Ionic transport across corneal endothelium

Exp.

Eyt- Res. (1981)

Ionic

32. 673479

and Water Transference Numbers of Descemet’s Membrane of the Bovine Cornea ATLE

Department

LYSLO,

KVERNES

of Ophthalmology, Faculty of fiZedicine, 7000 Trondheim, Norway AND SIGNE

Laboratory

SIGMUND

of Physicul

KJELSTRUP

University

RATKJE

Chemistry, The Norwegian 7034 Trondhedm, Norway

Institute

(Received 7 May 1980, New Transport properties the following cell :

of Descemet’s

Buffer

membrane

of Trondheim,

(DM)

from

of Technology,

York)

bovine

corneas

have been studied

in

MCI,, C, 1 j> MCI,. C* pH = 7.3 \ DM) Buffer pH = 7.3

measurements for gradients ML+ are metals Li+ to Rb+, Mg *+ to Sr*+, La3+ and Ce3+. E.m.f. (I1 :C, = 2 : 1 yield cation transference numbers, tMz+ decreasing in value with z. Changes in pH potential measurements give water transference or temperature have little effect ant,, + Streaming numbers, varying dramatically in magnitude with concentration and type of ion present. The data show that Descemet’s membrane has no ion selectivity for monovalent salts. For di- and trivalent salts DM behaves as an ion-exchange membrane with weak preference for anions. The water transfer is in the direction of the cation transfer, and the results arc similar to those reported for porous inorganic membranes. Key u~ds: Descemet’s membrane; ionic transport; water transport: e.m.f. : streaming potentials.

1. Introduction Basement membranes give mechanical support to endothelial cell walls. They are regular collageneous structures with large pores (see Kefalides, 1973 for a general review). Apart from a filtering action, the membrane has been considered as inert, making no restrictions on transport to and from the stroma. The endothelial cells of cornea are believed to control the water cvntents of strvma. and to actively regulate the transport of at least some ions to the interior of the cornea (Maurice, 1971: Fischharg, 1973; Barfort and Maurice, 1974: Hodson and Miller, 1976). A systematic study of cornea1 water and ionic transport should include an investigation of the transport properties of the basement membrane as well. Any selective function of Descemet’s Membrane can then be taken into account when the overall transport is considered. In this paper we propose that, the membrane has ion exchange properties under certain conditions.

2. Methods Electrical

transport

properties

of

DM were

MCI,,

C,

studied

i ~14.483~/~1/060673+07

MCI,.

DM

Buffer pH = 7.3

mainly

Buffer

in the

following

cell

C, pH

= 7.3

I 0

SOi.OO/O 673

1981 Academic

Press Inc. (London)

Limited

671

A. I,YSI,O.

S. KVERSES

ASJ)

S. KJ~I,STl~~~l

I Na+ > K+ > Ca2+ > Mg2+ > La3+. Little effect of pH on water transference numbers is found for NaCl solutions, pH = 55-85. Water transference number is measured for aqueous humour and found to be approximately 50 mol per Faraday. A systematically negative potential of a few hundred microvolts was recorded at the start of each experiment with equal solutions and conditions on both sides of the membrane. This potential lasted for several hours, but approached zero within 30 hr, indicating equilibrium membrane/electrolyte. All potentials are corrected for the initial value. In magnitude the initial potential was about 1 o/o of a concentration gradient potential. A systematic asymmetry in the streaming potentials was also found. A lower value was measured when pressure was applied to the stroma side, than when pressure was applied to the endothelial side. Th e d’ff I erence was about 15 %. The average value has been used to calculate the transference numbers. The electrical resistance of the cell with DM between the two cell compartments is practically the same as that of bulk solution.

IONIC

AKD

WATER

TRANSFERENCE

OF

DESCEMET’S

MEMBRANE

67-l

5. Discussion This work shows that Descemet’s membrane acts basically as an inert transport barrier, in agreement with earlier proposed functions (e.g. Kitfalides, 1973). Transference numbers of monovalent ions are almost identical to values achieved in bulk solutions (see Table I), and so is the concentration variation (not shown). The number of fixed charges in the membrane collagen must be low also because the change in transference number of Na+ and H,O with different values of pH is negligible, and because specific conductance of the membrane is the same as that of bulk solution. The divalent and especially the trivalent ions have significantly lower transference numbers in Descemet’s membrane than in bulk solution (Table II). This demonstrates TABLE

Competition

in

charge transfer.

MCI, KCI LiCl &cl, CaCl, L&l,

II

Ionic transference in the cell:

tMC1,

00223 00156 00286 0.0216 0.0285

numbers

of MCI,

measured

r:NA/i7M

1.6 2.3 1.3 1.7 1.3

an interaction between the basement membrane and the multivalent cations. The value obtained for t Na+ with aqueous humour in the cell chamber (Table I) is lower than for the buffered NaCl solution. This is probably due to charge transfer also by K+ in the first case. The relative amount of Na+ and K+ in aqueous humour is 140 : 20 (Graymore, 1970). Calculation of t, + from equation (4) using the result of mobility ratios in Table II yields t,+ = 003 in aqueous humour. The increased transference number of the sodium ion , t Na+, with increasing thickness of stroma on the membrane shown in Table 1 confirm the stroma as being negatively charged. The structure of the membrane is probably not changed between temperatures of 5 and 35 “C, since no temperature effects on the sodium transference numbers were found in this region. The high water transference numbers, particularly for low ion concentrations, suggest a membrane with high porosity in agreement with a measured water content 1955). Transference numbers of this of 79% by weight (Dohlman and Balazs, magnitude have been measured in a collodion membrane with 75 o/0 water content by Carr, McClinlock and Sollner (1962), who found the same order of decrease with monovalent ions as we do. The number of moles of water transported per Faraday is so high for monovalent ions that the water of hydration does not play any role in transport. The transfer must be electroconvective in nature. For Caa+, Mg2+ and La3+, the water transfer is around 10 mol per Faraday at 100 mM salt concentration. At this high concentration the reduced water transfer may be due to a reduction of the membrane pores by the multivalent cations, an effect in agreement with the reduction

67X

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of ion transference number for these ions. The water transference number is ~o.siti/lp in value. meaning that net water movement’ is in the direction of caation flow. The concentration of Na, K. Cl, La and Mg in intact bovine cornea is 140. 2 11 90. 3 and 1.5 meq kg-i H,O respectively (Graymore, 1970). The concentration of these ions in aqueous humour of rabbit (Graymore. 1970) is of the same order except for potassium, whirh is only 25% of cornea. Values for bovine cornea have not been measured as far as we know. We should therefore expect only a minor effect of the divalent ions on the structure of Descemet’s membrane in vivo. This has been confirmed by our measurement oft, with aqueous humour in the cell chambers (50 mol per Faraday). The aqueous humour provides a rich source for water transfer. The asymmetry of the streaming potentials (about 15 %) is regarded as a consequence of the known asymmetry of the Descemet’s membrane (Jakus, 1956; Kayes and Holmberg, 1964: McTiguw, 1968). Near the stroma, the membrane contains compacted laminae. Near the endothelial cells the membrane is more open. A change in sign of applied pressure gradient should give as.ymmetric changes in the total membrane.

6. Conclusion No surprising effects have been found in this straightforward study of Descemet’s membrane of bovine cornea. The membrane acts basically as an inert transport barrier with large water-filled pores. Multivalent cations plays a less important role in passive ion and water transfer. ACKNOWLEDGMENTS The students Gro Mjellem and Hans Jsrgen Hegg are thanked for assistance in the competition experiment. This work was supported financially by NTH’s fond and Norsk Hydros Forskningsfond. REFERENCES Barfort, P. and Maurice, D. (1974). Electrical potential and fluid transport across the cornea1 endothelium. Exp. Eye Res. 19, 11-19. Carr, C. W.., McClinlock, R. and Sollner, K. (1962). J. Electrochem. Sot. 109, 251-55. Dohlman, C. and Balazs, E. (1955). Clinical studies on Descemet’s membrane of the Bovine Cornea. Arch. Biochem. 57, 445-47. Fischbarg, J. (1973). Active and passive properties of the rabbit cornea1 endothelium. Exp. Eye Res. 15, 615-34. Fcrrland, T.. Thulin, L. and Ostvold, T. (1971). Concentration cells with liquid junction. J. Chem. Educ. 48, 741-44. Ferland, T. and Ostvold, T. (1974). The biological membrane potential. A thermodynamic approach. J. Membr. Biol. 16, 101-20. Graymore, C. N. (1970). Biochemistry of the Eye. P. 10; p. 116. Academic Press, London. Harned, H. S. and Owen, B. B. (1950). The Physical Chemistry of Electrolytic Solutions. P. 538. Reinhold, New York. Hodson, S. and Miller, F. (1976). The bicarbonate ion pump in the endothelium which regulates the hydration of rabbit cornea. J. Physiol. (London) 263, 536. Jakus, M. A. (1956). Studies on the cornea. II. Structure of Descemet’s Membrane. J. Biophys. Biochem. Cytol. (suppl.) 2, 243-52. Kayes, J. and Holmberg, A. (1964). The fine structures of the cornea in Fuch’s endothelial dystrophy. Invest. Ophthdmol. 3, 4747. Kefalides, N. (1973). Structure and biosynthesis of basement membranes. Znt. Rev. COWL. Tiss. Res. 6, 63-104.

1ONICAh'DWATERTRANSFEREP;CEOFDESCEMET'SMEMBRANE

tim

Kenny, T., Benya, P., Nimni, M. and Smith, R. (1979). A new technique for isolation of Descemet’s Membrane: Preliminary studies. Invest. Ophthalmol. Vis. Sci. 18, 52742. Maurice, D. (1972). The location of the fluid pump in the cornea. J. Physiol. (London) 221. 43. McTigue, J. W. (1968). The human cornea. A light and electron microscopic study of the normal cornea and its alterations in various dystrophies. Tr. Am. Ophth. Sot. 65,591-660. Trivijitkasem, P. and Ostvold, T. (1980). Water transport in ion-exchange membranes. Electrochem. Acta 25, 17 l-8.