Biomedical subjects

A Takai

Publications and source records attributed to A Takai.

At least 37 records · Page 2Linked to original sources

PubMed1995

Activity of smooth muscle phosphatases 1 and 2A in rabbit basilar artery in vasospasm.

BACKGROUND AND PURPOSE: Subarachnoid hemorrhage frequently leads to a long-term cerebral artery narrowing called vasospasm. Recently, the involvement of myosin light chain kinase has been found in experimental vasospasm in our laboratory. We therefore measured the activity of serine/threonine protein phosphatases 1 and 2A in the rabbit basilar artery in vasospasm and in vasocontraction to study their role, particularly in regard to vasospasm compared with vasocontraction. METHODS: Vasospasm was produced in the rabbit basilar artery by a two-hemorrhage method. Vasocontraction was induced by local application of KCl or serotonin to the rabbit basilar artery after a transclival exposure. The control animals were treated with saline instead of fresh blood. Serine/threonine protein phosphatase activity in the basilar artery was assayed with the use of [32P]phosphorylase-a as a substrate; protein phosphatase 1 activity was evaluated as protein phosphatase activity in the presence of 1 nmol/L okadaic acid, whereas protein phosphatase 2A activity was assessed as protein phosphatase activity inhibited by 1 nmol/L okadaic acid. RESULTS: Values of mean activity of protein phosphatase 1 in myofibrillar extract were 3.58 +/- 0.26 nmol/min per milligram in the control group, 3.22 +/- 0.12 nmol/min per milligram in the spastic group on day 2, and 3.01 +/- 0.16 nmol/min per milligram in the spastic group on day 4 (a significant decrease in protein phosphatase 1 activity in the spastic group on days 2 and 4). In contrast, these values did not show any significant changes in the KCl and serotonin groups. Values of mean activity of protein phosphatase 2A in cytosolic extract were 0.90 +/- 0.07 nmol/min per milligram in the control group, 0.75 +/- 0.10 nmol/min per milligram in the spastic group on day 2, and 0.62 +/- 0.17 nmol/min per milligram in the spastic group on day 4 (a significant reduction in protein phosphatase 2A in the spastic group on days 2 and 4). There was no evidence of significant changes of protein phosphatase 2A in cytosolic extract in the KCl and serotonin groups. CONCLUSIONS: Protein phosphatase 1 in myofibrillar extract is reported to catalyze the dephosphorylation of myosin light chain and calponin, whereas protein phosphatase 2A in cytosolic extract catalyzes the dephosphorylation of calponin and caldesmon. In addition, the phosphorylation of calponin and caldesmon results in the loss of their ability to inhibit smooth muscle contraction. Therefore, the significant decrease in activity of protein phosphatases 1 and 2A in vasospasm may result in uninterrupted vascular smooth muscle contraction by the preservation of phosphorylation of not only myosin light chain but also calponin and caldesmon.

Animals↗

PubMed1994

Affinity of okadaic acid to type-1 and type-2A protein phosphatases is markedly reduced by oxidation of its 27-hydroxyl group.

Okadaic acid (OA), a potent inhibitor of type-1 and type-2A protein phosphatases (PP1 and PP2A), has four hydroxyl groups at 2, 7, 24 and 27 positions (see Figure 1). By chemical treatment of OA we synthesized a derivative, in which the 27-hydroxyl group was specifically oxidized (27-dehydro-OA). The inhibitory effect of this OA derivative was examined on the activities of PP1 and PP2A, which were inhibited by intact OA with dissociation constants (Ki) of 150 nM and 32 pM respectively. We found that the affinity of OA was decreased 40-fold (Ki = 6 microM) with PP1 and 230-fold (Ki = 7.3 nM) with PP2A after oxidation of the 27-hydroxyl group. According to the model of the three-dimensional conformation of OA on the basis of X-ray analyses, the 27-hydroxyl group appears to be present in a position relatively free from intramolecular bonding formation, in comparison with the other three hydroxyl groups. The marked increases in the Ki values for PP1 and PP2A, which indicate the reduction of the absolute values of the free energy of binding by 9 kJ/mol and 14 kJ/mol respectively, may imply that the 27-hydroxyl group serves as a binding site with the phosphatase molecules.

Ethers, Cyclic↗

PubMed1994

[Tubercle bacilli and the defence factors for infection in sputum and bronchoalveolar lavage fluid].

The defence factors against infection in sputum and bronchoalveolar lavage fluid (BALF) of patients with pulmonary tuberculosis were measured. As the defence factors, lactoferrin, lysozyme and secretory IgA (sIgA) in sputum or BALF of patients with bacilli (+) or (-) tuberculosis were measured and compared. Lactoferrin in sputum was significantly higher in patients with sputum smear positive tuberculosis compared with patients with smear and culture negative tuberculosis. SIgA in sputum was significantly higher in smear negative and culture positive cases compared with culture negative cases. As to the lysozyme in sputum, significant difference was not proved between each group. The level of these factors in BALF did not show significant difference between bacilli (+) and (-) cases. Neither significant correlation was observed among the level of three defence factors in sputum or BALF, nor between the number of leucocyte and tubercle bacilli in sputum. In tubercle bacilli positive group, however, significant positive correlation between the number of leucocyte and lactoferrin in sputum was found. In vitro experiments, high concentration of lactoferrin or lysozyme inhibited the growth of standard strain of tubercle bacilli (H37Rv) and BCG. The results suggest that the measurement of lactoferrin, lysozyme and sIgA in sputum or BALF is useful to determine the clinical activity of tuberculosis.

Bronchoalveolar Lavage Fluid↗

PubMed1994

[Psychological test as a mass-screening system for depression].

A mass-screening system for depression is aimed at calculation of the prevalence rate, or at mental health checks in employees. But with the recent increased interest in mental health and QOL, depression has become an important issue at every stage of the life cycle. In this paper, we review psychological screening tests for child, adult and aged from the viewpoint of life cycle. Some tests, especially for children and the aged need further tests regarding reliability and validity. What is clarified and which approach in mental health is indicated by the screening test must be further examined. Further development of screening tests, appropriate to each life cycle, is needed.

Depression↗

PubMed1993

Okadaic acid-induced actin assembly in neutrophils: role of protein phosphatases.

Activation of neutrophils results in morphological and functional alterations including changes in cell shape and initiation of motile behavior that depend on assembly and reorganization of the actin cytoskeleton. Phosphoproteins are thought to be key intermediates in the regulation of cytoskeletal alterations and whereas much attention has been directed at the role of protein kinases, relatively little information is available on the importance of phosphatases. To elucidate the role of protein phosphatases, we studied the effects of the phosphatase inhibitors okadaic acid and calyculin A on the actin cytoskeleton of human neutrophils. Exposure of cells to okadaic acid resulted in assembly and spatial redistribution of actin, which peaked at 25 min and returned to baseline levels by 45 min, as assessed by flow cytometric analysis of NBD-phallacidin stained cells and confocal fluorescence microscopy, respectively. These effects correlated with an increase in protein phosphorylation, determined by incorporation of 32P into cellular proteins using SDS-PAGE and autoradiography. Similar but more rapid responses were observed in electropermeabilized cells treated with okadaic acid or calyculin A. The dose dependence of these effects was compatible with a role for phosphatase type 1 as the target enzyme. These findings also suggested the presence of constitutively active protein kinases capable of effecting actin polymerization. Phosphorylation of myosin light chain (MLC) has been postulated to promote actin assembly, but myosin light chain kinase (MLCK) appeared not to be involved because: (1) the effect of okadaic acid was not inhibited by the MLCK inhibitor KT5926 and (2) in permeabilized cells suspended in medium with free calcium [Ca2+] < 10 nM (conditions under which MLCK is inactive), the effect of okadaic acid persisted. The role of phosphatases in stimulus-induced actin assembly was assessed in cells preincubated with okadaic acid for 45 min, after F-actin levels had returned to baseline. Under these conditions, okadaic acid completely abrogated actin assembly induced by phorbol myristate acetate, platelet activating factor, and leukotriene B4, whereas the effects of the chemotactic peptide fMLP and opsonized zymosan (OpZ) were unaffected. We conclude that serine and threonine phosphatases exert a tonic negative influence on actin assembly and organization. Furthermore, divergent pathways seem to mediate the response to lipidic stimuli, on one hand, and fMLP and OpZ, on the other, as evidenced by the differential susceptibility to inhibition by okadaic acid.

Actins↗

PubMed1993

Evaluation of three occlusal examination methods used to record tooth contacts in lateral excursive movements.

Accurate and repeatable methods for recording tooth contacts are required for the clinical management of problems related to occlusion. A thorough understanding of the materials and procedures used in these methods is important to achieve desirable results in the treatment of such problems. This study compared three occlusal examination methods to determine the influence of materials and procedures on the number of tooth contacts recorded. Tooth contacts were analyzed at two lateral mandibular positions with each method. It was found that the method that uses black silicone recorded the highest number of tooth contacts. Thus the most frequent type of occlusal pattern observed was full-balanced occlusion. This study suggested that the disparities of results reported in literature on occlusal contact patterns could be the result of the different materials and methods used for occlusal registration.

Adult↗

PubMed1992

Estimation of the rate constants associated with the inhibitory effect of okadaic acid on type 2A protein phosphatase by time-course analysis.

As is often the case with tightly binding inhibitors, okadaic acid produces its inhibitory effect on type 2A protein phosphatase (PP2A) in a time-dependent manner. We measured the rate constants associated with the binding of okadaic acid to PP2A by analysing the time-course of the reduction of the p-nitrophenyl phosphate (pNPP) phosphatase activity of the enzyme after application of okadaic acid. The rate constants for dissociation of okadaic acid from PP2A were also estimated from the time-course of the recovery of the activity from inhibition by okadaic acid after addition of a mouse IgG1 monoclonal antibody raised against the inhibitor. Our results show that the rate constants for the binding of okadaic acid and PP2A are of the order of 10(7) M-1.s-1, a typical value for reactions involving relatively large molecules, whereas those for their dissociation are in the range 10(-4)-10(-3) s-1. The very low values of the latter seems to be the determining factor for the exceedingly high affinity of okadaic acid for PP2A. The dissociation constants for the interaction of okadaic acid with the free enzyme and the enzyme-substrate complex, estimated as the ratio of the rate constants, are both in the range 30-40 pM, in agreement with the results of previous dose-inhibition analyses.

Animals↗

PubMed1992

Inhibitory effect of okadaic acid derivatives on protein phosphatases. A study on structure-affinity relationship.

The effect of structural modifications of okadaic acid (OA), a polyether C38 fatty acid, was studied on its inhibitory activity toward type 1 and type 2A protein phosphatases (PP1 and PP2A) by using OA derivatives obtained either by isolation from natural sources or by chemical processes. The dissociation constant (Ki) for the interaction of OA with PP2A was estimated to be 30 (26-33) nM [median (95% confidence limits)]. The OA derivatives used and their affinity for PP2A, expressed as Ki (in brackets) were as follows: 35-methyl-OA (DTX1) [19 (12-25) pM], OA-9,10-episulphide (acanthifolicin) [47 (25-60) pM], 7-deoxy-OA [69 (31-138) pM], 14,15-dihydro-OA [315 (275-360) pM], 2-deoxy-OA [899 (763-1044) pM], 7-O-palmitoyl-OA [greater than 100 nM], 7-O-palmitoyl-DTX1 [greater than 100 nM], methyl okadate [much greater than 100 nM], 2-oxo-decarboxy-OA [much greater than 100 nM] and the C-15-C-38 fragment of OA [much greater than 100 nM]. The sequence of the affinity of these derivatives for PP1 was essentially the same as that observed with PP2A, although the absolute values of Ki were very different for the enzymes. The inhibitory effect of OA on PP2A was reversed by applying a murine monoclonal antibody against OA, which recognizes modifications of the 7-hydroxyl group of the OA molecule. It has been shown by n.m.r. spectroscopy and X-ray analysis that one end (C-1-C-24) of the OA molecule assumes a circular conformation. The present results suggest the importance of the conformation for the inhibitory action of OA on the protein phosphatases. The ratios of the Ki values for PP1 to that for PP2A, which were within the range 10(3)-10(4), tended to be smaller for the derivatives with lower affinity, indicating that the structural changes in OA impaired the affinity for PP2A more strongly than that for PP1.

Antibodies, Monoclonal↗

PubMed1992

Phorbol ester-induced actin assembly in neutrophils: role of protein kinase C.

The shape changes and membrane ruffling that accompany neutrophil activation are dependent on the assembly and reorganization of the actin cytoskeleton, the molecular basis of which remains to be clarified. A role of protein kinase C (PKC) has been postulated because neutrophil activation, with the attendant shape and membrane ruffling changes, can be initiated by phorbol esters, known activators of PKC. It has become apparent, however, that multiple isoforms of PKC with differing substrate specificities exist. To reassess the role of PKC in cytoskeletal reorganization, we compared the effects of diacylglycerol analogs and of PKC antagonists on kinase activity and on actin assembly in human neutrophils. Ruffling of the plasma membrane was assessed by scanning EM, and spatial redistribution of filamentous (F)-actin was assessed by scanning confocal microscopy. Staining with NBD-phallacidin and incorporation of actin into the Triton X-100-insoluble ("cytoskeletal") fraction were used to quantify the formation of (F)-actin. [32P]ATP was used to detect protein phosphorylation in electroporated cells. Exposure of neutrophils to 4 beta-PMA (an activator of PKC) induced protein phosphorylation, membrane ruffling, and assembly and reorganization of the actin cytoskeleton, whereas the 4a-isomer, which is inactive towards PKC, failed to produce any of these changes. Moreover, 1,2-dioctanoylglycerol, mezerein, and 3-(N-acetylamino)-5-(N-decyl-N-methylamino)-benzyl alcohol, which are nonphorbol activators of PKC, also promoted actin assembly. Although these effects were consistent with a role of PKC, the following observations suggested that stimulation of conventional isoforms of the kinase were not directly responsible for actin assembly: (a) Okadaic acid, an inhibitor of phosphatases 1 and 2A, potentiated PMA-induced protein phosphorylation, but not actin assembly; and (b) PMA-induced actin assembly and membrane ruffling were not prevented by the conventional PKC inhibitors 1-(5-isoquinolinesulfonyl)-2-methylpiperazine, staurosporine, calphostin C, or sphingosine at concentrations that precluded PMA-induced protein phosphorylation and superoxide production. On the other hand, PMA-induced actin assembly was inhibited by long-chain fatty acid coenzyme A esters, known inhibitors of nuclear PKC (nPKC). We conclude that PMA-induced actin assembly is unlikely to be mediated by the conventional isoforms of PKC, but may be mediated by novel isoforms of the kinase such as nPKC.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1992

Modulation of neutrophil activation by okadaic acid, a protein phosphatase inhibitor.

We determined the effects of okadaic acid (OA), a specific inhibitor of protein phosphatases 1 (PP1) and 2A (PP2A), on protein phosphorylation and on the activation of the NADPH oxidase in human neutrophils. In otherwise unstimulated cells, OA induced phosphoprotein accumulation, revealing the presence of constitutively active protein kinases. Pulse-chase experiments in electropermeabilized cells confirmed that this effect was due, at least in part, to inhibition of dephosphorylation. OA potentiated phosphoprotein accumulation induced by phorbol esters and by the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP). In phorbol ester-stimulated cells, OA prolonged the respiratory response after inhibition of protein kinase C (PKC) with staurosporine, consistent with a reduced rate of dephosphorylation of active phosphorylated components. Similarly, OA delayed the inactivation of the burst after displacement of FMLP from its receptor by a competitive antagonist. This suggests that the substrates of the protein kinases activated by FMLP are dephosphorylated by PP1 and/or PP2A. That phosphatases control the intensity and duration of the respiratory response is suggested by the finding that OA magnified and prolonged the oxidative burst elicited by FMLP. In contrast, pretreatment with OA produced a time-dependent inhibition of the phorbol ester-induced respiratory burst. Under conditions where inhibition of the phorbol ester response was nearly complete, activation by the chemoattractant peptide not only persisted but was in fact accentuated. These findings provide strong evidence that receptor-mediated stimulation of the NADPH oxidase can occur by pathways not involving PKC.

Cell Membrane Permeability↗

PubMed1991

Okadaic acid, a phosphatase inhibitor, induces activation and phosphorylation of the Na+/H+ antiport.

We determined the effect of okadaic acid (OA), a potent phosphoprotein phosphatase inhibitor, on the intracellular pH (pHi) of rat thymic lymphocytes and human bladder carcinoma cells. OA induced a rapid and sustained cytosolic alkalinization. This pHi increase was Na(+)-dependent and was inhibited by 5,N-disubstituted analogs of amiloride, indicating mediation by the Na+/H+ antiport. As described for other stimulants, such as mitogens and hypertonic challenge, activation of the antiport by OA is attributable to an upward shift in its pHi dependence. Accordingly, the alkalinization produced by the phosphatase inhibitor was not additive with that induced osmotically. Activation of the antiport by OA was accompanied by a marked increase in phosphoprotein accumulation, revealing the presence of active protein kinases in otherwise unstimulated cells. We considered the possibility that phosphorylation of the antiport itself or of an ancillary protein is responsible for activation of Na+/H+ exchange. Consistent with this notion, the alkalinization induced by OA was absent in ATP depleted cells. More importantly, immunoprecipitation experiments demonstrated increased phosphorylation of the antiport following treatment with OA. We conclude that, upon inhibition of phosphoprotein phosphatase activity, constitutively active kinases induce the activation of Na+/H+ exchange, possibly by direct phosphorylation of the antiport.

Adenosine Triphosphate↗

PubMed1991

Inhibitory effect of okadaic acid on the p-nitrophenyl phosphate phosphatase activity of protein phosphatases.

The phosphatase activities of type 2A, type 1 and type 2C protein phosphatase preparations were measured against p-nitrophenyl phosphate (pNPP), a commonly used substrate for alkaline phosphatases. Of the three types of phosphatase examined, the type 2A phosphatase exhibited an especially high pNPP phosphatase activity (119 +/- 8 mumol/min per mg of protein; n = 4). This activity was strongly inhibited by pico- to nano-molar concentrations of okadaic acid, a potent inhibitor of type 2A and type 1 protein phosphatases that has been shown to have no effect on alkaline phosphatases. The dose-inhibition relationship was markedly shifted to the right and became steeper by increasing the concentration of the enzyme, as predicted by the kinetic theory for tightly binding inhibitors. The enzyme concentration estimated by titration with okadaic acid agreed well with that calculated from the protein content and the molecular mass for type 2A phosphatase. These results strongly support the idea that the pNPP phosphatase activity is intrinsic to type 2A protein phosphatase and is not due to contamination by alkaline phosphatases. pNPP was also dephosphorylated, but at much lower rates, by type 1 phosphatase (6.4 +/- 8 nmol/min per mg of protein; n = 4) and type 2C phosphatase (1.2 +/- 3 nmol/min per mg of protein; n = 4). The pNPP phosphatase activity of the type 1 phosphatase preparation shows a susceptibility to okadaic acid similar to that of its protein phosphatase activity, whereas it was interestingly very resistant to inhibitor 2, an endogenous inhibitory factor of type 1 protein phosphatase. The pNPP phosphatase activity of type 2C phosphatase preparation was not affected by up to 10 microM-okadaic acid.

4-Nitrophenylphosphatase↗

PubMed1991

Effects of Ca2+ removal and of tetraethylammonium on membrane currents induced by carbachol in isolated cells from the rat parotid gland.

In freshly dispersed rat parotid acinar cells, 10 microM carbachol increased outward currents at 0 mV and also inward currents at -70 mV recorded with the wholecell clamp method using patch pipettes containing 1 mM EGTA. When EGTA in the pipette was increased to 2.4 mM, carbachol increased only outward currents and a further increase of EGTA to 4 mM blocked the carbachol response. Effects of changes in external K+ and Cl- concentrations suggested that outward currents were carried by K+ and inward by Cl-. Effects of Ca2+ removal from the medium differed between experiments with 0 and 5 mM ATP in the patch pipettes. When pipettes contained no ATP, responses evoked by repeated applications of 10 microM carbachol (0.5-1 min) at 1.5-4 min intervals decreased only slowly after Ca2+ removal, outward currents being reduced to 90 +/- 6% and inward currents to 47 +/- 11% (n = 6) in 10 min. On the other hand, when 5 mM ATP was included in the electrodes, Ca2+ removal abolished the carbachol responses in about 5 min (n = 4). It was also found that tetraethylammonium (5 mM) strongly reduced both currents, by blocking muscarinic receptors, while Ba2+ (2.4 mM) inhibited only the outward K+ current.

Adenosine Triphosphate↗

PubMed1991

Okadaic acid, a protein phosphatase inhibitor, increases the calcium transients in isolated ferret ventricular muscle.

Okadaic acid is a protein phosphatase inhibitor which has been found to produce a marked positive inotropic effect in isolated cardiac muscle. Using aequorin-injected ferret papillary muscles, we demonstrate that the increase in tension seen with okadaic acid is accompanied by a simultaneous increase in the amplitude of the calcium transients. By comparison with the effects of changing the extracellular calcium concentration, it is shown that the increase in calcium transient amplitude can account for the inotropic effect of okadaic acid.

Animals↗

PubMed1991

Okadaic acid, a phosphatase inhibitor, decreases macrophage motility.

Cellular locomotion results from a series of spatially and temporally integrated reactions. The coordinated regulation of these reactions requires sensitive intracellular signaling mechanisms. Because protein phosphorylation reactions represent important signaling mechanisms in mammalian cells, we investigated the effect of okadaic acid, a phosphoprotein phosphatase inhibitor, on protein phosphorylation and macrophage motility. Okadaic acid was applied to rat alveolar macrophages, and motility was quantitated by a directed chemotaxis assay. Okadaic acid inhibits macrophage motility in a dose-dependent fashion; the concentrations for 50 and 100% inhibition were 3 and 25 microM, respectively. Protein phosphorylation studies demonstrated a 2.5-fold increase in total protein phosphorylation in macrophages treated with 25 microM okadaic acid. These experiments also demonstrated a dose-dependent increase in the phosphorylation of the 20-kDa light chain of myosin. Moreover, 25 microM okadaic acid 1) maximally increased myosin light chain phosphorylation by 6.6-fold, 2) raised the level of myosin associated with the cytoskeleton from a basal level of 47.0 to 96.7% of the total myosin, and 3) induced profound morphological changes as visualized by scanning electron microscopy. These data correlate an increase in protein phosphorylation with a decrease in macrophage motility. Furthermore, they suggest that phosphoprotein phosphatase inhibition may prevent motility by uncoupling coordinated processes, such as cytoskeletal reorganization, that are essential for macrophage motility.

Animals↗

PubMed1990

Mucoepidermoid carcinoma of the esophagus: report of two cases and review of the literature.

The clinicopathological manifestation of mucoepidermoid carcinoma of the esophagus are reported in two cases. Barium swallow and endoscopy revealed infiltrating and constricting appearances. The tumors were grossly indurated, with or without ulceration. Histologically, the tumors were composed of a mixture of glandular cells which contained signet ring cells and squamous cells. In two cases, the tumor cells invaded into the adventitia, with lymph node metastasis. Two patients died of wide-spread metastasis within 1 year after operation. Our results and previous reports suggest that mucoepidermoid carcinoma of the esophagus is extremely aggressive and has a poor prognosis.

Carcinoma↗

PubMed1990

Coping styles to basic disorders among schizophrenics.

Coping efforts to subjectively experienced basic disorders were investigated by self-report in 60 chronic schizophrenics in Japan, and the relationships to clinical and demographic variables, intelligence, personality, and attitude toward their illness were analyzed. Self-reports on coping efforts were obtained for more than half of all subjectively experienced basic disorders, and the predominant technique was reported to be the approach oriented towards problem-solving. Within each coping style, the behavioral change, divertive problem-solving approach correlated negatively with hospitalization and symptoms, and the struggle, problem-solving efforts without any effective results correlated positively with symptoms. Suggestions regarding integration of these self-efforts into psychosocial treatment of schizophrenia are discussed.

Adaptation, Psychological↗

PubMed1989

Regulation of Ca2+-dependent K+-channel activity in tracheal myocytes by phosphorylation.

Isoprenaline is a beta-adrenergic agonist of clinical importance as a remedy for asthma. In airway smooth muscle its relaxant action is accompanied by hyperpolarization of the membrane and elevation of the level of intracellular cyclic AMP. Hyperpolarization and relaxation are also induced by drugs such as forskolin, theophylline and dibutyryl cAMP, indicating that cAMP-dependent phosphorylation is involved in producing the electrical response. Cyclic AMP-dependent protein kinase (protein kinase A) has been reported to activate Ca2+-dependent K+ channels in cultured aortic smooth muscle cells and snail neurons. The membrane of tracheal smooth-muscle cells is characterized by a dense distribution of Ca2+-dependent K+-channels. We have now examined the effect of isoprenaline and protein kinase A on Ca2+-dependent K+-channels in isolated smooth muscle cells of rabbit trachea, using the patch-clamp technique. Our results show that the open-state probability of Ca2+-dependent K+-channel of tracheal myocytes is reversibly increased by either extracellular application of isoprenaline or intracellar application of protein kinase A. We also show that this effect is significantly enhanced and prolonged in the presence of a potent protein phosphatase inhibitor, okadaic acid.

Animals↗