Modulation of volume and intracellular pH in the ciliary body epithelial cells

X ICER Abstracts

Wednesday, Sep 23, 1992 La Palms/C EPITHELIUM

CODE: E-l

SEPTEMBER

23/WEDNE!SDAY

OVERVIEW Civan. M.M, Department Philadelphia,

OOM: LA PALM& MAIRPERSONS;

NUMBERI

9:oo

of Cell vos Civan (USA) n of Volume and

9:20 N.A.

3

odv Farahhakhsh

lntracellulaa

Q&.&lX& (USA)

9:40 J. Fischbarg. 1. Li (USA)

4

(USA) (USA)

PRESENTATION

M.M. 2

JOSE A. ZADUNAISKY MORTIMER M. ClVAN

10:00

contraction -Meshwork I. Zadunaisky, W.G. Robison

M. Echevarria,

P. Iserovich

and

and Area Chance of Human Cells in Tissue K. Spring, 1. Sellers. and S. Cardona (USA)

C~&uw

434

1

OF CELL VOLUME

REGULATION

of Physiology, University PA 19104-6085, USA

of Pennsylvania,

Cell volume regulation is critical for transporting epithelial cells. Volume control reflects changes in free solute content of the cells, with water passively following. At least 10 mechanisms for solute transfer may be involved in regulatory responses to swelling and shrinkage. Organic intracellular solutes may play a prominent role in these responses. Following the redistribution of solute, water may cross the membranes of some cells through channels, as well as nonspecifically across the phospholipid barrier. The cytoskeleton itself may play a role, but probably not in the initial regulatory response to anisosmotic swelling. Despite knowledge of the mechanisms of solute and water transfer, the sensors and signalling pathways involved in integrating the regulatory volume responses have not yet been identified. The aims of the present symposium are to provide an insight into the: (1) mechanisms involved in inorganic and organic solute and water transfer across the cell membranes, (2) transcriptional and translational events involved in organic osmolyte regulation, and (3) possible signalling pathways responsible for integrating cell volume regulation.

435

2 l4ODULATION OF VOLlnm mm BODY BPITEBLIAL CBLLE.

Farah&&bsh. Department UCLA School

INTBAcELLmAR

pH IB THE CILIaRY

N.A. of Ophthalmology, Jules Stein Eye of Medicine, Los Angeles, California,

Institute, U.S.A.

We have combined DIC, fluorescent microscopy and image processing techniques to investigate the presence and role of ion transporting mechanisms in the intact ciliary epithelial cells. Measurable changes in cell volume and intracellular Ph (pIti) could be induced by varying the composition and/or osmolality of the medium bathing the tissue. This could be accomplished either by changing the concentration of ions present in the superfusate (e.g. Na*, HOCs') , or by addition of known transport blockers (e.g. ouabain, amiloride, Ba"). In some cases changes in cell volume and pH, were transient indicating secondary activation of volume or pH regulatory mechanisms. Thus, combination of such maneuvers could be used to perturb the volume and/or pH, in a controlled fashion to help identify the ion transporting mechanisms present in this epithelium. Some applications of this technique will be presented. We have found that acid loading the cells (either directly with acetate pulse or ammonium prepulse, or by readmission of IiC03- to the medium after a prolonged exposure of tissue to HCO,'-free solution), induced an amiloride-blockable transient swelling of cells, accompanied by a biphasic change in pH, apparently as a result of stimulation of Na+/H* exchange. Similarly, (100 nM) led to cell block of sodium pump by ouabain shrinkage, while Ba (10 mM) induced swelling in some cells. Supported by NIH grant EY06969. l

3

436

WATER CHANNELS IN CORNEAL ENDOTHELIAL CELLS. Fischbarn. Echevarria.Departments ofPhysiology andophtlmlmology,Coll. ofphysiciansand Surgeons, ColumbiaUniversity,NewYork,USA. Culturedbovinecomealendothelialcells(CBCEC)transportfluidfromthebasal to the apical compartment. We have found evidence for the presence of water channels in these cells. We utilized the intensity (Is) of light scattered by plated CBCEC to determine both the osmotic (Pf) and diffusional (Pd) cell membrane pmneabilitiestowater. WecalculatedPfand Pd fromthe kineticconstantsforthe osmotic (If) and diffusional (kd) time-transient changes in Is after either anisotonic challenge oranexchangeoftheoutsidemed~:mforanisotonicsolutionwith50% v/v of D20. At 20 "C, kf=O.60?0.05 set (n=9) and Pf = 78?7 pm/s, while kd-0.038+0.0X? secml (n-7) and Pd = 5.0+ 0.3 pm/s, hence Pf/pd-16. The snlfbydryl reagent pCMBS 1 mh4 reduced Pf by 75%; such inhibition could be revetted by 5 mM ditbiotbreitol. In other experiments (at 37 T), pbloretin (0.1 n&f) reduced kf reversibly from 0.75t0.02 to 0.56&0.01, and cytocbalasin B from 0.52+0.045 to 0.47+0.03 se=-‘. For the range 10-37 “C, the activation energy 6) of Pfwas 4.7+0.7 Kcal/mole (n=5). From the comparatively high value of Pf, its low Ea. the inhibitory effects of the protein ligands pCMBS and phloretin, and a ratio Pf/Pd >> 1, we conclude that protein(s) probably exist that form water channels across CBCEC plasma membranes. The main role would be played by B yet unidentified protein(s), with s smaller contribution by glucose transporters. Assuming thatthe water channels forming the main population are single-file, PfiF’d (16) would equal their occupancy number. We calculate a length of 31 A for such a file of H-bonded water molecules. Supp.: Natl.Eye Inst.,NIH; Kidney Intnl.Found.;Juvenile Diabetes Found.

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