Biomedical subjects

R E Yantorno

Publications and source records attributed to R E Yantorno.

5 recordsLinked to original sources

PubMed1992

Regulatory volume decrease by cultured non-pigmented ciliary epithelial cells.

Cells (ODM C1-2/SV40) derived from human non-pigmented ciliary epithelial cells were studied by electronic cell sizing. The time course of the cell volume (vc) was monitored after suspending cells in paired experimental and control, isosmotic and hyposmotic solutions of identical ionic composition. Following anisosmotic cell swelling, the cells displayed the regulatory volume decrease (RVD) previously described. The RVD primarily reflects loss of cell KCl since: (1) the K(+)-channel blockers quinidine and Ba2+ both inhibit the RVD; and (2) replacement of external Cl- with gluconate or addition of the Cl- channel blocker NPPB also inhibits the RVD. Bicarbonate has previously been reported to speed the RVD. This action likely reflects pH dependence of the channels since: (1) increasing the external pH speeds the RVD, whether or not HCO3- is present; and (2) DIDS (a blocker of Cl- channels and of Cl-/HCO3- exchange) is an effective inhibitor of the RVD, even after blocking Cl-/HCO3- exchange by removing external HCO3-. The RVD could also be inhibited by reducing the availability of Ca2+, either by omitting Ca2+ from the external medium or by blocking mobilization of intracellular Ca2+ with TMB-8. Furthermore, the RVD was slowed and incomplete in the presence of the calcium/calmodulin blocker trifluoperazine. We conclude that anisosmotic swelling triggers a series of events, mediated at least in part by calcium/calmodulin, leading to the extrusion of KCl through parallel K+ and Cl- channels.

Bicarbonates↗

PubMed1992

Whole cell patch clamping of ciliary epithelial cells during anisosmotic swelling.

Anisosmotic cell swelling triggers a regulatory volume decrease (RVD) in cell lines derived from human nonpigmented ciliary epithelium. Measurements of cell volume have indicated that the RVD reflects activation of K+ and/or Cl- channels. We have begun to characterize the putative channels by whole cell patch clamping. The results obtained by altering the external K+ and Cl- concentrations and by adding 20-50 microM quinidine or 1 mM Ba2+ indicate that K+ conductances contribute substantially and Cl- conductances contribute very little to the total membrane conductance (GT) under baseline isotonic conditions. Reducing the external osmolality by 20-50% reversibly and reproducibly increased GT by an order of magnitude. Data obtained from ion substitutions and the channel blockers quinidine and 5-nitro-2-(3-phenylpropylamino)-benzoate indicate that most of the hypotonicity-induced conductance reflects stationary Cl(-)-channel activity. The contribution of new K(+)-channel activity was small at intracellular free Ca2+ concentrations of 10 or 200 nM. We conclude that the RVD triggered by bath hypotonicity primarily reflects increased Cl(-)-channel activity.

Chlorides↗

PubMed1989

Volume regulation of cultured, transformed, non-pigmented epithelial cells from human ciliary body.

Electronic cell sizing has been used to measure the volume of cells suspended in isosmotic and in hyposmotic solutions of identical ionic composition. Without inhibitors, the cells displayed a regulatory volume decrease (RVD) following anisosmotic cell swelling with a time constant (tau) of 6.3 +/- 0.9 min (mean +/- S.E.). The RVD was markedly impaired by substituting gluconate for external Cl-, and tau was prolonged by: (i) reducing the chemical gradient favoring K+ loss (by elevating the external [K+] and blocking the Na. K-exchange pump), (ii) blocking the K+ channels with Ba2+, (iii) blocking Cl- channels and Cl-/HCO3 = exchange with DIDS, and (iv) removing external HCO3-. Withdrawing HCO3- may have altered the RVD either directly by inhibiting a Cl-/HCO3- antiport, or indirectly by affecting intracellular pH. The regulatory volume response of ODM Cl-2/SV40 cells is in several respects qualitatively similar to that of non-pigmented epithelial cells of the intact ciliary body. These common characteristics suggest that the cultured cells can serve as a useful model for studying solute and fluid transport across the human ciliary epithelium. The basis for the RVD is likely to be activation of separate K+ and Cl- channels, with or without the parallel operation of coupled K+/H+ and Cl-/HCO3- antiports.

Cell Line, Transformed↗

PubMed1984

Comparative study of the kinetic and structural properties of monomeric and oligomeric forms of sarcoplasmic reticulum ATPase.

Sarcoplasmic reticulum (SR) isolated from rabbit muscle was treated with N-ethyl-maleimide (NEM) to specifically inhibit the dephosphorylation step of the Ca2+,Mg2+-dependent ATPase reaction. However, when this membrane was solubilized with dodecyl octaethyleneglycol monoether (C12E8), rapid decomposition of the phosphoenzyme (EP) was observed both in the absence and presence of Mg2+. When the detergent was removed from the reaction mixture, the inhibition of EP decomposition by NEM was observed again. These results support our previous suggestion (1,2) that in the presence of high concentrations of C12E8, EP may be hydrolyzed to produce P1 in a manner different from the reaction in the native SR ATPase. Gel filtration of the solubilized ATPase was performed in the presence of low concentrations of C12E8 to elute ATPase aggregates of various sizes. Two distinct fractions were selected after column chromatography and their physical and kinetic properties were compared. The molecular weights of the ATPase proteins of these two fractions were determined to be about 150 and 360K daltons with Stokes radii of about 5.5 and 8.0 nm, respectively. The Stokes radii agreed with the values obtained from polarization decay measurement data of N-1-pyrene maleimide (N-1-P)-labeled ATPase aggregates separated on the same column. The rate of EP decomposition was determined for the two column fractions described above. After the addition of EDTA the EP decomposition rate of the smaller-sized ATPase was much higher than the EP decomposition rate of the larger-sized ATPase.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1983

Energy transfer between fluorescent dyes attached to Ca2+,Mg2+-ATPase in the sarcoplasmic reticulum.

Sarcoplasmic reticulum (SR) isolated from rabbit skeletal muscle was solubilized with a nonionic detergent, dodecyl octaethyleneglycol monoether (C12E8), at a weight ratio of detergent to protein of greater than 10, so that the Ca2+, Mg2+ dependent ATPase existed mainly in a monomeric form (7). The solubilized ATPase was reacted with 10 microM N-1-P or 5 microM DACM in the presence of 5 mM CaCl2, 0.4 M KCl, 20% glycerol and 50 mM TES at pH 7.5 and 20 degrees C. Under these conditions, about 1 mol of N-1-P was incorporated into 10(5) g SR protein on 10 min incubation and 1 mol of DACM was incorporated into the same amount of SR on 5 min incubation. Analysis of the tryptic digest of the N-1-P- or DACM-labeled. ATPase on SDS polyacrylamide gel revealed that almost all the fluorescence was associated with the 30K m.w. subfragment of the ATPase protein. Even when the amount of the probe incorporated into SR-ATPase was increased from 1 to 3 mol per 10(5) g SR protein, all was incorporated into the 30K subfragment. Both the activities of formation and decomposition of the phosphorylated intermediate (EP) were unaffected by these modifications. When the separately labeled ATPases were mixed together in the presence of C12E8 and the detergent was removed by incubation with Bio-Beads SM-2, a significant amount of fluorescence energy transfer was observed between N-1-P and DACM. However, energy transfer did not occur when the labeled ATPases were mixed after removal of C12E8.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗