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A Hazama

Publications and source records attributed to A Hazama.

At least 37 records · Page 2Linked to original sources
PubMed1994

Transport of cycasin by the intestinal Na+/glucose cotransporter.

The medicinal and food use of seed from the cycad plant (Cycas spp.), which contains the neurotoxin cycasin, is a proposed etiological factor for amyotrophic lateral sclerosis/Parkinsonism dementia complex (ALS/PDC), a prototypical neurodegenerative disease found in the western Pacific. Cycasin, the beta-D-glucoside of methylazoxymethanol might enter neurons and other cells via a glucose transporter. Since the intestinal brush-border Na+/glucose cotransporter plays a major role in the absorption of monosaccharides, the following studies were conducted to determine if cycasin, the beta-D-glucoside of methylazoxymethanol, is a substrate for the transporter. We measured the ability of cycasin to (i) inhibit Na+/glucose uptake into rabbit intestinal brush-border membrane vesicles, and (ii) to generate current by the cloned Na+/glucose cotransporter (SGLT1) expressed in Xenopus laevis oocytes. The results show that cycasin inhibits Na(+)-dependent sugar transport in the vesicles, and cycasin generates phlorizin-sensitive currents in oocytes. We conclude that cycasin is a substrate for the intestinal brush-border Na+/glucose cotransporter, albeit with a lower affinity than D-glucose. This suggests that cycasin may be absorbed from the gut lumen by the cotransporter, and as a result either cycasin or the aglycone is presented to the blood-brain barrier for uptake into the brain.

Animals↗
PubMed1994

Properties of volume-sensitive Cl- channels in a human epithelial cell line.

A regulatory volume decrease is accomplished by parallel activation of Ca(2+)-dependent K+ channels and Ca(2+)-independent Cl- channels in cultured human intestinal epithelial cells (Intestine 407). The anion selectivity of whole-cell currents recorded in osmotically swollen cells falls into the Eisenman type I sequence corresponding to a low-field anion channel. The volume-sensitive Cl- channel has an intermediate unitary conductance. Both the whole-cell and single-channel Cl- currents exhibit unique voltage-dependency. The Cl- current can be maintained in the activated state in the physiological voltage range. However, at very large depolarizations (over +50 mV), the current is quickly inactivated. The Cl- current shows moderate outward rectification. The whole-cell Cl- current is sensitive to Cl- channel blockers such as SITS and NPPB as well as to cis unsaturated fatty acids such as arachidonic acid and oleic acid. The whole-cell current is totally independent of Ca2+ and cyclic AMP, but inhibited by increases in cytosolic free Mg2+ ions. Removal of intracellular ATP, but not Mg2+, abolishes the Cl- current. The ATP role can be substituted for non-hydrolyzable ATP analogs. Therefore, it is likely that intracellular ATP maintains the channel activity through non-hydrolytic binding.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed1993

Relaxation kinetics of the Na+/glucose cotransporter.

An important class of integral membrane proteins, cotransporters, couple solute transport to electrochemical potential gradients; e.g., the Na+/glucose cotransporter uses the Na+ electrochemical potential gradient to accumulate sugar in cells. So far, kinetic analysis of cotransporters has mostly been limited to steady-state parameters. In this study, we have examined pre-steady-state kinetics of Na+/glucose cotransport. The cloned human transporter (hSGLT1) was expressed in Xenopus oocytes, and voltage-clamp techniques were used to monitor current transients after step changes in membrane potential. Transients exhibited a voltage-dependent time constant (tau) ranging between 2 and 10 ms. The charge movement Q was fitted to a Boltzmann relation with maximal charge Qmax of approximately 20 nC, apparent valence z of 1, and potential V0.5 of -39 mV for 50% Qmax. Lowering external Na+ from 100 to 10 mM reduced Qmax 40%, shifted V0.5 from -39 to -70 mV, had no effect on z, and reduced the voltage dependence of tau. Qmax was independent of, but tau was dependent on, temperature (a 10 degrees C increase increased tau by a factor of approximately 2.5 at -50 mV). Addition of sugar or phlorizin reduced Qmax. Analyses of hSGLT1 pre-steady-state kinetics indicate that transfer upon a step of membrane potential in the absence of sugar is due to two steps in the reaction cycle: Na+ binding/dissociation (30%) and reorientation of the protein in the membrane field (70%). The rate-limiting step appears to be Na+ binding/dissociation. Qmax provides a measure of transporter density (approximately 10(4)/microns 2). Charge transfer measurements give insight into the partial reactions of the Na+/glucose cotransporter, and, combined with genetic engineering of the protein, provide a powerful tool for studying transport mechanisms.

Animals↗
PubMed1992

Exocytosis upon osmotic swelling in human epithelial cells.

Upon osmotic swelling human epithelial cells exhibited significant increases in the membrane capacitance. Evidence for exocytosis includes its dependency on temperature, cytosolic Ca2+ and ATP as well as its sensitivity to guanosine 5'-O-(3-thio)triphosphate (GTP gamma S) and N-ethylmaleimide (NEM). A role of the osmotic exocytosis in the subsequent cell volume regulation is suggested.

Adenosine Triphosphate↗
PubMed1992

Membrane capacitance increases induced by histamine and cyclic AMP in single gastric acid-secreting cells of the guinea pig.

During acid secretion, gastric parietal cells undergo profound morphological changes including formation of the apical secretory membrane. To examine the mechanism of histamine-induced increases in the apical membrane area at the single cell level, we monitored the membrane capacitance by applying a time-resolved phase-sensitive detection method to singly isolated parietal cells of guinea pig. A real-time increase in the membrane capacitance was detected within several min after stimulation with histamine. An H2-blocker (cimetidine), but not an H1-blocker (pyrilamine), inhibited the histamine response. Dibutyryl cyclic AMP mimicked the histamine effect. The capacitance response to histamine was sensitive to cytosolic Ca2+, temperature and N-ethylmaleimide. The histamine response was inhibited by intracellular application of a non-hydrolyzable ATP analog (AMP-PNP) and an isoquinolinesulfonamide derivative that works as an inhibitor of protein kinase A (H-8). These results indicate that in parietal cells, elevation of intracellular cyclic AMP induces exocytotic insertion of intracellular membranes into the plasma membrane, presumably by activating protein kinase A.

Animals↗
PubMed1991

Evidence for a cytosolic inhibitor of epithelial chloride channels.

It has been known for several years that the outwardly rectifying 30-pS chloride channel, the regulation of which has been reported to be defective in cystic fibrosis, can be activated by excision of a membrane patch from a cell. This suggested that the cytosol contains an inhibitory factor, which diffuses away after excision, thereby releasing the channel block. To test for such an inhibitor we have isolated cytosol from two epithelial cell lines, and in larger quantities from pig kidney cortex. Kidney cortex was chosen because published and unpublished evidence suggested that proximal tubular cells might also have a tonically suppressed Cl- conductance in the brush-border membrane, which is activated during isolation of membrane vesicles. The inhibitory effect of the cytosol preparations was assessed by: (a) measuring conductive Cl- fluxes on renal proximal tubular brush-border membrane vesicles preloaded with or without cytosol, and (b) recording single Cl- channel currents from excised membrane patches of nasal polyp epithelia and CFPAC-1 cells in the presence and absence of cytosol. All cytosol preparations tested were found to inhibit both conductive Cl- flux in membrane vesicles and single Cl- channels in patch-clamp experiments. In the latter case a type of flicker block was observed with a reduction of channel open probability. Stepwise dilution of the cytosol consistently reduced the inhibitory potency. Since the inhibition was preserved after boiling the cytosol for 10 min, we conclude that the inhibitor is a heat-stable substance. Whether it is identical with the postulated intracellular regulator that couples the defective function of the cystic fibrosis gene product to Cl- channel inhibition cannot be decided at present.

Animals↗
PubMed1990

Atrial natriuretic peptide reduces the basal level of cytosolic free Ca2+ in guinea pig cardiac myocytes.

The cytosolic free Ca2+ concentration ([Ca2+]i) was monitored in quiescent atrial and ventricular myocytes isolated from guinea-pig hearts by the fura-2 fluorescence ratio technique. Recombinant human atrial natriuretic peptide (ANP) was found to reduce their basal [Ca2+]i level in a dose-dependent manner. Dibutyryl-cGMP mimicked the effect of ANP. Neither the prior application of caffeine nor removal of extracellular Na+ impaired the ANP effect. ANP had no inhibitory effect on voltage-gated Ca2+ currents measured by a whole-cell patch clamp technique. The ANP-induced [Ca2+]i decrease was abolished by orthovanadate. Thus, it is concluded that ANP reduces the basal [Ca2+]i presumably through the cGMP-mediated activation of the plasma membrane Ca2(+)-pump in cardiac myocytes.

Animals↗
PubMed1990

Involvement of Ca2(+)-induced Ca2+ release in the volume regulation of human epithelial cells exposed to a hypotonic medium.

Exposure of cultured human epithelial cells (Intestine 407) to a hypotonic solution results in initial osmotic swelling and in a subsequent volume decrease near to the original level. The regulatory volume decrease was inhibited by reduction of the extracellular free Ca2+ concentration to 90 nM. Single epithelial cells responded to a hypotonic challenge with a biphasic increase in the cytosolic free Ca2+ level from about 90 to 200 nM. Both phases of the Ca2+ rise were abolished by reducing the extracellular Ca2+ to 90 nM. In the presence of caffeine (20 mM), the second-phase Ca2+ response to a hypotonic challenge occurred earlier immediately after the first-phase response. The second-phase Ca2+ response was selectively impaired by adenine (10 mM), procaine (1 mM) or ryanodine (5 to 10 microM). These blockers for Ca2(+)-induced Ca2+ release channels inhibited volume regulation after osmotic swelling. It is concluded that Ca2(+)-induced Ca2+ release from a ryanodine-sensitive store is a prerequisite for the volume regulation of human intestinal epithelial cells under hypotonic conditions.

Calcium↗
PubMed1990

Biphasic rises in cytosolic free Ca2+ in association with activation of K+ and Cl- conductance during the regulatory volume decrease in cultured human epithelial cells.

During exposure to a hypotonic solution (55% osmolarity), cultured human epithelial (Intestine 407) cells exhibit a regulatory volume decrease after osmotic swelling. This process is known to involve parallel activation of volume-regulatory K+ and Cl- conductances. Biphasic increase in the cytosolic free Ca2+ concentration ([Ca2+]i) were observed by microspectrofluorometry, in fura-2-loaded cells upon hypotonic stress. Electrophysiological studies with Ca2(+)-selective and conventional microelectrodes indicated that a biphasic [Ca2+]i increase was associated with a biphasic hyperpolarization, whereas an interposing [Ca2+]i decrease coincided with a transient depolarization. A Ca2+ ionophore, ionomycin, produced a sustained Ca2+ increase and a prolonged hyperpolarization which was sensitive to the K+ channel blocker, quinine. A subsequent hypotonic challenge gave rise to a depolarization, which was sensitive to a stilbene-derivative Cl- channel blocker, without inducing further changes in [Ca2+]i. Normal cell volume regulation in a hypo-osmotic medium could take place even in the presence of ionomycin. It is concluded that a biphasic [Ca2+]i increase is closely associated with activation of the volume-regulatory K+ conductance, and that the interposing [Ca2+]i decrease is neither a causative factor for activation of the volume-regulatory Cl- conductance nor a prerequisite for regulatory volume decrease in epithelial cells exposed to a hypotonic solution.

Biological Transport↗
PubMed1988

Ca2+ sensitivity of volume-regulatory K+ and Cl- channels in cultured human epithelial cells.

1. During exposure to a hypotonic solution, cultured human epithelial cells (Intestine 407) exhibited a regulatory volume decrease (RVD) after initial osmotic swelling. 2. The volume readjustment was slowed by elevating the extracellular K+ concentration and facilitated by reducing the extracellular Cl- concentration. Not only putative K+ channel blockers, quinine and Ba2+, but also a stilbene derivative Cl- channel blocker (SITS) inhibited the RVD. 3. The volume recovery of hypoosmotically swollen cells was very much suppressed by the deprivation of extracellular Ca2+ ions or by chelation of cytosolic Ca2+ ions with Quin-2 loaded within the cells. 4. Biphasic membrane potential changes were associated with the RVD process at low extracellular K+ and Cl- concentrations. The initial hyperpolarizing response was inhibited by quinine and Ba2+, whereas the late depolarizing response was inhibited by SITS. The deprivation of extracellular Ca2+ inhibited the initial hyperpolarizing phase but not the late depolarizing phase. 5. Two-microelectrode voltage clamp studies showed that the initial hyperpolarization and late depolarization were associated with quinine-sensitive outward currents and SITS-sensitive inward currents, respectively. The reversal potentials estimated from the current-voltage curves were about -80 mV for the initial response and -27 mV for the late response. Tenfold changes in the K+ and Cl- concentrations shifted these reversal potentials by 50 mV for the initial response and by 42 mV for the late response. 6. Under whole-cell recordings, similar current changes were observed in the cells exposed to a hypotonic solution, when the intracellular Ca2+ ions were moderately buffered with 1 mM-EGTA in the dialysing solution filled in a patch pipette. When most Ca2+ ions were chelated with 10 mM-EGTA in the pipette solution, the initial outward current as well as the corresponding hyperpolarization was suppressed, but the late current associated with the depolarizing phase was preserved. 7. Intracellular Ca2+ injections induced an increase in the quinine-sensitive K+ conductance but failed to activate the Cl- conductance. 8. It is concluded that both K+ and Cl- channels are involved in the regulatory volume decrease, and that the former channel is exclusively activated by elevation of the cytosolic Ca2+ concentration in the epithelial cells.

Action Potentials↗
PubMed1985

HeLa cells have histamine H1-receptors which mediate activation of the K+ conductance.

HeLa cells responded to exogenous histamine with a transient hyperpolarization due to increased membrane conductance to K+. After successive applications of histamine, the cell membrane became virtually unresponsive (desensitized). The responses were blocked by pyrilamine but not by cimetidine. Thus, it appears that HeLa cells have H1-receptors which mediate an increase in the K+ conductance.

Cell Membrane↗
PubMed1981

Parainfluenza 3 virus: plaque-type variants lacking neuraminidase activity.

Virus clones lacking detectable neuraminidase activity (SC-YN and M-YN) as well as those possessing it (LT-910N and LT-YN) were isolated from bovine strains of parainfluenza 3 virus. LT-910N and LT-YN viruses produced large turbid plaques in MDBK cells, and SC-YN virus produced small clear plaques. Incorporation of a bacterial neuraminidase in agar overlay medium made SC-YN virus form large turbid plaques, whereas it made M-YN virus form large clear plaques. However, M-YN virus formed only pinhole plaques or no plaques in the absence of neuraminidase. The exogenous neuraminidase had little effect on the plaque formation of LT-910N and LT-YN viruses. M-YN virus induced extensive syncytial formation, and SC-YN virus produced less extensive syncytial formation. The exogenous neuraminidase enhanced replication of SC-YN and M-YN viruses and reduced syncytial formation by these viruses. The enzyme had little effect on replication and cytopathic effect of LT-910N and LT-YN viruses. The reason for these effects of the exogenous neuraminidase is discussed.

Animals↗