathies caused by ANO5 defects are poorly understood. To understand the role of ANO5 in skeletal muscle development and function, we silenced the ANO5 gene in C2C12 myoblasts and evaluated whether it impairs myogenesis and myotube function. ANO5 knockdown (ANO5-KD) by shRNA resulted in clustered or aggregated nuclei at the body of myotubes without affecting differentiation or myotube formation. Nuclear positioning defect of ANO5-KD myotubes was accompanied with reduced expression of Kif5b protein, a kinesin-related motor protein that controls nuclear transport during myogenesis. ANO5-KD impaired depolarization-induced [Ca²⁺]i transient and reduced sarcoplasmic reticulum (SR) Ca²⁺ storage. ANO5-KD resulted in reduced protein expression of the dihydropyridine receptor (DHPR) and SR Ca²⁺-ATPase subtype 1. In addition, ANO5-KD compromised co-localization between DHPR and ryanodine receptor subtype 1. It is concluded that ANO5-KD causes nuclear positioning defect by reduction of Kif5b expression, and compromises Ca²⁺ signaling by downregulating the expression of DHPR and SERCA proteins.

Visceral Hypersensitivity and Altered Colonic Motility in Type 2 Diabetic Rat

BACKGROUND/AIMS: Abnormal visceral sensitivity and disordered motility are common in patients with diabetes mellitus. The purpose of the present study was to investigate whether visceral sensation and bowel motility were altered in a rat model of type 2 diabetes mellitus accompanied by weight loss. METHODS: A type 2 diabetic rat model in adulthood was developed by administrating streptozotocin (STZ; 90 mg/kg, i.p.) to neonatal rats. Eight weeks after STZ administration, rats with blood glucose level of 200 mg/dL or higher were selected and used as diabetic group (n = 35) in this study. Abdominal withdrawal reflex and arterial pulse rate were measured to examine visceral nociception induced by colorectal distension (0.1-1.0 mL). The amplitude, frequency, and area under the curve (AUC) of spontaneous phasic contractions of colonic circular muscles were recorded in vitro to examine colonic motility. RESULTS: STZ-treated diabetic rats gained significantly less weight for 8 weeks than control (P < 0.01). Forty-eight percent of the diabetic rats showed enhanced visceral nociceptive response to colorectal distension. Diabetic rats did not differ from control rats in colorectal compliance. However, the frequency and AUC, not the amplitude, of colonic spontaneous contraction in vitro was significantly decreased in diabetic rats compared to control rats (P < 0.01 in frequency and P < 0.05 in AUC). CONCLUSIONS: These results demonstrate visceral hypersensitivity and colonic dysmotility in a rat model of type 2 diabetes mellitus accompanied by weight loss.

Mechanism of Relaxation Via TASK-2 Channels in Uterine Circular Muscle of Mouse

Plasma pH can be altered during pregnancy and at labor. Membrane excitability of smooth muscle including uterine muscle is suppressed by the activation of K+ channels. Because contractility of uterine muscle is regulated by extracellular pH and humoral factors, K+ conductance could be connected to factors regulating uterine contractility during pregnancy. Here, we showed that TASK-2 inhibitors such as quinidine, lidocaine, and extracellular acidosis produced contraction in uterine circular muscle of mouse. Furthermore, contractility was significantly increased in pregnant uterine circular muscle than that of non-pregnant muscle. These patterns were not changed even in the presence of tetraetylammonium (TEA) and 4-aminopyridine (4-AP). Finally, TASK-2 inhibitors induced strong myometrial contraction even in the presence of L-methionine, a known inhibitor of stretchactivated channels in myometrium. When compared to non-pregnant myometrium, pregnant myometrium showed increased immunohistochemical expression of TASK-2. Therefore, TASK-2, seems to play a key role during regulation of myometrial contractility in the pregnancy and provides new insight into preventing preterm delivery.

CD40 Co-stimulation Inhibits Sustained BCR-induced Ca2+ Signaling in Response to Long-term Antigenic Stimulation of Immature B Cells

Regulation of B cell receptor (BCR)-induced Ca2+ signaling by CD40 co-stimulation was compared in long-term BCR-stimulated immature (WEHI-231) and mature (Bal-17) B cells. In response to long-term pre-stimulation of immature WEHI-231 cells to alpha-IgM antibody (0.5~48 hr), the initial transient decrease in BCR-induced [Ca2+]i was followed by spontaneous recovery to control level within 24 hr. The recovery of Ca2+ signaling in WEHI-231 cells was not due to restoration of internalized receptor but instead to an increase in the levels of PLCgamma2 and IP3R-3. CD40 co-stimulation of WEHI-231 cells prevented BCR-induced cell cycle arrest and apoptosis, and it strongly inhibited the recovery of BCR-induced Ca2+ signaling. CD40 co-stimulation also enhanced BCR internalization and reduced expression of PLCgamma2 and IP3R-3. Pre-treatment of WEHI-231 cells with the antioxidant N-acetyl-L-cysteine (NAC) strongly inhibited CD40-mediated prevention of the recovery of Ca2+ signaling. In contrast to immature WEHI-231 cells, identical long-term alpha-IgM pre-stimulation of mature Bal-17 cells abolished the increase in BCR-induced [Ca2+]i, regardless of CD40 co-stimulation. These results suggest that CD40-mediated signaling prevents antigen-induced cell cycle arrest and apoptosis of immature B cells through inhibition of sustained BCR-induced Ca2+ signaling.

PKC-Independent Stimulation of Cardiac Na+/Ca2+ Exchanger by Staurosporine

[Ca2+]i transients by reverse mode of cardiac Na+/Ca2+ exchanger (NCX1) were recorded in fura-2 loaded BHK cells with stable expression of NCX1. Repeated stimulation of reverse NCX1 produced a long-lasting decrease of Ca2+ transients ('rundown'). Rundown of NCX1 was independent of membrane PIP2 depletion. Although the activation of protein kinase C (PKC) was observed during the Ca2+ transients, neither a selective PKC inhibitor (calphostin C) nor a PKC activator (PMA) changed the degrees of rundown. By comparison, a non-specific PKC inhibitor, staurosporine (STS), reversed rundown in a dose-dependent and reversible manner. The action of STS was unaffected by pretreatment of the cells with calphostin C, PMA, or forskolin. Taken together, the results suggest that the stimulation of reverse NCX1 by STS is independent of PKC and/or PKA inhibition.

Sodium-activated Potassium Current in Guinea pig Gastric Myocytes

This study was designed to identify and characterize Na+ -activated K+ current (I(K(Na))) in guinea pig gastric myocytes under whole-cell patch clamp. After whole-cell configuration was established under 110 mM intracellular Na+ concentration ([Na+]i) at holding potential of -60 mV, a large inward current was produced by external 60 mM K+([K+] degree). This inward current was not affected by removal of external Ca2+. K+ channel blockers had little effects on the current (p>0.05). Only TEA (5 mM) inhibited steady-state current to 68+/-2.7% of the control (p<0.05). In the presence of K+ channel blocker cocktail (mixture of Ba2+, glibenclamide, 4-AP, apamin, quinidine and TEA), a large inward current was activated. However, the amplitude of the steadystate current produced under [K+]degree (140 mM) was significantly smaller when Na+ in pipette solution was replaced with K+ - and Li+ in the presence of K+ channel blocker cocktail than under 110 mM [Na+]i. In the presence of K+ channel blocker cocktail under low Cl- pipette solution, this current was still activated and seemed K+ -selective, since reversal potentials (E(rev)) of various concentrations of [K+]degree-induced current in current/voltage (I/V) relationship were nearly identical to expected values. R-56865 (10-20 microgram), a blocker of IK(Na), completely and reversibly inhibited this current. The characteristics of the current coincide with those of IK(Na) of other cells. Our results indicate the presence of IK(Na) in guinea pig gastric myocytes.

Ca2+-dependent Long-term Inactivation of Cardiac Na+/Ca2+ Exchanger

Using BHK cells with stable expression of cardiac Na+/Ca2+ exchanger (BHK-NCX1), reverse mode (i.e. Ca2+ influx mode) of NCX1 current was recorded by whole-cell patch clamp. Repeated stimulation of reverse NCX1 produced a cytosolic Ca2+-dependent long-term inactivation of the exchanger activity. The degrees of inactivation correlated with NCX1 densities of the cells and were attenuated by reduced Ca2+ influx via the reverse exchanger. The inactivation of NCX1 was attenuated by (i) inhibition of Ca2+ influx with reduced extracellular Ca2+, (ii) treatment with NCX1 blocker (Ni2+), and (iii) increase of cytoplasmic Ca2+ buffer (EGTA). In BHK-NCX1 cells transiently expressing TRPV1 channels, Ca2+ influx elicited by capsaicin produced a marked inactivation of NCX1. We suggest that cytoplasmic Ca2+ has a dual effect on NCX1 activities, and that allosteric Ca2+ activation of NCX1 can be opposed by the Ca2+-dependent long-term inactivation in intact cells.

Regulation of L-type Calcium Channel Current by Somatostatin in Guinea-Pig Gastric Myocytes

To study the direct effect of somatostatin (SS) on calcium channel current (IBa) in guinea-pig gastric myocytes, IBa was recorded by using whole-cell patch clamp technique in single smooth muscle cells. Nicardipine (1microM), a L-type Ca2+ channel blocker, inhibited IBa by 98+/-1.9% (n=5), however IBa was decreased in a reversible manner by application of SS. The peak IBa at 0 mV were decreased to 95+/-1.1, 92+/-1.9, 82+/-4.0, 66+/-5.8, 10+/-2.9% at 10-10, 10-9, 10-8, 10-7, 10-5 M of SS, respectively (n=3~6; mean+/-SEM). The steady-state activation and inactivation curves of IBa as a function of membrane potentials were well fitted by a Boltzmann equation. Voltage of half-activation (V0.5) was -12+/-0.5 mV in control and -11+/-1.9 mV in SS treated groups (respectively, n=5). The same values of half-inactivation were -35+/-1.4 mV and -35+/-1.9 mV (respectively, n=5). There was no significant difference in activation and inactivation kinetics of IBa by SS. Inhibitory effect of SS on IBa was significantly reduced by either dialysis of intracellular solution with GDPbetaS, a non-hydrolysable G protein inhibitor, or pretreatment with pertussis toxin (PTX). SS also decreased contraction of guinea-pig gastric antral smooth muscle. In conclusion, SS decreases voltage-dependent L-type calcium channel current (VDCCL) via PTX- sensitive signaling pathways in guinea-pig antral circular myocytes.

Five subtypes of muscarinic receptors are expressed in gastric smooth muscles of guinea pig

Muscarinic receptors play key roles in the control of gastrointestinal smooth muscle activity. However, specific physiological functions of each subtype remain to be determined. In this study, the nonselective cation channel activated by carbachol (ICCh) was examined in circular smooth muscle cells of the guinea pig gastric antrum using patch-clamp technique. 4-DAMP inhibited ICCh dose- dependently with IC50 of 1.1 +/- 0.1 nM (n = 6). GTPgS- induced current, however, was not inhibited by 10 nM 4-DAMP. ICCh was not recorded in pertussis- toxin (PTX)-pretreated smooth muscle cells of gastric antrum. ICCh values in response to 10 mM CCh at a holding potential of 60 mV were -330 32 pA (n=4) and -15 +/- 3 pA (n = 6) in the control and PTX-treated cells, respectively (P<0.01). Sensitivities to nanomolar 4-DAMP and PTX suggest the possible involvement of m4 subtype. Using sequence information obtained from cloned guinea pig muscarinic receptor genes, it is possible to amplify the cDNAs encoding m1-m5 from guinea pig brain tissue. Single cell RT-PCR experiments showed that all five subtypes of muscarinic receptor were present in circular smooth muscle cells of the guinea pig gastric antrum. Together with our previous results showing that Go protein is important for activation of ACh-activated NSC channels, our results suggest that ICCh might be activated by acetylcholine through m4 subtype as well as m2 and m3 subtypes in guinea-pig stomach.

Mechanism of an increase in concentration of intracellular calcium by carbachol in human gastric smooth muscle cell / 대한내과학회지

BACKGROUND: The contraction of smooth muscle depends on an increase in the concentration of intracellular calcium ion and the source of this increase to various stimuli is different according to organs or species. Nevertheless, there have been only a few studies on human stomach smooth muscle. This study was designed to identify the source of the calcium utilized in the muscle contraction induced by carbachol, which is an important factor among various stimuli. METHODS: After the administration of carbachol with various conditions in cultured human stomach smooth muscle cells, fura-2-acetoxymethyl ester was used to measure the increase in the intracellular calcium concentration. RESULTS: (1) The carbachol-induced increase in the intracellular calcium concentration was not attenuated after removal of extracellular calcium. (2) Carbachol induced a small increase in the intracellular calcium concentration even after the depletion of intracellular calcium store. (3) Repeated histamine administration blocked the carbachol-induced increase in the intracellular calcium concentration in calcium-free extracellular solution. CONCLUSION: The main source of calcium utilized in human stomach smooth muscle contraction by carbachol is intracellular calcium store, particularly inositol triphosphate(IP3)-sensitive calcium stores. However, extracellular calcium also contributes to the carbachol-induced increase in the intracellular calcium concentration.

Effects of Propofol on the Cytosolic Calcium Concentration of the Astrocytoma Cells / 대한마취과학회지

BACKGROUND: Propofol is a widely-used intravenous anesthetic with a rapid onset, short duration of action and rapid elimination but the molecular mechanisms of action are not completely understood. Not only neurons but astrocytes are potential substrates for anesthetics, specifically for propofol. Intracellular calcium ion ([Ca2 ]i) is known to play a key role in the transduction and propagation of various chemical signals in astrocytes. METHODS: In the present study, the effects of propofol on the intracellular calcium concentration of astrocytoma cells by using a fura-2 fluorescence spectroscopy was investigated. RESULTS: In an isotonic standard solution, propofol (50 and 500microM) produced a transient increase in [Ca2 ]i while the intralipid did not change [Ca2 ]i. In several cells (20%), a transient increase in [Ca2 ]i was followed by sustained elevation which was sensitive to depletion of external calcium. A propofol-induced increase in [Ca2 ]i was not altered by an L-type calcium channel blocker (nifedipine 2microM). In cells bathed in a Ca2 -free external solution, a transient increase in [Ca2 ]i was observed. After the pretreatment of cyclopiazonic acid (CPA), an endoplasmic reticulum Ca2 -ATPase blocker, propofol 500microM did not produce any significant increase in [Ca2 ]i. Carbachol, which is known to release calcium from the inositol 1,4,5-triphosphate (IP3)-induced calcium release (IICR) stores, prevented the [Ca2 ]i increase by propofol and vice versa. High concentrations of caffeine (10 mM), which release calcium from the calcium-induced calcium release (CICR) stores, had no effect on [Ca2 ]i. CONCLUSIONS: From the above results, it is suggested that an increase in [Ca2 ]i by propofol in astrocytoma cells is mainly due to calcium release from the IICR stores.

Neurotensin enhances gastric motility in antral circular muscle strip of guinea-pig

Many reports suggest that neurotensin (NT) in the gastrointestinal tract may play a possible role as a neurotransmitter, a circulating hormone, or a modulator of motor activity. NT exerts various actions in the intestine; it produces contractile and relaxant responses in intestinal smooth muscle. This study was designed to investigate the effect of NT on motility of antral circular muscle strips in guinea-pig stomach. To assess the role of Ca2+ influx in underlying mechanism, slow waves were simultaneously recorded with spontaneous contractions using conventional intracellular mircoelectrode technique. At the concentration of 10-7 M, where NT showed maximum response, NT enhanced the magnitude (863 +/- 198%, mean +/- SEM, n = 13) and the frequency (154 +/- 10.3%, n = 11) of spontaneous contractions. NT evoked a slight hyperpolarization of membrane potential, tall and steep slow waves with abortive spikes (278 +/- 50%, n = 4). These effects were not affected by atropine (2 micrometer), guanethidine (2 micrometer) and tetrodotoxin (0.2 micrometer). NT-induced contractile responses were abolished in Ca2+-free solution and reduced greatly to near abolition by 10 micrometer of verapamil or 0.2 mM of CdCl2. Verapamil attenuated the effects of NT on frequency and amplitude of the slow waves. Taken together, these results indicate that NT enhances contractility in guinea-pig gastric antral circular muscle and Ca2+ influx through the voltage-operated Ca2+ channel appears to play an important role in the NT-induced contractile mechanism.

Muscarinic receptor subtype controlling the carbachol-induced muscle contraction in guinea pig gastric antrum

Stimulation of muscarinic receptors by carbachol (CCh) in the circular smooth muscle of the guinea pig gastric antrum causes muscle contraction. In the present study, muscarinic receptor subtype controlling the muscle contraction in response to CCh was studied using putative muscarinic receptor antagonists. Isometric force of the isolated circular muscle strips was measured in an organ bath. CCh contracted the muscle in a dose-dependent way, and each of the three muscarinic receptor antagonists, 4-diphenylacetoxy-N-methylpeperdine methiodide (4-DAMP), methoctramine and pirenzepine shifted the concentration-response curves to the right without significantly reducing the maximum force. The affinities of the muscarinic antagonists (pA2 values) obtained from Schild plot analysis were 10.15, 7.05 and 6.84 for 4-DAMP, methoctramine and pirenzepine, respectively. These results suggest that the M3-subtype mainly mediate the muscle contraction in response to CCh in guinea pig gastric antrum.

Evidence for adenosine triphosphate (ATP) as an excitatory neurotransmitter in guinea-pig gastric antrum

We explore the question of whether adenosine 5'-triphosphate (ATP) acts as an excitatory neurotransmitter in guinea-pig gastric smooth muscle. In an organ bath system, isometric force of the circular smooth muscle of guinea-pig gastric antrum was measured in the presence of atropine and guanethidine. Under electrical field stimulation (EFS) at high frequencies (>20 Hz), NO-mediated relaxation during EFS was followed by a strong contraction after the cessation of EFS (a "rebound-contraction"). Exogenous ATP mimicked the rebound-contraction. A known P2Y-purinoceptor antagonist, reactive blue 2 (RB-2), blocked the rebound-contraction while selective desensitization of P2x-purinoceptor with alpha, beta-MeATP did not affect it. ATP and 2-MeSATP induced smooth muscle contraction, which was effectively blocked by RB-2 and suramin, a nonselective P2-purinoceptor antagonist. Particularly, in the presence of RB-2, exogenous ATP and 2-MeSATP inhibited spontaneous phasic contractions, suggestingthe existence of different populations of purinoceptors. Both the rebound-contraction and the agonist-induced contraction were not inhibited by indomethacin. The rank orders of agonists' potency were 2-MeSATP > ATP gtoreq UTP for contraction and alpha, beta-MeATP gtoreq beta, gamma-MeATP for inhibition of the phasic contraction, that accord with the commonly accepted rank order of the classical P2Y-purinoceptor subtypes. Electrical activities of smooth muscles were only slightly influenced by ATP and 2-MeSATP, whereas alpha, beta-MeATP attenuated slow waves with membrane hyperpolarization. From the above results, it is suggested that ATP acts as an excitatory neurotransmitter, which mediates the rebound-contraction via P2Y-purinoceptor in guinea-pig gastric antrum.

Regulation of Smooth Muscle Excitability

No abstract available.

Effects of prostaglandin E2 on the spontaneous contractions and electrical activities of the antral circular muscle in guinea-pig stomach

The spontaneous contractions of gastric smooth muscles are regulated by slow waves, which are modulated by both nervous system and humoral agents. This study was designed to examine the effects of Prostaglandin E2 (PGE2) on the contractile and electrical activities of antral smooth muscles in guinea-pig stomach, using an intracellular recording technique. To elucidate the underlying mechanism for its effect on contractility, ionic currents were also measured using a whole-cell patch clamp method. The basal tone by PGE2 was variable, whereas the magnitude of phasic contractions was reduced (19.0 +/- 2.1%, n=19). The resting membrane potentials were hyperpolarized (-4.4+/-0.5 mV, n=10), and plateau potentials were lowered (-2.9+/-0.5 mV, n=10). In most cases, however, the initial peak potentials of slow waves were depolarized more by PGE2 than those of control. The frequency of the slows wave was increased from 5.7+/-0.2 cycles/min to 6.5+/-0.2 (n-22). Voltage-operated Ca2+ currents were decreased by PGE2 (n=5). Voltage-operated K+ currents, both Ca-dependent and Ca-independent, were increased (n-5). These results suggest that PGE2 plays an important role in the modulation of gastric smooth muscle activities, and its inhibitory effects on the contractility and activities of slow waves are resulted from both decrease of Ca2+ currents and increase of K+ currents.

Two types of voltage-dependent outward potassium currents in smooth muscle cells of rabbit basilar artery

We have investigated the two types of voltage-dependent outward potassium (K) currents, i.e. delayed rectifier K current (I-K(V)) and 'A-like' transient outward K current (I-to) with patch-clamp technique in single smooth muscle cells (SMCs) isolated from rabbit basilar artery, and investigated the characteristics of them. The time-courses of activation were well fitted by exponential function raised to second power (n-2) in I-K(v) and fourth power (n-4) in I-to. The activation, inactivation and recovery time courses of I-to were much faster than that of I-K(V). The steady-state activation and inactivation of I-K(V) was at the more hyperpolarized range than that of I-to contrary to the reports in other vascular SMCs. Tetraethylammonium chloride (TEA; 10 mM) markedly inhibited I-K(V) but little affected 1-to. 4-Aminopyridine (4-AP) had similar inhibitory potency on both currents. While a low concentration of Cd-2+ (0.5 mM) shifted the current-voltage relationship of I-to to the positive direction without change of maximum conductance, Cd-2+ did not cause any appreciable change for I-K(V).