Aldo C. Zamudio

Acetylcholine modulation of the short-circuit current across the rabbit lens

Rabbit lenses were bathed within a bicameral Ussing-type chamber under short-circuit conditions. In this situation the short-circuit current (Isc) reflects, across the anterior aspect, the presence of anteriorly facing K+ conductance(s) plus the Na(+)-K+ pump current. Across the posterior surface the Isc is primarily carried by the movement of Na+ from the posterior bathing solution to the lens. Addition of acetylcholine (ACh) to the posterior hemichamber did not affect the translens electrical parameters; but, its introduction to the anterior bath at 1 microM immediately reduced the Isc from 8.91 +/- 1.47 to 5.84 +/- 1.28 microA cm-2 and increased the translens resistance from 1.50 +/- 0.08 to 1.59 +/- 0.09 K omega cm2 (+/- S.E.S; P < 0.05 as paired values, n = 25 lenses). The suppressed Isc gradually recovered and reached 75% of the control value 5 min after the introduction of the neurotransmitter. In six cases the recovery was nearly complete (> or = 95% of control) within this time. The preaddition of 0.1 microM atropine prevented an effect by 1 microM ACh. When atropine was added within 1 min of ACh, the suppressed Isc immediately recovered. The ACh-elicited Isc suppression was averted in lenses pre-exposed to either K+ channel blockers (quinidine or barium) or to the endoplasmic reticular Ca(2+)-ATPase inhibitor thapsigargin (Tg: 0.1 microM), which in itself produced Isc inhibitions similar to those seen with ACh under control conditions. Similarly comparable were the ACh-evoked Isc inhibitions garnered upon introduction of the agonist to lenses bathed in the absence of extracellular Ca2+. In these cases, however, the Isc recovered fully within 2-3 min. This condition also revealed that the anterior removal of medium Ca2+ increased the Isc by about 50%, a completely reversible phenomenon; Ca2+ restoration in the presence of the Ca2+ channel blocker, nifedipine (10 microM), blunted markedly the reversal to the control Isc. Overall, these results suggest that ACh receptor activation induces the release of intracellularly stored Ca2+, which in turn leads to the temporary deactivation of a K+ conductance(s); in addition, secondary Ca2+ inflow may further extend the observed inhibition. During this study, the Isc of about 30% of the lenses used spontaneously oscillated (common duration of 30 min, with a mean peak frequency of 0.76 +/- 0.32 cycle min-1 and mean amplitude of 4.07 +/- 2.65 microA cm-2; +/- S.D.S, n = 24). Experiments attempted to determine the sensitivity of the oscillatory activity to ACh. Tg, nifedipine, and the phorbol ester PMA.(ABSTRACT TRUNCATED AT 400 WORDS)

Surface change of the mammalian lens during accommodation

Classical theories suggest that the surface area of the crystalline lens changes during accommodation while the lens volume remains constant. Our recent work challenged this view by showing that the lens volume decreases as the lens flattens during unaccommodation. In this paper we investigate 1) the magnitude of changes in the surface of the in vitro isolated cow lens during simulated accommodation, as well as that of human lens models, determined from lateral photographs and the application of the first theorem of Pappus; and 2) the velocity of the equatorial diameter recovery of prestretched cow and rabbit lenses by using a custom-built software-controlled stretching apparatus synchronized to a digital camera. Our results showed that the in vitro cow lens surface changed in an unexpected manner during accommodation depending on how much tension was applied to flatten the lens. In this case, the anterior surface initially collapsed with a reduction in surface followed by an increase in surface, when the stretching was applied. In the human lens model, the surface increased when the lens unaccommodated. The lens volume always decreases as the lens flattens. An explanation for the unexpected surface change is presented and discussed. Furthermore, we determined that the changes in lens volume, as reflected by the speed of the equatorial diameter recovery in in vitro cow and rabbit lenses during simulated accommodation, occurred within a physiologically relevant time frame (200 ms), implying a rapid movement of fluid to and from the lens during accommodation.

Sildenafil stimulates aqueous humor turnover in rabbits.

Sildenafil citrate increases ocular blood flow and accelerates the rate of anterior chamber refilling after paracentesis. The latter effect could have resulted from a reduction in outflow facility or from an increase in aqueous humor (AH) production. In this study, we used scanning ocular fluorophotometry to examine the effects of sildenafil on AH turnover, and thus, AH production in eyes of live normal rabbits. For this, the rate of aqueous humor flow (AHF) was quantified with a commercially available fluorophotometer that measured the rate of fluorescein clearance from the anterior segment, which predominantly occurs via the trabecular meshwork. After ≈2 h of control scans to determine the baseline rate of AHF, the rabbits were fed 33 mg of sildenafil and allowed ≈45 min for the drug to enter the systemic circulation. Thereafter, fluorescence scans were retaken for an additional 90-120 min. Sildenafil ingestion increased AHF by about 36%, from 2.31 μL/min to 3.14 μL/min (P < 0.001, as two-tailed paired data, n = 20 eyes). This observation indicates that sildenafil citrate, which is a phosphodiesterase type-5 inhibitor currently marketed as a vasodilator (e.g., Viagra, Revatio), stimulates AHF in rabbits. Our results seem consistent with reports indicating that the drug dilates intraocular arteries and augments intraocular vascular flow. These physiological responses to the agent apparently led to increased fluid entry into the anterior chamber. As such, the drug might have utility in patients with ocular hypotony resulting from insufficient AH formation.

Interaction between Mechanical and Osmotic Forces in the Isolated Rabbit Lens

Based on our previous work showing that cow and rabbit lenses isolated with their accommodation anatomical components intact change volume during simulated accommodation in vitro, and that hyposmolality and hyperosmolality also produce volume changes, we tested the idea that exerting these forces simultaneously may add or counteract each other. Further, we attempted to find a point at which osmotic and mechanical forces may cancel each other. Using previously described methodology, we found that combined stretching and anisotonic conditions applied to a lens always produced less of a volume change than that observed on its paired lens from the fellow eye that was only subjected to anisotonic conditions. Our results suggest that a stretching force that increases the equatorial diameter by 0.4% and reduces the lens volume by 1.8% could be canceled by a hyposmotic force of about -20 to -30 mOsM. Counter-intuitively, lenses that were subjected to stretching and hyperosmolality had less volume decrease than their paired lenses only exposed to hypertonicity. This latter observation is likely due to the prevention by the mechanical stretching forces of the shortening of the equatorial diameter, which normally occurs in hypertonic media.

Effects of Ca2+ on rabbit translens short-circuit current: evidence for a Ca2+ inhibitable K+ conductance

PURPOSE To characterize the effects of medium Ca2+ levels on rabbit lens electrical properties. Early studies with wholly submerged lenses had shown that Ca2+ removal from the bath resulted in an increased Rb+ efflux, a consequence of an increased Na+ Permeability and lens depolarization. METHODS Lenses were bathed with Ussing-type chambers under short-circuited conditions, an arrangement in which the translens short-circuit current (Isc) is carried out across the posterior lens surface mainly by an influx of Na+, and across the anterior face largely by a K+ efflux. RESULTS Under the present conditions in which the effects of Ca2+ were characterized unilaterally, the above established effects could only be ascribed to the posterior surface. When Ca2+ removal was limited to the anterior face, the Isc increased from 11.87 +/- 1.17 to 17.04 +/- 1.52 microA/cm2 (means +/- SEs, n = 18; an accompanying translens resistance (Rt) decrease of 0.23 +/- 0.049 K omega.cm2 was also recorded). Conversely, increasing the control, anterior-bath [Ca2+] from 1.8 to 3.6 mM reduced the K+ efflux-dependent Isc from 10.54 +/- 1.09 to 8.93 +/- 1.02 (n = 10, with an Rt increase of 0.11 +/- 0.013). These changes were reversible Na(+)-independent, and fully inhibited by the presence of K+ channel blockers (quinidine or Ba2+). Inhibitions of the Ca2+ effects were also obtained with strontium, a Ca2+ surrogate. The Isc was less responsive to changes in the Ca2+ content of the posterior bath. Removal of the cation caused a gradual 1.65 +/- 0.72 microA/cm2 increase (n = 9, with an Rt decrease of 0.090 +/- 0.021 K omega.cm2). In the absence of posterior Na+, Ca2+ withdrawal resulted in highly variable responses, with some specimens exhibiting salient current increases, suggesting that an outwardly directed, posterior efflux of an anion could also have been affected. During the course of this study it was consistently observed that the removal of Na+ from the anterior bath led to an Isc decrease of 2.62 +/- 0.22 microA/cm2 (n = 32, with an Rt increase of 0.35 +/- 0.029 k omega.cm2). This change occurred in both the presence of ouabain and the absence of Ca2+, suggesting that it did not result from an inhibition of the Na(+)-K+ pump current nor from a reversal in putative Na+/Ca2+ exchange activity. Small Isc increases upon anterior Na+ withdrawal (1.68 +/- 0.17, n = 7), consistent with Na+ efflux from the lens, could only be observed with K+ channels inhibited with Ba2+. Also congruent with the observations of a relatively limited anterior Na+ permeability, was the finding that the induction of nonspecific cation channels with amphotericin B reduced the Isc by following Na+ from the anterior bath to enter the lens. Thus, changes in lens Isc can differentiate changes in K+ permeability across the native anterior epithelium from changes in Na+ permeability. CONCLUSIONS Overall, these results suggest that lens Ca2(+)-mobilizing agents (e.g. acetylcholine) could trigger the inhibition of epithelial K+ conductance(s) by the direct action of Ca2+ on K+ channels.

Mechanical stretching forces oppose osmotic lens swelling.

In earlier work we demonstrated, with isolated lenses under in vitro conditions mimicking accommodation, that changes in lens volume occur in accord with the lens shape changes (Gerometta et al., 2007; Zamudio et al., 2008). In vivo, human lenses become rounder for closer vision and flatter for distant vision, a process involving a completely reversible change in shape. Our data indicated that lens volume was larger in the rounder, accommodative state than when the lens was in the flatter conformation (Gerometta et al., 2007). More recently, we determined that under hypo-osmotic challenge the lens not only changes volume but also its shape by becoming rounder (Kong et al., 2009). In both the rounded accommodative state and the hypo-osmotic swelling conditions, the observed increase in lens “circularity” occurred because of an increase in the length of the axis between the anterior and posterior poles (A-P length). Osmotically induced lens swelling does not markedly affect the equatorial diameter (ED) indicating that the swollen lens exhibits a distinctive shape change. In the accommodative process, ED is increased as the lens is stretched, thereby shortening A-P length, with lens “circularity” restored by reversal of these changes. These observations suggested to us that the mechanical stretching force that flattens the lens and shortens A-P length might oppose the force of osmotic swelling, which increases A-P length. In this short communication, we present data consistent with this idea. Moreover, in principle, a defined hypotonicity might exist that would not elicit a net gain in lens volume when lenses are in the stretched conformation. This situation could occur if the stretching force were adequate to prevent the lens from adopting a more rounded shape, and if the capsule were sufficiently inelastic so that the lens cannot gain volume. Increases in lens volume can only occur if either the lens changes its shape, and/or increases its surface area. If the capsule were completely inelastic, volume increases would only occur by the lens becoming more spherical, a shape allowing maximal volume for a given surface area. In practice, we also observed that tonicity-evoked changes in lens volume develop very gradually, and that relatively large tonicity shifts (i.e., ± 90 mOsM) are necessary in order to assuredly detect lens volume changes within a relatively short time frame (≈ 10-20 min) (Kong et al., 2009). Given these observations, we presently aimed to test the idea that the stretched, flattened lens might resist the swelling effect of a −90 mOsM, hypotonic solution. Based on a lens topology similar to a torus, we developed a technique that allows volume determination from the lens cross-sectional area (CSA). The CSA was obtained from photographs taken perpendicularly to the lenticular A-P axis and computed with software. From the same digital images, we also measured the A-P length between the polar surfaces and the ED. This approach was described in detail in Gerometta et al., (2007), and later used to determine rabbit and cow lens volumes with time of exposure to anisotonic conditions (Kong et al., 2009). As discussed in the latter publication, our method enables measurements of rapid changes in lens volume, because sequential digital photographs can be captured quickly. A minor inconvenience is that the lens volume must be meticulously calculated from each of the captured images. However, our technique has a high resolution that readily enables detection of volume changes greater than 1% (Kong et al., 2009), and importantly, the lens can be left untouched within a bathing chamber throughout the protocol. In the present experiments, the lenses were bathed within the stretching apparatus that was described in detail for the rabbit lens (Zamudio et al., 2008). The stretching device contained eight motors evenly mounted on a circular module. These motors were synchronized to simulate un-accommodation and accommodation by respectively transmitting radial stretching forces and relaxation to the lens as described in detail before (Zamudio et al., 2008). The only difference was that the rabbit lens was now photographed perpendicularly to the A-P axis, as done earlier to calculate volume changes in the cow lens during accommodation (Gerometta et al., 2007). Such lateral photographs of the rabbit lens captured both the length of the A-P axis and ED in the same image, which is a requisite of the volume calculation. In a protocol done on 6 rabbit lenses, our approach entailed monitoring the lens volume under control conditions, then stretching it, exposing the stretched lens to a hypotonic medium for 20 min, followed by releasing the stretching force in the hypotonic solution. The measured lens parameters from these experiments are compiled in Table 1. Table 1 Effects of Hypotonic Conditions on the Anterior-Posterior (A-P) Length, Equatorial Diameter (ED), A-P to ED ratio, and Volume of Isolated Rabbit Lenses Held in a Stretched Conformation. Because rabbit lenses do not stretch as well as lenses from higher primates, a small stretching force was applied until a significant reduction in A-P length could be observed (from 8.02 mm to 7.93 mm, Table 1). This degree of lens flattening caused the lenses to lose 6 μL (from 454 to 448 mm3, Table 1). After 10 min in 200 mOsM solution (t= 15 min of the protocol), the flattened lenses gained 11 μL (from 448 to 459 mm3). After an additional 10 min (t= 25 min), the lenses gained only two more microliters, bringing lens volume to 461 mm3. At this point, the stretching force was released (t= 26 min) causing the lenses to gain an additional 8 μL within 1 min, suggesting that the stretching force had impeded the inflow of fluid into the lenses by preventing them from becoming rounder. Earlier we defined the ratio of the A-P length to ED as a measure of “circularity” and showed that this ratio increased significantly in rabbit lenses exposed to 200 mOsM solution for 20 min (Kong et al., 2009). In contrast, in the present experiments, in which the 20 min of swelling occurred while the lenses were under a stretching tension, thereby inhibiting an unimpeded increase in A-P length, the “circularity” ratio did not change significantly under the hypotonic conditions. This ratio remained at a value between 0.75 and 0.76 throughout the experimental protocol for the 6 lenses shown in Table 1, an observaton also suggesting that the stretching tension had prevented the lenses from swelling freely. Furthermore, if the 290-to-200 mOsM toncity shift were independent of an opposing stretching force that impedes fluid uptake, then the changes in volume of lenses swollen in the stretched and relaxed conformations would be the same. Data in Table 2 suggest that the stretching force reduced the relative degree of swelling exhibited by the lenses. If stretching had no influence on the degree of lens swelling, the percentage changes in lens volume compared to the t= 0, control values should be similar for the two groups, but our results suggest otherwise. Table 2 Comparison of Control Rabbit Lens Volume to Volume after swelling for 20 min at 200 mOsM in the Stretched versus Relaxed Conformation. Finally, our earlier data indicating that lens volume was larger in the rounder, accommodative state than when the lens was in the flatter conformation were consistent with a possible net flow of fluid to and from the lens during the accommodative process (Gerometta et al., 2007). The present results are also in accord with net movements of fluid between the lens and the bathing medium in response to mechanical forces; i.e., stretching forces and hypo-osmotic forces have oppositely directed influences on lens volume. Putatively, stretching may inhibit the length of the A-P axis from freely increasing during swelling.

Application of JC1 for non-toxic isolation of cells with MDR transporter activity by flow cytometry

The DNA intercalating dye Hoechst 33342 or its close analog DCV are actively removed from cells by the multidrug resistance transporter ABCG2, a protein overexpressed in metastatic cells and somatic stem cells. In bivariate blue-red flow cytometry fluorescent plots active Hoechst or DCV efflux combined with a concentration dependent bathochromic shifts of these nuclear dyes leads to the segregation of the transporter-rich cells into a distinct cell cohort tilted towards the shorter wavelength axis of the plot, the cohort is generically known as the side population (SP). This feature has facilitated the surface marker-independent isolation of live stem cells. A drawback, though, is the known toxicity of Hoechst dyes. In this study we show that JC1, a bathochromic mitochondrial membrane potential-sensitive dye applied at proper concentration, can yield flow cytometry fluorescent emission bivariate plots containing a low JC1 accumulation (JC1low) cohort. Using a combination of multiple cell lines, ABC-transporter inhibitors and viral vector-driven insertion of the ABCG2 gene or ABCG2 and ABCB1 shRNAs we demonstrate that JC1low can be generated by either of the two aforementioned multidrug resistance transporters. Complete wash out of mitochondrial bound JC1 required more than 24 h. In spite of this tight binding, the dye did not affect either the mitochondrial membrane potentials or the proliferation rate. In contrast, contemporaneous with its nuclear accumulation, Hoechst 33342 or DVC, caused changes in the fluorescent emission of mitochondrial membrane potential sensitive dyes resembling the effects caused by the mitochondrial uncoupler FCCP. In a number of cell lines exposure to Hoechst resulted in marked slow-down of proliferation and abolition of ABCG2 transport activity during the subsequent 2 days but in K562 cells the exposure induced cell extended death. Overall, its lack of toxicity vis. a vis. the toxicity and genotoxicity of the DNA intercalating dyes makes JC1 an ideal tool for isolating live cells expressing high multidrug resistance transport activity.