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Y Ohya

Publications and source records attributed to Y Ohya.

At least 163 records · Page 9Linked to original sources
PubMed1989

Modulation of single slow (L-type) calcium channels by intracellular ATP in vascular smooth muscle cells.

Involvement of ATP in the regulation of slow (L-type) Ca2+ channels of vascular smooth muscle cells was investigated by recording single Ca2+ channel currents (single-channel conductance of 18 pS) using a patch clamp technique. In the cell-attached configuration, intracellular composition was modified by permeabilizing the cell membrane with mechanical disruption at one end of the cell. Single cells were freshly isolated from guinea-pig portal vein by collagenase treatment. For the channel recordings, the pipette solution contained 100 mM Ba2+ and the bath contained K+-rich solution (with 5 mM EGTA) to depolarize the membrane to near 0 mV. The channel activity decreased usually within 3 min after permeabilizing the cell end and exposure to ATP-free bath solution. If ATP (1-5 mM) was applied to the bath (access to cell interior) before complete disappearance of channel activity, channel activity was partially recovered. ATP did not change the current amplitude (i) or the mean open time of the channels, whereas the number of channels available for opening and/or the probability of their being open (NPo) were increased by ATP. A non-hydrolyzable analogue of ATP, AMP-PNP, did not exert an ATP-like effect; ATP-gamma-S had a weak effect. With 1 microM Bay-K-8644 (Ca2+ channel agonist) in the pipette, the activity of the Ca2+ channel was high; such activity persisted for more than 10 min after permeabilizing the cell and exposing to ATP-free solution containing KCN (1 mM) and 2-deoxy-D-glucose (10 mM).(ABSTRACT TRUNCATED AT 250 WORDS)

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1989

Fast Na+ and slow Ca2+ channels in single uterine muscle cells from pregnant rats.

Whole cell voltage-clamp method was applied to single smooth muscle cells freshly isolated from the longitudinal layer of 18-day pregnant rat uterus. Inward currents were isolated after outward currents were minimized by use of high Cs+ in the pipette solution and 4-aminopyridine (3 mM) in the bath solution. Depolarizing pulses, applied from a holding potential of -90 mV, evoked two types of inward current, fast and slow. The fast inward current decayed and disappeared within 30 ms and depended on extracellular Na+ concentration. This fast current was inhibited by tetrodotoxin (TTX) dose dependently (KD = 27 nM). These results suggest that the fast inward current was a TTX-sensitive Na+ channel current. In contrast, the slow inward current decayed slowly, dependent on extracellular Ca2+ (or Ba2+) concentration, and was inhibited by the Ca2+ channel blocker, nifedipine, dose dependently (10 nM-10 microM). These results suggest that the slow inward current was a Ca2+ channel current. A fast-inactivating Ca2+ channel current was not evident when Ba2+ was the charge carrier. We conclude that the major ion channels in the cell membrane of pregnant rat uterus, which generate inward currents, are TTX-sensitive fast Na+ channels and dihydropyridine-sensitive slow Ca2+ channels (L-type, high-threshold type).

Animals↗
PubMed1989

ATP regulation of the slow calcium channels in vascular smooth muscle cells of guinea pig mesenteric artery.

Effects of intracellularly perfused ATP, and extracellularly applied cyanide and 2-deoxy-D-glucose, on fast and slow Ca2+ channel currents of isolated single vascular smooth muscle cells were investigated by a whole-cell voltage-clamp method combined with an intracellular perfusion technique. Single smooth muscle cells were prepared by collagenase treatment from guinea pig small mesenteric arteries (diameter of less than 300 micron). With Cs+-rich solution in the pipette and isotonic Ba2+ solution (100 mM) in the bath, depolarizing pulses evoked two types of the Ca2+ channel current. Depolarizing pulses from the holding potential of -80 mV to over -30 mV evoked a fast Ca2+ channel current. This fast component was inhibited by shifting the holding potential in a positive direction. With a holding potential of -40 mV, the fast component was almost inhibited. In contrast, the slow current was evoked by command potentials to above -10 mV, and its full amplitude was preserved at the holding potential of -40 mV. Without ATP in the pipette, the fast current was dominant. Increase in the ATP concentration in the pipette (0.3 to 5 mM) enhanced the slow current but did not affect the fast current. Maximum enhancement of the slow current was observed at 5 mM ATP. Increase in ATP concentration, however, did not modify the shape of the current trace and the steady state inactivation curve of the slow current. Maximum amplitudes of the fast current and slow current recorded with 5 mM ATP averaged 17.4 pA (SD of 10.4 pA, n = 30; observed at -10 mV to +10 mV) and 141.8 pA (SD of 27.1 pA, n = 30; observed at +30 mV to +40 mV), respectively. Presence of CN- and 2-deoxy-D-glucose (without glucose) in the bath, and absence of ATP in the pipette, abolished the slow current within 10 minutes; in contrast, it took more than 10 minutes to depress the fast current. The inhibitory effect of CN- and 2-deoxy-D-glucose on the slow current was reduced by intracellular application of ATP. In summary, the activation of the slow Ca2+ channel required physiological concentration of ATP, whereas the fast channel current was preserved, even under ATP-free conditions. These results indicate that only the slow current is a metabolically dependent Ca2+ channel current in these vascular smooth muscle cells.

Adenosine Triphosphate↗
PubMed1988

Operon structure of flagellar genes in Salmonella typhimurium.

In Salmonella typhimurium, more than 40 genes have been shown to be involved in flagellar formation and function and almost all of them have been assigned to three regions of the chromosome, termed region I, region II, and region III. In the present study, a large number of transposon-insertion mutants in these flagellar genes were isolated using Tn10 and Mud1. The flaV gene was found to be a strong hot spot for Tn10 insertion. Complementation analysis of the polarity effects exerted by the transposon-insertion mutants defined 13 different flagellar operons; 3 in region I, 4 in region II, and 6 in region III. These results are compared with the reported arrangement of the corresponding genes in Escherichia coli.

DNA Transposable Elements↗
PubMed1988

Ethanol-induced gastric mucosal blood flow and vascular permeability changes in the rat.

Ethanol-induced gastric mucosal injury is accompanied by complete cessation of blood flow in the lesion area. An in vivo microscopy technique was used in the rat to determine whether this cessation of blood flow could be due to increased microvascular permeability with marked plasma exudation and a resultant increase in blood viscosity. The topical application of 100% ethanol to the mucosa caused complete stasis of mucosal blood flow within 1 min. Fluorescent in vivo microscopy revealed that topical ethanol also caused a prompt increase in mucosal microvascular permeability to albumin. This, however, did not explain the slowing and cessation of mucosal microvascular blood flow as these preceded the permeability change.

Animals↗
PubMed1988

Effects of inositol phosphates on the membrane activity of smooth muscle cells of the rabbit portal vein.

The effects of intracellular perfusion of inositol 1,4,5-trisphosphate (InsP3) or inositol 1,3,4,5-tetrakisphosphate (InsP4) on electrical responses of smooth muscle cell membranes of the rabbit portal vein were studied using the whole cell voltage clamp technique. Depolarisation to 0 mV from a holding potential of -60 mV, evoked inward Ca (Ica), transient outward (ISO), oscillatory outward (IOO) and sustained outward (ISO) currents. Generation of IOO was dependent on the [Ca]o, but it was also generated in 0 mM Ca solution for over 10 min. From amplitude histograms, IOO was divided into two components. Reduction in [Ca]o inhibited the appearance of but not the amplitudes of both IOO components. However, the larger component of IOO was more resistant to a reduction in [Ca]o than the smaller one. InsP3 (10 microM) increased the frequency of both IOO components to a greater extent than their amplitude, but the larger component was more sensitive to InsP3 than the smaller one. The increase in the occurrence of IOO induced by InsP3 did not occur following pretreatment with 3 mM caffeine or 1 nM A23187. In normal PSS, InsP3 was evoked by a depolarising pulse positive to -40 mV, whereas following perfusion with InsP3 (10 microM), IOO was evoked at -60 mV. In normal PSS, intracellular perfusion with 10 microM InsP4 changed neither the frequency nor the amplitude of IOO, and the amplitudes of ICa, ITO and ISO were also unchanged. However, in 10 mM Ca solution, 10 microM InsP4 generated IOO at a membrane potential of -60 mV.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Regulation of calcium current by intracellular calcium in smooth muscle cells of rabbit portal vein.

Effects of concentrations of intracellular calcium, [Ca2+]i, on the voltage-dependent Ca2+ current (ICa) recorded from dispersed single smooth muscle cells of the rabbit portal vein were studied, using a whole cell voltage clamp method combined with an intracellular perfusion technique. Outward currents were minimized by replacement of Cs+ -rich solution in the pipette and 20 mM tetraethylammonium in the bath. The ICa was evoked by command pulses of above -30 mV, and the maximum amplitude was obtained at about 0 mV. This ICa was dose dependently inhibited by increases in the [Ca2+]i above 30 nM. The Kd value of the [Ca2+]i required to inhibit the ICa was about 100 nM. The Ba2+ current was also inhibited by increases in the [Ca2+]i. Conversely, perfusion of Ba2+ into the cell up to 100 microM did not suppress the ICa. Changes in the [Ca2+]i did not modify the steady-state inactivation curve. The inhibition of the ICa evoked by the test pulse is most prominent when the preceding influx of Ca2+ during the conditioning pulse was large, as estimated using a double pulse protocol. This inhibition was proportionally reduced by increases in the concentration of the Ca2+ chelator, ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). Therefore, the Ca2+ -dependent inactivation of the Ca2+ channel may contribute toward regulating [Ca2+]i in smooth muscle cells of the rabbit portal vein.

Animals↗
PubMed1987

Purification and biochemical properties of calmodulin from Saccharomyces cerevisiae.

Calmodulin from the yeast Saccharomyces cerevisiae was purified to complete homogeneity by hydrophobic interaction chromatography and HPLC gel filtration. The biochemical properties of the purified protein as calmodulin were examined under various criteria and its similarity and dissimilarity to other calmodulins have been described. Like other calmodulins, yeast calmodulin activated bovine phosphodiesterase and pea NAD kinase in a Ca2+-dependent manner, but its concentration for half-maximal activation was 8-10 times that of bovine calmodulin. The amino acid composition of yeast calmodulin was different from those of calmodulins from other lower eukaryotes in that it contained no tyrosine, but more leucine and had a high ratio of serine to threonine. Yeast calmodulin did not contain tryptophanyl or tyrosyl residues, so its ultraviolet spectrum reflected the absorbance of phenylalanyl residues, and had a molar absorption coefficient at 259 nm of 1900 M-1 cm-1. Ca2+ ions changed the secondary structure of yeast calmodulin, causing a 3% decrease in the alpha-helical content, unlike its effect on other calmodulins. Antibody against yeast calmodulin did not cross-react with bovine calmodulin, and antibody against bovine calmodulin did not cross-react with yeast calmodulin, presumably due to differences in the amino acid sequences of the antigenic sites. It is concluded that the molecular structure of yeast calmodulin differs from those of calmodulins from other sources, but that its Ca2+-dependent regulatory functions are highly conserved and essentially similar to those of calmodulins of higher eukaryotes.

Amino Acids↗
PubMed1987

D600 blocks the Ca2+ channel from the outer surface of smooth muscle cell membrane of the rabbit intestine and portal vein.

The voltage dependent Ca2+ inward current in single smooth muscle cells dispersed from the longitudinal muscle layer of the rabbit ileum and rabbit portal vein was recorded using the whole-cell voltage clamp technique. D600 added to the bathing solution inhibited the Ca2+ current, while the intracellular perfusion of this agent did not reduce the amplitude of this current. Thus, D600 probably acts from the outer surface of the membrane. The nature of the Ca2+ channel in smooth muscle cells seems to differ from that in cardiac muscle cells.

Animals↗
PubMed1987

Modulation of ionic currents in smooth muscle balls of the rabbit intestine by intracellularly perfused ATP and cyclic AMP.

The effects of intracellularly perfused ATP and cyclic-AMP (c-AMP) on ionic currents recorded from fragmented smooth muscle cells (smooth muscle ball; SMB) were investigated, using the single electrode whole cell voltage clamp method. The Ca2+ current was distinguished from K+ currents, using pipette solution containing Cs+, TEA+ and 4 mM EGTA. ATP enhanced the Ca2+ current dose-dependently between 0.3 and 10 mM, and slightly slowed the slow component of the decay of the Ca2+ current, while the steady-state inactivation curve remained unaffected. Intracellular application of 5'-adenylyl-imidodiphosphate (AMP-PNP; 1 mM) inhibited the Ca2+ current by competition with ATP, but c-AMP (up to 300 microM) had no effect. With a high-K+ solution containing 0.3 mM EGTA and ATP in the pipette and physiological salt solution in the bath, a net inward current with transient (Ca2+ dependent) and delayed (Ca2+ independent) K+ outwart currents were evoked. Increased concentrations of ATP (above 1 mM) but not c-AMP (up to 100 microM) in the pipette enhanced the transient K+ outward current. Neither agent had any effect on the delayed outward current. When repetitive stimulations of intervals shorter than 5 s were applied, the amplitude of the transient outward current was markedly reduced, and 100 microM c-AMP partially prevented this attenuation. ATP may act on the Ca2+ channel either by phosphorylating the channel protein or by other ATP requiring mechanisms, independently from those induced by the action of c-AMP.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1987

Cloning and expression of the gene(s) for chromosome-mediated beta-lactamase production of Proteus vulgaris in Escherichia coli.

The gene(s) for chromosome-mediated beta-lactamase production of Proteus vulgaris GN7919 was cloned into a unique EcoRI site of pACYC184 as an insert of a 14.2-kb fragment, which was further digested into two fragments with EcoRI, 4.9 and 9.3 kb. The restriction enzyme digestion pattern of the recombinant plasmid, designated pMS182, had no similarity to those of other chromosomal beta-lactamase genes cloned from gram-negative bacteria. Plasmid pMS182 enabled host Escherichia coli ML4953 to inducibly produce beta-lactamase which was identical to that of the parent P. vulgaris in substrate profile, molecular weight, and reactivity to antiserum raised against P. vulgaris GN7919 beta-lactamase. The pMS182-harboring E. coli were highly resistant to beta-lactam antibiotics, possibly based on inducible production of beta-lactamase.

Chromosomes, Bacterial↗
PubMed1987

Nucleotide sequence of the CLS4 (CDC24) gene of Saccharomyces cerevisiae.

The nucleotide sequence of the CLS4 gene controlling Ca2+ regulatory process of bud emergence, which was cloned previously [Ohya et al., J. Bacteriol. 165 (1986) 28-33], was determined. The CLS4 (CDC24) locus encodes a protein consisting of 736 amino acid (aa) residues with an Mr of 83,970. By primer extension mapping, the mRNA start point was located 139 bp upstream from the translation start codon. The predicted CLS4 protein was hydrophilic with two serine + threonine-rich domains in the middle and C-terminal regions. It has two putative Ca2+-binding regions, one being partly homologous to the Ca2+-binding domain of the S-100a protein and the other that of alpha-lactalbumin.

Base Sequence↗
PubMed1987

Cellular calcium regulates outward currents in rabbit intestinal smooth muscle cell.

The nature of transient and oscillatory outward currents (ITO and IOO) in fragmented smooth muscle cells (smooth muscle ball, SMB) from the longitudinal muscle layer of the rabbit ileum, was studied using a single electrode voltage clamp technique. With a high K+ solution containing 0.3 mM ethyleneglycol-bis(beta-aminoethylether)-N,N'-tetraacetic acid (EGTA) in the pipette and physiological salt solution (PSS) in the bath, the Ca inward current was followed by a large transient outward current (ITO) and spontaneous oscillations of the outward current (IOO) on the sustained outward current (ISO) were elicited by a depolarizing pulse, positive to -30 mV (holding potential of -60 mV). When the internal fluid of the SMB was replaced with Cs+-tetraethylammonium+ (TEA+) solution, or when the concentration of EGTA in the pipette was increased to 4 mM, using the intracellular perfusion technique, both ITO and IOO were abolished. In Mn2+ solution both currents were also inhibited. Bath application of TEA+, procaine or A23187 completely blocked both ITO and IOO. Caffeine (0.3-1 mM) enhanced the amplitude of ITO and generations of IOO, and concentrations of caffeine over 3 mM transiently enhanced, but finally suppressed both these currents. These results suggest that the generation of ITO is closely related to the Ca2+ influx, whereas the generation of IOO may be initiated by an increment in the intracellular concentration of Ca2+, possibly released from store sites.

Animals↗
PubMed1987

Guanidino compounds in hyperargininemia.

Plasma and cerebrospinal fluid (CSF) concentrations and urinary excretion of guanidino compounds were investigated in a patient with hyperargininemia during the treatment with low-protein diet, oral administration of an essential amino acid mixture and sodium benzoate, or enzyme replacement therapy such as exchange transfusion or erythrocyte transfusion. In the patient, alpha-keto-delta-guanidinovaleric acid (GVA), N-alpha-acetylarginine (NAA), argininic acid (ArgA) and homoarginine concentrations in plasma were elevated as well as arginine. Urinary excretion of GVA, ArgA, NAA and gamma-guanidinobutyric acid (GBA) were also increased. CSF concentrations of ArgA, homoarginine and arginine were also elevated. On the other hand, guanidinosuccinic acid (GSA), which is usually detected in all samples, was not detected in plasma, CSF and urine of the patient. The present results suggest that in patients with hyperargininemia other factors such as arginine and its metabolites including GVA, GAA, ArgA and homoarginine may cause the neurological symptoms. Furthermore, it is suggested that in patients with hyperargininemia, arginine may be catabolized via other pathways and nitrogen may be excreted partially in urine in the form of some of guanidino compounds.

Arginase↗
PubMed1987

Actions of flunarizine, a Ca++ antagonist, on ionic currents in fragmented smooth muscle cells of the rabbit small intestine.

Actions of flunarizine on the Ca++ inward and K+ outward currents were investigated using fragmented smooth muscle cells (smooth muscle ball) prepared from the longitudinal muscle layer of the rabbit ileum. Flunarizine dose dependently inhibited the Ca++ inward current (ID50 = 1.4 microM). The decay of the inward current consisted of two exponentials and flunarizine had no effect on these time constants. When command pulses (100 msec; stepped up to 0 mV from -60 mV) were applied every 20 sec, the peak amplitude of the inward current remained unchanged. Flunarizine above 0.3 microM slowly inhibited the peak amplitude of inward current, in a voltage- and use-dependent manner. Intracellular perfusion of flunarizine, up to 100 microM, did not modify the peak amplitude of the inward current. This Ca++ antagonist also inhibited the K+ outward current, in a dose-dependent manner (ID50 = 5.8 microM) and accelerated inactivations of this current. When the command pulses (300 msec; stepped up to +20 mV from -60 mV) were applied repetitively every 20 sec, amplitudes of the K+ outward current were not affected. However, flunarizine, above 1 microM, reduced the peak amplitude of the K+ outward current slowly. These results indicate that although flunarizine possesses the property of a Ca++ antagonist, it also inhibits the K+ outward current, in a manner different from that observed on the Ca++ inward current.

Animals↗
PubMed1986

Isolation and characterization of Ca2+-sensitive mutants of Saccharomyces cerevisiae.

Thirty Ca2+-sensitive (cls: calcium sensitive) mutants of Saccharomyces cerevisiae were isolated by replica-plating. These mutants, which each had a single recessive chromosomal mutation, were divided into 18 complementation groups. Some cls mutants showed a phenotype of specific sensitivity to Ca2+, while others showed phenotypes of sensitivities to several divalent cations. From measurements of the calcium contents and initial rates of Ca2+ uptake of the cls mutants, 16 of the 18 cls complementation groups were classified into four types: type I mutants (cls5, cls6, cls13, cls14, cls15, cls16, cls17, and cls18) had both elevated calcium contents and increased uptake activities. A type II mutant (cls4) had a normal calcium content and normal uptake activity; type III mutants (cls1, cls2 and cls3) had elevated calcium contents but normal initial rates of Ca2+ uptake; type IV mutants (cls8, cls9, cls10 and cls11) had normal calcium contents but increased initial rates of Ca2+ uptake. Two of the mutants (cls7 and cls12) had intermediate biochemical properties. The primary defects of these four types of cls mutants were considered in terms of the Ca2+ transport system(s). Both type I and type III mutants, which had elevated calcium contents, simultaneously showed a trifluoperazine-sensitive phenotype, suggesting a close correlation of this phenotype with elevated calcium content. In addition, all type IV mutants were unable to utilize nonfermentable sugars. One CLS gene, CLS7, was located on the left arm of chromosome V.

Calcium↗