(Hypertension. 1996;28:440-443.)
© 1996 American Heart Association, Inc.
Articles |
Altered Lens Short-Circuit Current in Adult Cataract-Prone Dahl Hypertensive Rats
Research and Medical Services, San Juan VA Medical Center, Department of Pharmacology and College of Health Related Professions, University of Puerto Rico, Medical Sciences Campus, and Departments of Ophthalmology and Physiology & Biophysics, Mount Sinai School of Medicine, New York, NY.
Correspondence to C. Rodriguez-Sargent, PhD, Research Services (151), San Juan Veterans Medical Center, One Veterans Plaza, San Juan, PR 00927-5800. E-mail rodriguez-sargent, c@san juan.
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Abstract |
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We assessed components of lenticular short-circuit current in adult hypertensive Dahl salt-sensitive rats (DS) during chronic control (0.4% sodium) versus high (3% sodium) dietary NaCl intake begun at the age of 4 weeks until rats were studied. We also evaluated the influence of barium, a potassium channel blocker, and ouabain, a specific inhibitor of Na+,K+-ATPase activity, by adding them to the anterior lens surface, thus measuring barium-sensitive, ouabain-sensitive, and barium- and ouabain-insensitive short-circuit currents. During control NaCl intake, short-circuit current in DS and their control group, Dahl salt-resistant rats (DR), did not differ significantly. DS were subclassified into cataract-prone rats and rats unlikely to develop cataracts on the basis of their initial pressor response to the change from a normal to high NaCl diet during the first weeks of age. Although only transparent lenses were studied, total lens short-circuit current was already markedly decreased in the cataract-prone subgroup compared with DS unlikely to develop cataracts and control DR. This was in sharp contrast to the increase in short-circuit current previously reported in Sprague-Dawley rats and now observed in control DR in response to high dietary NaCl. The decrease in lens short-circuit current in cataract-prone rats was associated with lower absolute values of barium- and ouabain-sensitive short-circuit currents as well as with low barium- and ouabain-insensitive short-circuit current. Although the barium- and ouabain-sensitive components of the short-circuit current were similar in DS unlikely to develop cataracts and DR, the barium- and ouabain-insensitive component of the short-circuit current was lower in DS unlikely to develop cataracts than values in DR. Interestingly, this component of lens short-circuit current also increased in DR during chronic high NaCl, whereas the opposite change occurred in cataract-prone DS and DS unlikely to develop cataracts. Thus, the barium- and ouabain-insensitive short-circuit current may be a mechanism that protects the normal lens from developing cataracts. Possible candidates for this short-circuit current component are voltage-dependent potassium channels, calcium-activated potassium channels, or both. Our studies show altered lens short-circuit current in response to high NaCl intake in cataract-prone DS and suggest the possibility of altered lens potassium transport during sustained hypertension but before loss of lens transparency.
Key Words: cataract • lens, crystalline • potassium channels • sodium chloride, dietary
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Introduction |
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We previously described a high frequency of cataract formation in the adult hypertensive DS, whereas control DR consistently had transparent lenses.1 Subsequently, we were able to identify those DS that will eventually develop cataracts (DSC) based on the initial pressor response to a high NaCl diet in the weanling rat.2 3 This permitted detailed lens studies before cataract formation in DS during sustained hypertension. We found that adult hypertensive DSC showed decreased lenticular ouabain-sensitive 86Rb uptake before cataract formation, suggesting that inhibition of lens Na+,K+-ATPase activity may participate in cataractogenesis associated with hypertension in DS.2 Nevertheless, other lenticular ionic defects may also participate in cataract formation in this model of salt-sensitive hypertension. Altered lens K+ transport in DS might be one such ion transport defect in cataractogenesis. Indeed, our initial report of cataracts in DS showed an abnormality in aqueous K+ concentration, suggesting aberrant lens K+ transport as a possible mechanism of cataractogenesis.1 Consequently, in the present studies, we evaluated lenticular K+ transport in adult DSC using the short-circuiting technique.
The Isc across the isolated lens has been previously determined to represent across the anterior face (ie, the transporting epithelial basolateral surface) ionic flows generated by a rheogenic Na+-K+ pump plus net efflux of K+ via K+ channels. This has been shown with lenses from the toad,4 rabbit,5 and rat.6 Indeed, the largest K+ channel conductance in lenses of several species including the rat is specifically inhibited by barium.7 In rat lens, however, about 18% of the Isc remains insensitive to ouabain (a specific Na+,K+-ATPase inhibitor) in combination with barium. Thus, this Isc component appears to represent K+ efflux via other channels.4 6
Our early studies also demonstrated that the development of cataracts in DS depends on the level of dietary NaCl intake,8 suggesting the possibility that the magnitude of lenticular ion transport defects in DS may vary with differences in NaCl intake. Taken together with our recent observation of increased barium-sensitive as well as increased barium- and ouabain-insensitive lens Isc values in normal Sprague-Dawley rats in response to chronic high dietary NaCl intake,6 this led us to assess the influence of dietary NaCl on lens transport properties in adult DS. Therefore, we assessed components of lenticular Isc in DS during chronic control versus high dietary NaCl intake. Inasmuch as the lens is a nonuniform spherical syncytium in which the epithelium that lines only the anterior lens surface is the most metabolically active site, we evaluated the influence of barium and ouabain by addition exclusively to the solution bathing the isolated anterior lens surface. During the present studies, PDt and Rt were concurrently determined in the same lenses.
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Methods |
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A total of 60 weanling DS and 20 age- and sex-matched DR (both derived from Brookhaven outbred Dahl rats) were placed in a room kept at an ambient temperature of 26°C with a 12-hour light/dark cycle. SBP was measured in all conscious, restrained rats by tail-cuff plethysmography. The arithmetic mean of 10 consecutive measurements in each rat was accepted as a single SBP value. Ten DS and 10 DR were maintained on a standard chow diet (Purina Mills; 0.4% sodium, 0.65% chloride), and the remaining rats were given a high NaCl diet (Teklad; 3% sodium, 4.75% chloride). All rats were provided with tap water ad libitum. The dietary regimens were maintained throughout the studies. SBP was measured weekly as described above up to the age of 8 weeks in DS and DR given the high NaCl regimen to permit subclassification of DS into DSC and DS unlikely to develop cataracts (DSNC) with the use of criteria we previously reported.2 3 This subdivision is based on the initial pressor response to a high NaCl intake, permitting effective identification of subgroups by the age of 6 to 7 weeks: DS with blood pressures greater than 160 mm Hg were classified as DSC and those with blood pressures less than 130 mm Hg were classified as DSNC. DS with intermediate SBP did not fall into either subgroup2 ; therefore, they were not studied further. Subsequently, SBP was measured only every 3 to 4 weeks in all rats regardless of NaCl intake. Lens transparency was assessed in each rat by ophthalmoscopic examination and monthly slit-lamp biomicroscopy until rats reached adulthood. Upon assessment of lens transparency, only rats free of cataracts were studied further. Once all rats reached adulthood, they were decapitated randomly in staggered sequence for lenticular electrophysiological study by the short-circuiting technique (described below). All studies were approved by the local Institutional Review Board and complied with the National Institutes of Health publication Guide for the Care and Use of Laboratory Animals. In addition, all studies complied with the Association for Research in Vision and Ophthalmology Resolution. Data analyses included unpaired t test (one-tailed) between groups and paired t test (two-tailed) within groups. All values represent the arithmetic mean±SE.
For Isc studies, rats were anesthetized with sodium pentobarbital (50 mg/kg body wt) and decapitated. We previously determined that such anesthesia does not influence lens transport properties (unpublished observations, 1991). Immediately after death, the rats' eyes were removed and lenses dissected via a posterior approach, with a thin ring of equatorial zonular fibers carefully left as a reference point for subsequent lens transfers and mounting. Throughout the dissection and incubation period, lenses were maintained in a control solution (mmol/L: NaCl 124, KCl 5, NaHCO3 16, HEPES 10, CaCl2 1.0, MgCl2 0.5, dextrose 5.0). Upon dissection, each lens was incubated at 37°C for 1 hour to allow recovery of ionic permeabilities. Upon completion of the initial 1-hour incubation, lenses were visually examined for transparency, and only those completely free of trauma were inserted in a tunnel-like cylindrical aperture of a flat Lucite disc with lens position supported by an O-ring.6 9 The disc-lens assembly was mounted as a partition between two modified Ussing-type hemichambers, thus effectively isolating the anterior and posterior lenticular surfaces.10 Hemichambers were filled with 10 mL of bath solution (mmol/L: NaCl 124, KCl 5, sodium gluconate 16, hemisodium HEPES 10, acid HEPES 5.0, CaCl2 1.0, MgCl2 0.5, dextrose 5.0). Solutions were maintained at 37°C and bubbled with humidified air throughout the studies to assure a constant pH (7.3). Bridges for electrical connections and measurements as well as recording instruments were as previously described.10 An automatic voltage-clamp device was used to keep lenses short-circuited.11 The entire modified Ussing apparatus, voltage-clamp device, and electrodes were obtained from Mount Sinai School of Medicine, NY. Current pulses 2 seconds long were used for determination of Rt and PDt. Once basal values were stabilized, experimental basal lens electrical parameters were recorded. Afterwards, barium (BaCl2; final concentration, 5 mmol/L) followed by ouabain (final concentration, 1 mmol/L) was added to the anterior bathing solution, and measurements were made after 10 minutes of exposure to each pharmacological agent. Both barium and ouabain in the anterior solution cause distinct independent changes regardless of the order of addition.9 In this manner, sequential influences of barium and ouabain on Isc as well as on PDt and Rt were assessed. The barium-sensitive component of Isc was determined as the difference between basal (total) Isc and Isc in response to barium. Ouabain-sensitive Isc was calculated as the difference between Isc after addition of barium and Isc recorded after serial addition of ouabain. The Isc measured after addition of both agents constitutes the barium- and ouabain-insensitive Isc component.
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Results |
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During chronic control NaCl intake, DSC cannot be identified, so all DS on this dietary regimen were studied as a single population. Under these conditions, SBP was sharply increased in DS compared with DR, as anticipated (Table 1
). Slit-lamp biomicroscopy revealed transparent lenses in all DS and DR. These studies did show prominent Y sutures in most DS and DR, as we have previously noted but have not reported in these and other normal strains. Total lens Isc, barium-sensitive Isc, and ouabain-sensitive Isc were comparable in DS and DR (Fig 1). There was a tendency for barium- and ouabain-insensitive Isc to be increased in DS during chronic control NaCl intake, but this was not statistically significant. PDt was also similar in DS (basal: 12.2±1.4 mV; barium: 5.1±0.9; and ouabain: 2.5±1.0) and DR (10.8±0.9, 5.3±0.5, and 3.4±0.5, respectively) under each short-circuited condition studied.
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During chronic high NaCl intake, SBP was sharply and similarly increased in adult DSC and DSNC compared with DR (Table 1
). Only rats with transparent lenses were studied further; lens transparency in these rats was confirmed by slit-lamp biomicroscopy. Total lens Isc was markedly low in DSC, whereas a modest decrease was observed in DSNC compared with DR (Fig 2). This decreased Isc in DSC was due to lower barium-sensitive, ouabain-sensitive, and barium- and ouabain-insensitive Isc values. In contrast, the tendency for lower total lens Isc in DSNC was the exclusive result of a striking and significantly lower barium- and ouabain-insensitive Isc (Fig 2). The lower Isc values in DSC were paralleled by decreases in PDt (Table 2). Similarly, the lower barium- and ouabain-insensitive Isc observed in DSNC was associated with decreased PDt (Table 2). Rt was similar among DSC (basal: 243±25 
·cm2; barium: 255±27; and ouabain: 264±28), DSNC (249±13, 266±14, and 269±16, respectively), and DR (233±16, 244±18, and 246±18, respectively) throughout the lens studies.
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significant differences compared with values from DS unlikely to develop cataracts (DSNC) (P<.05). BA indicates barium; OUAB, ouabain; and DSc, cataract-prone DS.
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Discussion |
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The development of sustained hypertension in DS maintained on a control NaCl diet was not surprising because this has been previously reported.12 Similarly, the comparably increased SBP in adult DSC versus DSNC during chronic high dietary NaCl was anticipated. Our previous studies consistently showed that during chronic sodium loading under conditions identical to those in the present study, DSC and DSNC achieved similar levels of SBP by the age of 16 weeks,1 indicating that lens opacification in DS cannot be ascribed to increased arterial blood pressure per se. Consequently, the differences in lens ionic transport observed between the two DS subgroups are also probably independent of high SBP. As such, the data presented herein suggest the possibility that cataractogenesis in DS may depend on the degree of salt sensitivity and conceivably may result from some specific mechanism or mechanisms that participate in the initiation of salt-sensitive hypertension. Alternatively, but not mutually exclusively, the present data together with previously reported data are consistent with a possible genetic basis for cataracts and hypertension in DS.
The lens is known to have several K+-selective currents, of which at least three are apparently common to most species.13 14 One of these is a lenticular K+ current resulting from inwardly rectifying channels. This lens current, found in all species studied (including normal rat), is highly sensitive to barium. Another K+ current described in lenses from chicks, humans, and rabbits is due to Ca2+-activated K+ channels that are blocked by a number of agents, including barium. The third current is outwardly rectifying, depends on transmembrane voltage, and is relatively insensitive to barium. The barium-sensitive Isc was strikingly low in adult DSC before cataract formation during chronic NaCl loading, whereas normal values were observed in DSNC. Therefore, these data suggest a decrease in lens K+ inwardly rectifying channels.
A common observation for both DS subgroups during chronic NaCl loading was the values of lens barium- and ouabain-insensitive Isc. This Isc component was decreased in both DSC and DSNC, although to a lesser extent in DSNC; it was the only lens ionic transport change observed in the latter group. Consequently, evaluation of voltage-dependent K+ channels and Ca2+-activated K+ channels in this genetic model of hypertension merits future attention.
In addition to this change, the ouabain-sensitive Isc was also somewhat decreased, suggesting that the Na+,K+-ATPase was inhibited. This is consistent with our earlier report of decreased lens ouabain-sensitive 86Rb uptake in adult DSC maintained on a high NaCl diet and studied before cataract formation.2 Decreased Na+,K+-ATPase activity has been shown in several tissues of adult DS and other models of genetic hypertension in rats.15 16 17 The mechanism(s) or causes of such observations are not altogether clear. The presence of circulating ouabain-like factors is now established in volume-overload states as well as in the human neonate.18 19 20 Perhaps these factors, largely of adrenal and/or hypothalamic origin, have a role in the present observations. Furthermore, recent studies have shown the presence of a digitalis-like substance in human senile cataractous lens nuclei.21 This substance inhibits both Na+,K+-ATPase in rat brain microsomes and [3H]ouabain binding. The substance also exhibits digoxin-like immunoreactivity. In addition, the same or a similar substance is present in normal bovine and rat lenses.21 Clearly, any link between these observations and the present reduction of the ouabain-sensitive Isc of DSC remains to be determined.
Overall, the most notable observation of the present study was the finding that lenses from DSC did not express as much barium-sensitive K+ channel activity as those from the other groups. In recent years, it has become evident that the hypotensive effects of agents used as smooth muscle relaxants occur as a consequence of the opening of K+ channels,22 23 24 25 an action opposite to the K+ channel closure and cell depolarization that lead to contraction. In addition, endogenous K+ channel openers have been identified (eg, NO, vascular intestinal peptide), and their regulatory actions are presently the subject of investigation.23 25 The unknown factors that reduce lens K+ channel activity and eventually lead to cataracts in DS may be independent of high SBP during adulthood but might be dependent on the duration of sustained hypertension, which we have shown to be longer in DSC compared DSNC.3
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This work was supported by Merit Review funds of the Department of Veterans Affairs, RR-03051, and EY 00160 and EY 01867. The data in this manuscript will be submitted by N. Fernandez as partial fulfillment of the requirements for a PhD degree in Pharmacology. We acknowledge the excellent technical assistance of Evelio Marrero as well as the fine secretarial assistance of Magda Palerm de Prats.
Received June 30, 1995;
first decision August 21, 1995; first decision March 28, 1996;
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