Chlorpromazine effects upon rabbit lens water and electrolyte balance

Eqo. Eye Res.(1983)35,

55!&566

Chlorpromazine

Effects upon Rabbit Electrolyte Balance

Lens

Water

and

CATHRYN C. WILSON, NICHOLAS A. DELAMERE AND CHRISTOPHER A. PATERSON Department

of Ophthalmology,

of Colorado CO 80262, U.S.A.

University

(Received 8 July 1982 and

Health

30 November

accepted

Sciences Center, Deruuer,

1982, London)

Chlorpromazine (CPZ) concentrations of 5 x 10-j JI or greater were found to disrupt lens electrolyte and water balance significantly. Lens sodium and calcium levels increased while potassium decreased. These changes were accompanied by water accumulation. Such effects of CPZ were not dependent upon exposure to ultraviolet light. Electrical measurements revealed that CPZ induced depolarization of the lens potential but little change of overall lens conductance. 86Rb efflux from the lens was unaltered by a 1 hr exposure to CPZ. The active sodium pump in the lens was inhibited by CPZ, as evidenced by a reduction in P;a- K-ATPase activity. Key words: chlorpromazine; cataract; rabbit lens; sodium; potassium; calcium; lens hydration; sodium pump; permeability.

1. Introduction Chlorpromazine (CPZ) is used extensively for the management of psychotic disorders and is, more recently, being employed as an inhibitor of intestinal fluid secretion in the treatment of cholera (Holmgren, 1981). Greiner and Berry (1964) described lens opacities in psychotic patients receiving chronic doses of chlorpromazine, and this finding has since been confirmed by other investigators (Howard, McDonald, Dunn and Creasey, 1969). The lens changes are generally seen as anteriorly located, brown, punctate opacities which eventually spread to give rise to anterior stellate cataracts. Chlorpromazine-induced cataracts have also been produced experimentally in guineapigs (Howard et al., 1969) and mice (Smith, Gavitt and Karmin, 1966). The precise etiology of CPZ-induced cataracts is not known. Since CPZ is a photosensitive agent, it has been suggested that lenticular changes are dependent upon exposure to ultraviolet radiation. This is supported by the observation that lens opacities in man are frequently found within the pupillary area, and that lens opacities in guinea-pigs fed CPZ are exacerbated by UV light exposure (Howard et al., 1969). Biophysical studies have shown that CPZ or its photoproducts might result in increased permeability of biological membranes (Schaubman and Felmeister, 1968; Felmeister and Schaubman, 1969), and it has been suggested that CPZ may have a detergent-like influence upon cell membranes that would be expected to result in permeability changes (Kochevar and Lamola, 1979). A preliminary study by Michon and Lambert (1968) demonstrated alteration of 86Rb fluxes in the CPZ-treated lens in vitro. It was: therefore; the purpose of this study to determine whether CPZ caused alterations in rabbit lens cell membrane permeability and ion transport properties, and whether such changes might cont’ribute to CPZ-induced cataract. To this end we Address for reprints and Denver: CO 80262, U.S.A.

0014-4835/83/040559+07

correspondence:

$03.00/O

Dr

C. A. Paterson,

0

1983

Box

Academic

B205,

UCHSC,

Press

Inc.

4200

(London)

E. 9th

Ave,

Limited

C. C. WILSON

560

ET ;\L

examined the effect of varying concentrations of CPZ upon lens electroIyte levels, lens water content, electrophysiological properties, Na- K-ATPase activity and 86Rb efllux. We also investigated whether any such effects were dependent upon exposure to UV light. 2. Materials

and

Methods

Bdult New Zealand white rabbits. weighing 1-2 kg, were killed by a blow on the head and the eyes enucleated. The lens was removed from the globe by a posterior approach and transferred to a sealed glass incubation vial containing a modified Tyrode’s solution (145 miw-NaCl, 6 mM-KCl, 2.4 m;M-CaCl,, I.2 miw-MgCl,, 5-5 miu-dextrose, 5 mM-Hepes buffer), buffered to a pH of 7.2 and maintained at 37V. Test solutions were prepared by adding chlorpromazine hydrochloride (Sigma) to the Tyrode’s solution at given concentrations. Following incubation, the lens was dried to determine water content and then digested in 2 ml of 30 y0 nitric acid. The lens digest was diluted appropriately for sodium, potassium and calcium analysis by atomic absorption spectrophotometry (Perkin Elmer 272). Electrophysiological measurements were performed with the lens seated in a plastic chamber through which Tyrode’s solution at 37OC was constantly flowing. The lens potential was determined with a mieroelectrode introduced into the posterior lens cortex (Delamere and Paterson, 1979). Lens conductance was measured using a microelectrode technique described previously for the amphibian lens (Delamere, Duncan and Paterson. 1980). The lens conductance and lens potential are measurements which relate closely to the membrane permeability of the lens. Because the complexity of lens conductance characteristics (Delamere et al., 1980; Mathias, Rae and Eisenberg, 1979) precludes a detailed analysis of specific membrane conductance values in the large rabbit lens, the conductance data are presented as gross measurements of the overall electrical (and thus ionic) leakiness of the lens. To measure the efflux rate constant of 8GRb in the presence of CPZ, the lens was loaded in normal Tyrode’s solution containing 86Rb (1 pCi/ml) for 20 hr. The lens was then placed in a special efflux chamber (Paterson, Delamere and Holmes, 1980). After establishing the efflux rate constant in normal Tyrode’s solution, the lens was exposed to CPZ (1O-4 M). The efflux rate constant was calculated as described previously (Delamere and Paterson, 1978). Lens ATPase determinations, using the whole lens. were performed by the calorimetric phosphate assay technique described in detail by Neville, Paterson and Hamilton (1978). ATPase activity was described in terms of moles of phosphate released per hour per g wet weight of lens. Sodium-potassium-stimulated ATPase (Na- K-ATPase) activity was defined as the difference between the ATPase activities determined in the presence and absence of 10m4 M-ouabain. The Na- K-BTPase activity in the presence of CPZ was defined as the difference between the ATPase activity observed in the presence of a given concentration of CPZ and that activity observed in the presence of ouabain ( 10m4 Y) together with a similar concentration of CPZ. 3. Results Lens electrolyte

and water con,tent

Incubation of the lens in the presence of a 5 x 10e5 M or greater concentration of CPZ resulted in a pronounced increase of the lens sodium concentration and depletion of the potassium concentration [Fig. i(a)]. 0 ver t’he 20 hr incubation time period used in this study, the sodium and potassium concentrations and water content of the lens were unaffected by exposure to CPZ at concentrations less than or equal to 2 x lop5 M. The disturbance of the lens sodium and potassium content upon CPZ treatment was accompanied by a marked increase in the lens calcium content [Fig. l(b)] and by an accumulation of lens water (Fig. 2). Lenses which had gained water as a result of CPZ treatment were observed to have small fluid droplets present in the subcapsular regions, resulting in marked loss of clarity.

CPZ

AND

LEKS

I

1

10-6

10-5 CPZ

,561

ELECTROLYTES

I

,

10-4

10-3

Concentration(M)

FIG. I. Xet changes in the concentration of (a) sodium (O), potassium (W) and (b) calcium (A) in the rabbit lens following incubation for 20 hr in the presence of various concentrations of chlorpromazine (CPZ). The experiments were performed using paired lenses. For each CPZ concentration, the data represent the mean difference between ion concentrations measured in six CPZ-treated lenses and those determined in six control lenses. The vertical bars represent the standard error of the mean. The control (untreated) values of K-a; K and Ca in the lens following 20 hr incuba.tion were 18.4kO.7, 138.9+%1 and 0.65 2 0.02 mm/kg lens water respectively.

16

0-

O-

10-6

10-5

CPZ

10-4 concentration

10-3 (M)

FIG. 2. Change in the lens water content following incubation for 20 hr in the presence of various concentrations of chlorpromazine (CPZ). The experiments were performed using paired lenses. For each CPZ concentration. the data represent the mean percentage change in water content measured in six CPZ-treated lenses and those determined in six control lenses. The vertical bars represent the standard error of the mean. The control (untreated) value of lens water content following incubation for 20 hr was 828 _+ 0.5 “/’ respectively.

562

C. C. WILSON

ET

AL.

UV light dependence The potential influence of UV light upon the action of CPZ was investigated by comparing the electrolyte content of lenses incubated in a sub-effective concentration of CPZ (10e5 M) under conditions of darkness with the lens electrolyte content following incubation in 1O-5 M-CPZ in room light together with a UV light source (Mineralight Model UVSL 25; peak wavelength of 362 nm, with an energy of 280 ,uW/cm2). No influence of CPZ upon the lens electrolyte or water balance was seen in either case (Table I). Similarly, conditions of light or darkness were without significant influence upon the changes of the lens electrolyte or water content observed following incubation for 20 hr in the presence of an effective concentration of CPZ (1O-4 M) (Table I). TABLE

The in$uence

of

I

UV light on lens ion and water balance chlorpromazine

in the presence of

Change in ion and water content after 20 hr Dark 10-J x-CPZ xia K Ca H,O 10-d x-CPZ Na K Ca Hz0

+ 1.9 -1.2 0 0

(* 1.8) (1.3.9) (kO.03) (?O.l)

+73.4 (k7.1) - 59.3 ( + 3.6) +1.9(+0.12) + 84 ( * 1.0)

+1,9(f36) + 1.0 (* 1.7) - 004 ( f 0.06) +0.2 (f 1.0) + 69.4 - 60.0 +2.33 +9,1

( + 2.9) ( + 55) (+@14) (k2.1)

The data are expressed as the mean f S.E. from determinations on six lenses for each experiment. Ion data are expressed in mm/kg lens water. Change in lens water content is expressed as percentage change. The control values of lens Na, K and Ca were 15+3+25, 142.1 k3.1 and 066+0.02 mm/kg lens water respectively; the water content was 63.6 & @06 %.

Electrophysiological

parameters

Exposure of the lens to lop4 M-CPZ resulted immediately in depolarization of the lens ; after 1 hr, the electrical potential of the lens was diminished to - 47.0 + 2.4 mV from a control value of -7@5+ 1.5 mV (four lenses). In contrast, the electrical conductance of the lens was not significantly altered by CPZ treatment. Lens of 10-J M-CPZ was conductance determined after 1 hr in the presence 1.64+0*14 x lop3 S compared to a control (pre-CPZ) value of 1.81+0.2 x low3 S (four lenses).

Following an efflux period of 150 min in normal Tyrode’s solution, a steady efflux rate constant of 10.79+1.42 x 1O-3 mine1 was determined in seven lenses. Upon exposure to 10m4 M-CPZ there was no significant change in the ef?lux rate constant. After 60 min in the presence of CPZ, the mean efllux rate constant in the same seven lenses was 1912 f 1.68 X 10e3 min-‘.

CPZ

Na- K-ATPase

AND

LEXS

ELECTROLYTES

563

activity

The influence of CPZ upon lens active cation transport was evaluated by examining its effect on lens Na- K-ATPase. In the presence of 10-j M-CPZ, the activity of Na- K-ATPase was 1.57 + 0.15kmol PO, released/hr/g wet wt ; this was not different from the control value of 1.56 f 0.12 pmol PO, released/hr/g wet wt (mean f S.E., eight lenses). However, when the concentration of CPZ was increased to lop4 M, the ?\‘a- K-ATPase activity was reduced to 1~05+0~18~mol PO, released/hr/g wet wt, and this difference was significant (P < 0.001). Thus it is evident that CPZ has an inhibitory effect on lens Na- K-ATPase at the same concentration at which cation balance is markedly impaired. 4. Discussion The substantial changes in the lens electrolyte and water balance which took place upon incubation in 2 5 x lO-5 M-CPZ indicate that, at such concentrations, CPZ exerts a pronounced intluence upon lenticular ion balance mechanisms. At lesser concentrations, however, CPZ was without measurable effect upon the lens electrolyte balance over an incubation period of 20 hr. In the presence of 2 5 x lop5 M-CPZ, the incubated lens lost potassium while accumulating sodium and calcium. Following incubation for 20 hr in the presence of 10W3 M-CPZ, the lens calcium level, expressed as a concentration, exceeded that of the bathing solution. No evidence was found to relate UV light to the disruptive influence of CPZ upon lens electrolyte and water balance. However, this does not rule out the possibility that UV light may potentiate a disturbance of the lens ion balance over much longer time periods when the lens is exposed, in vivo, to an otherwise sub-effective, low concentration of CPZ. Jose and Yielding (1978) have demonstrated a deleterious effect of photoactivated CPZ on the rat lens as evidenced by alteration of DNA in the lens epithelium. Photoactivation was achieved at the same wavelength of LJV light as was used in the present study. In the red blood cell, CPZ has been shown to have a photosensitive effect, whereby it caused cell lysis due to a detergent-like action upon t’he cell membrane (Kochevar and Lamola, 1979). The electrolyte imbalance observed following CPZ treatment was due in part to inhibition of active cation transport. CPZ (lop4 M) markedly reduced the lens Na- KmATPase activity. Such an effect of CPZ on Na- K-ATPase activity has been demonstrated in other tissues (Keeffe, Blankenship and Scharschmidt, 1980; Roed and Brodal, 1981). It is most unlikely that partial inhibition of the lens sodium pump by CPZ would produce the severity of electrolyte imbalance observed. Therefore, the possibility that CPZ also perturbs the passive permeability properties of the lens was investigated. Lens conductance, which reflects the degree of lens membrane permeability, was largely unaffected by CPZ at a concentration (10e4 M) which disturbs the lens ion balance significantly. This finding suggests that the overall degree of ion permeability, or leakiness, of the lens is not increased by CPZ. However, the electrical potential (PD) of the lens was altered substantially by 10m4 M-CPZ; the lens was depolarized by approximately 24 mV. A small part ( < 10 mV) of the depolarization may be accounted for by inhibition of the electrogenic sodium pump (Paterson, Neville, Jenkins and Cullen, 1975) However, the much larger CPZ-induced depolarization indicates that an additional mechanism may contribute to lens depolarization. The movement of the

564

C. C. WILSON

ET

AL.

lens PD towards the equilibrium potential of sodium suggests that, while the net permeability (as shown by conductance) of the lens may be unaltered, the permeability constant of the lens to sodium was increased at the expense of the potassium permeability constant. In fact, a reduction in the potassium permeability constant can be inferred from the 86Rb efflux experiments. CPZ (lo-* M) subst’antially depolarizes the lens, but the expected increase in s6Rb efflux rate resulting from the depolarization (Delamere and Paterson, 1978) did not take place. These early changes in sodium and potassium permeability and transport would be expected to result ultimately in the type of electrolyte imbalance observed following extended CPZ exposure. The mechanisms which result in the accumulation of calcium by the lens during exposure to CPZ cannot be defined from the present experiments. However, CPZ is known to interfere with calcium extrusion mechanisms, possibly due to its interaction with the calcium-binding protein calmodulin (Vincenzi, Hinds and Raess; 1980). Hightower and associates (Hightower and Reddy, 1981; Hightower, Leverenz and Reddy, 1980) have presented evidence for a calcium extrusion pump in the rabbit lens, and inhibition of this mechanism by CPZ would no doubt result in an increase of lens calcium. A CPZ concentration of > 5 x 10W5 M, det,ermined here to disrupt the rabbit lens ion and water balance in vitro, is likely to be higher than the concentration of CPZ that a human lens would be exposed to during systemic treatment with CPZ. Xevertheless, CPZ probably accumulates in the lens (Potts, 1962), and thus extended systemic treatment with CPZ, as is the case in the treatment of psychotic disorders, may well result in locally high CPZ concentrations in close proximity to the lens membranes. It remains to be determined whether cataracts induced by long-term CPZ therapy do indeed show disturbances of electrolyte and water content that are similar to those which result from short-term in vitro CPZ exposure.

ACKNOWLEDGMENT We thank Elizabeth Paterson for technical assistance in various aspects of the study. This study was supported by U.S.P.H.S. Research Grant No. EY 00506 from the National Eye Institute. REFERENCES Delamere, N. A., Duncan, G. and Paterson, C. A. (1980). Characteristics of voltage-dependent conductance in the membranes of a non-excitable tissue: the amphibian lens. J. Physiol. 308, 49-59. Delamere, N. A. and Paterson C. A. (1978). The influence of calcium-free EGTA solution upon membrane permeability in the crystalline lens of the frog. J. G’en. Physiol. 71,581-93. Delamere, N. A. and Paterson, C. A. (1979). The influence of calcium-free solutions upon permeability characteristics of the rabbit lens. Exp. Eye Res. 28, 4&53. Felmeister, A. and Schaubman, R. (1969). Photoinduced interaction of phenothiazine drugs

with a lecithin

monomolecular

film. J. P/Mw~. Sci. 58, 64-7.

C. and Berry, K. (1964). Skin pigmentation and cornea1 and lens opacities with chlorpromazine therapy. Can. Med. Assoc. J. 90, 663-5. K. R.; Leverenz, V. and Reddy, V. N. (1980). Calcium transport in the lens. Ophthalmol. Vis. Sci. 19, 105966. K. R. and Reddy, V. N. (1981). Metabolic studies on calcium transport in mammalian lens. Curr. Eye Res. 1, 197-204. Holmgren, ,J. (1981). Actions of cholera toxin and the prevention and treatment of cholera. Nature 292, 413-17. Greiner. A. prolonged Hightower. Invest. Hightower,

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AND

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,355

Howard, R. O., McDonald, C. J., Dunn, B. and Creasey, W. A. (1969). Experimental chlorpromazine cataracts. Invest. Ophthalmol. 8. 413-21. Jose, ,J. Cr. and Yielding. K. L. (1978). Photosensitive cataractogens, chlorpromazine and methoxypsoralen, cause DNA repair synthesis in lens epithelial cells. Invest. Ophthalmol.

17, 687-91. Keeffe, E. B., Blankenship, N. M. and Scharschmidt, B. F. (1980). Alteration of rat liver plasma membrane fluidity and ATPase activity by chlorpromazine hydrochloride and its metabolites. Gastroenterology 79. 222-31. Kochevar, I. E. and Lamola, A. A. (1979). Chlorpromazine and protriptyline phototoxicity: photosensitized, oxygen independent red cell hemolysis. Photochem. Photobiol. 29, 791-6. Mathias, R. T.; Rae, J. L. and Eisenberg, R. S. (1979). Electrical properties of structural components of the crystalline lens. Biopkys. J. 25, 181-201. Xchon, J., Jr and Lambert, B. W. (1968). The effects of chlorpromazine on the lens. Presented at meeting of the Association for Research in Ophthalmology, April 30, 1968, Tampa, Florida. Invest. Ophthalmol. 7, 118. Neville, M. C., Paterson, C. A. and Hamilton, P. M. (1978). Evidence for two sodium pumps in the crystalline lens of the rabbit eye. Exp. Eye Res. 27, 637-48. Paterson, C. A., Delamere: N. A. and Holmes, D. L. (1980). Calcium and lens membrane permeability characteristics. In Age&g of the Lens (Eds. Regnault, F., Hockwin, 0. and Courtois, Y.). Pp. 121-30. Elsevier/North-Holland Biomedical Press, Amsterdam. Paterson, C. A., Neville, M. C.: Jenkins, R. M. and Cullen, J. P. (1975). An electrogenic component of the potential difference in the rabbit lens, Biochim. Biophys. Acta 375. 30916. Potts, A. M. (1962). The concentration of phenothiazines in the eye of experimental animals. Invest. Ophthdm,ol. 1, 522-30. Roed, A. and Brodal, B. (1981). Inhibition of sarcolemma ATPases by some membranestabilizing drugs. Acta Pharmacol. Toxicol. 48, 65-8. Schaubman, R. and Felmeister, A. (1968). Interaction of a photosensitizer with dipalmitoyl lecithin. J. Pharm. Sei. 57, 178-80. Smith. A. A., Gavitt, J. A. and Karmin, ,M. (1966). Lenticular opacities induced in mice by chlorpromazine. Arch. Ophthalmol. 75, 99-101. Vincenzi, F. F., Hinds, T. R. and Raess, B. U. (1980). Calmodulin and the plasma membrane calcium pump. Ann. N. Y. Acad. Sci. 356, 232-44.