Volume regulation of cultured, transformed, non-pigmented epithelial cells from human ciliary body

Electronic cell sizing has been used to measure the volume of cells suspended in isosmotic and in hyposmotic solutions of identical ionic composition. Without inhibitors, the cells displayed a regulatory volume decrease (RVD) following anisosmotic cell swelling with a time constant (tau) of 6.3 +/- 0.9 min (mean +/- S.E.). The RVD was markedly impaired by substituting gluconate for external Cl-, and tau was prolonged by: (i) reducing the chemical gradient favoring K+ loss (by elevating the external [K+] and blocking the Na. K-exchange pump), (ii) blocking the K+ channels with Ba2+, (iii) blocking Cl- channels and Cl-/HCO3 = exchange with DIDS, and (iv) removing external HCO3-. Withdrawing HCO3- may have altered the RVD either directly by inhibiting a Cl-/HCO3- antiport, or indirectly by affecting intracellular pH. The regulatory volume response of ODM Cl-2/SV40 cells is in several respects qualitatively similar to that of non-pigmented epithelial cells of the intact ciliary body. These common characteristics suggest that the cultured cells can serve as a useful model for studying solute and fluid transport across the human ciliary epithelium. The basis for the RVD is likely to be activation of separate K+ and Cl- channels, with or without the parallel operation of coupled K+/H+ and Cl-/HCO3- antiports.     

Barrier qualities of the mouse eye to topically applied drugs

The mouse eye displays unusually rapid intraocular pressure (IOP) responses to topically applied drugs as measured by the invasive servo-null micropipette system (SNMS). To learn if the time course reflected rapid drug transfer across the thin mouse cornea and sclera, we monitored a different parameter, pupillary size, following topical application of droplets containing 40 microM (0.073 microg) carbachol. No miosis developed from this low carbachol concentration unless the cornea was impaled with an exploring micropipette as used in the SNMS. We also compared the mouse IOP response to several purinergic drugs, measured by the invasive SNMS and non-invasive pneumotonometry. Responses to the previously studied non-selective adenosine-receptor (AR) agonist adenosine, the A(3)-selective agonist Cl-IB-MECA and the A(3)-selective antagonist MRS 1191 were all enhanced to varying degrees, in time and magnitude, by corneal impalement. We conclude that the thin ocular coats of the mouse eye actually present a substantial barrier to drug penetration. Corneal impalement with even fine-tipped micropipettes can significantly enhance entry of topically-applied drugs into the mouse aqueous humor, reflecting either direct diffusion around the tip or a more complex impalement-triggered change in ocular barrier properties. Comparison of invasive and non-invasive measurement methods can document drug efficacy at intraocular target sites even if topical drug penetration is too slow to manifest convincing physiologic effects in intact eyes.     

Effect of bicarbonate on intracellular potential of rabbit ciliary epithelium.

Extracellular HCO3- hyperpolarizes the intracellular potential and makes the aqueous medium negative with respect to the stromal surface of the rabbit ciliary epithelial syncytium. The bases for these observations have been unclear. We have been studying the bicarbonate-induced hyperpolarization (BIH) with sustained intracellular recordings for periods as long as 1-2 hrs. The BIH was observed [6.0 +/- 0.4 mV (mean +/- SE, N = 22)] even when the external pH was clamped constant by appropriately changing the CO2 tension. External HCO3- was required since aeration with CO2 at low external pH did not replicate the BIH. DIDS [4,4'-diisothiocyano-2,2'-disulfonic acid] did not abolish the effect. The hyperpolarization is unlikely to reflect the pH dependence of K+ channels alone, since the effect was not reduced by either 2 mM Ba2+ alone or 2 mM Ba2+ together with 50-100 microM quinidine. The BIH depends directly or indirectly on external Na+, since the sign of the polarization response was reversed either by replacing Na+ with N-methyl-D-glucamine or by blocking the Na+,K(+)-exchange pump with 50-100 microM ouabain. Replacement of external Cl- with NO3- or application of the Cl(-)channel blocker NPPB [5-nitro-2-(3-phenylpropylamino)-benzoate] depolarized the membrane and reversed the sign of the BIH. The response of the ciliary epithelium to HCO3- is complex and may arise from several mechanisms. We suggest that one important element is an anion channel whose conductance is reduced by bicarbonate and whose reversal potential is indirectly dependent on the operations of the Na+,K(+)-pump and a Cl(-)-linked symport.     

Swelling-activated chloride channels in aqueous humour formation: on the one side and the other

Aqueous humour is secreted by the ciliary epithelium comprising pigmented and non-pigmented cell layers facing the stroma and aqueous humour respectively. Net chloride secretion likely limits the rate of aqueous humour formation and proceeds in three steps: stromal chloride entry into pigmented cells, diffusion through gap junctions and final non-pigmented cell secretion. Swelling-activated chloride channels function on both epithelial surfaces. At the stromal surface, swelling- and cyclic adenosine monophosphate-activated maxi-chloride channels can recycle chloride, reducing net chloride secretion. At the aqueous-humour surface, swelling- and A3 adenosine receptor-activated chloride channels subserve chloride release into the aqueous humour. The similar macroscopic properties of the two non-pigmented cell chloride currents suggest that both flow through a common conduit. In addition, measurements of intraocular pressure (IOP) in living wild-type and mutant mice have confirmed that A3 adenosine receptor-activated agonists and antagonists increase and lower IOP respectively. Isolated ciliary epithelial cells are commonly perfused with hypotonic solution to probe and characterize chloride channels, but the physiological role of swelling-activated channels has been unclear without knowing their epithelial distribution. Recently, hypotonic challenge has been found to stimulate the chloride-sensitive short-circuit current across the intact bovine ciliary epithelium, suggesting that the net effect of the swelling-activated chloride currents is oriented to enhance aqueous humour formation. Taken together, the results suggest that swelling-activated chloride channels are predominantly oriented to enhance aqueous humour secretion, and these chloride channels at the aqueous surface may be identical with adenosine receptor-activated chloride channels which likely modulate aqueous inflow and IOP in the living mouse.     

Noninvasive intraocular pressure measurements in mice by pneumotonometry

PURPOSE: To develop a reliable, noninvasive, continuous, and easily implemented system for measuring intraocular pressure (IOP) in mice. METHODS: Pneumotonometry was adapted for measurement of mouse IOP. Measurements were compared with those obtained with the servo-null micropipette system (SNMS) and with direct anterior chamber cannulation. Heart rate was monitored by the precordial pulse, EKG, or tail pulse in anesthetized mice. The characteristic ocular hypotensive response to mannitol was assessed as an additional validation of the METHOD: RESULTS: Measurements of IOP obtained using pneumotonometry agreed closely with values measured by SNMS or by direct cannulation. IOP oscillations were synchronous with the heart rate, with a coherence peak between them of approximately 2 Hz, equal to the pulse frequency. Hypertonic mannitol reduced IOP from 13.7 +/- 0.9 mm Hg by 7.7 +/- 0.7 mm Hg after 15 minutes. CONCLUSIONS: Pneumotonometry is a reliable and noninvasive method for the measurement of IOP in mice and may permit comparisons of IOP to hemodynamic factors. This system is simpler and more adaptable for glaucoma research than previously reported methodologies for measuring IOP in mice.     

Adult brightness vs. luminance as models of infant photometry: variability,biasability, and spectral characteristics for the two age groups favor the luminance model

When infants fail to make chromatic discriminations, do the characteristics of their performance minima coincide more closely with the properties of adult luminance matches or heterochromatic brightness matches? In addition to their spectral properties, adult luminance matches are typically characterized by relatively small individual differences, whereas brightness matches are believed to be both more variable and more biasable. Two complementary experiments were carried out on adults and 8-week-old infant subjects. Both groups were tested with small (1.5 degrees to 4 degrees ) red and blue test fields of varying luminances, embedded in a white surround. In adults, heterochromatic brightness matches were measured. Individual differences spanned about 0.5 log units, and brightness matches could be biased by as much as 0.8 log units by varying the range of test field luminances. In infants, the locations of performance minima were measured. Individual differences spanned less than 0.1 log units, the mean performance minima coincided with predictions based on V10(lambda), and the location of the performance minimum was nearly unaffected by the range of test field luminances used. Thus by all three criteria, these data suggest that infants' performance minima are mediated by luminance rather than by brightness signals. To date there remains no evidence that the infant visual system computes a brightness signal.     

Diffusion-weighted MR imaging findings of acute necrotizing encephalopathy

Multiple, symmetrical brain lesions affecting the bilateral thalami and cerebral white matter, which often show a concentric structure on CT and MR images, characterize acute necrotizing encephalopathy (ANE) of childhood. We describe the imaging findings of a 2-year-old child with ANE obtained with diffusion-weighted MR imaging. We discuss the significance of these findings, as well as the pathophysiology of ANE lesions, with reference to the appearance of the disease as revealed by diffusion-weighted MR imaging.