Kenichi Kishida

Sodium and chloride transport across the isolated rabbit ciliary body

Unidirectional Na+ and Cl- fluxes were determined in the isolated rabbit ciliary body under short-circuited conditions. A statistically significant net Cl- flux towards aqueous was detected in the standard bathing solution (HCO3- = 115 mM), but no net Na+ flux was demonstrated in this solution. The net Cl- flux exceeded the short-circuit current, suggesting the existence of a net flux of other ion(s). In a bathing solution containing 5 mM HCO3-, the net Cl- flux was abolished, suggesting that HCO3- in the bathing solution enhances the net Cl- flux.

Induction of cytochrome P-450 in the rabbit eye by phenobarbital, as detected immunohistochemically

The present study was aimed to demonstrate that cytochrome P-450 was induced by drug administration in particular tissues in the eye. Phenobarbital was used as an inducer. We showed immunohistochemically that cytochrome P-450 was induced in the cornea, conjunctiva and ciliary epithelium of rabbits after four days of intraperitoneal administration of phenobarbital at a dose of 80 mg per kg per day. We did not detect significant immunofluorescence in other ocular tissues. Prolonged administration caused degeneration of the ciliary epithelium, but not pathological change was seen in other ocular tissues. In this case, immunofluorescence was not detected in the ciliary epithelium but in the cornea, conjunctiva and lens.

Electrical characteristics of the isolated dog ciliary body

Transepithelial potential difference and short-circuit current of the dog ciliary body were measured in vitro in an Ussing-type chamber using HEPES-buffered solution containing bicarbonate. Their values were -1.35 +/- 0.08 mV (mean +/- S.E.M., n = 52) and -23.6 +/- 1.7 microA/cm2, respectively. At 10(-4) M, ouabain reduced the short-circuit current upon to either side of the chamber. On the other hand, at 10(-5) M of furosemide to the aqueous side, the short-circuit current was reduced. However even at 10(-3) M, this drug had no effect when applied to the stromal side. Behavior of the dog ciliary body towards these two inhibitors was compared with that of the toad and rabbit tissues.

Cytochrome P-450 and related components of the microsomal electron transport system in the bovine ciliary body

Microsomes were prepared from bovine ciliary bodies. The contents of cytochrome P-450 and related components of the microsomal electron transport system were determined. The cytochrome P-450 content was 32 pmoles/mg protein, which was about 4% that in rat liver. The cytochrome b5 content was 59 pmoles/mg protein. The NADH-cytochrome c reductase and NADPH-cytochrome c reductase activities were 268 and 18 nmoles/min/mg protein, respectively. The ethoxyresorufin deethylase activity was 2.1 pmoles product formed/min/mg protein.

Reactive Oxygen Species Involved in Phenazine-Methosulfate-Induced Rat Lens Opacification

The excised rat crystalline lens opacified when incubated aerobically with phenazine methosulfate, but no opacification was observed under anaerobic conditions. Morphological studies revealed development of opacification in the cortex. The opacification resembled that often seen in the early period of senile cataract as well as in naphthalene-induced and UV cataract. Both an increase in hydration and in electrolyte imbalance accompanied this opacification. NaK-ATPase activity of the opacified lens was found to decrease. In order to investigate if activated oxygen is involved in these processes, we conducted an electron spin resonane study by means of a spin trapping technique. When the lens homogate was incubated with phenazine methosulfate, OH radicals were generated under aerobic but not under anaerobic conditions. Reduced pyridine nucleotides must be involved in the process, because the mixture of nicotinamide adenine dinucleotide phosphate [NAD(P)] and phenazine methosulfate did not generate OH radicals, but the mixture of NAD(P)H and phenazine methosulfate generates OH radicals, indicating that reduced phenazine methosulfate was involved in the OH radical generation. Probably, the generated OH radicals inactivated NaK-ATPase residing in the epithelium of the lens, which eventually caused opacification of the lens. The present experiment system may be used for the elucidation of lens opacification (cataract) involved with reactive oxygen species.

Differential effects of fibronectin-derived oligopeptides on the attachment of rabbit lens epithelial cells in vitro

Among the Arg-Gly-Asp (RGD)-containing sequences that are known to be cell-binding domains of fibronectin, 500 migrograms/ml of Arg-Gly-Asp-Ser (RGDS) and Gly-Arg-Gly-Asp-Ser showed 100% inhibition of the attachment of cultured lens epithelial cells (TOTL-86 cells), when they were added to culture medium and coincubated with the cells for 24 h whereas RGD at concentrations of 500, 1,000 and 2,000 micrograms/ml had no such activity. After 48 h of cocultivation of 800, 400 or 200 micrograms/ml of RGDS with TOTL-86 cells, the percentage of floating cells was 100, 30.1 or 11.1%, respectively. After 144 h of cocultivation with RGDS, the percentage of floating cells was 1.6, 2.4 or 1.9%, respectively, indicating that RGDS was not cytotoxic to lens epithelial cells. However, replacing the medium with fresh medium containing new RGDS peptide resulted in floating of cells. We also studied the inhibitory effect of two other amino acid sequences that are found in cell-binding sites of the fibronectin molecule: Glu-Ile-Leu-Asp-Val-Pro-Ser-Thr (EILDVPST) and Arg-Glu-Asp-Val (REDV). At 500 and 1,000 micrograms/ml, respectively, neither EILDVPST nor REDV has an inhibitory effect on the attachment of TOTL-86 cells, while RGDS at a concentration of 500 micrograms/ml completely inhibited the attachment of the cells in 24 h of incubation.

2-Substituted 1, 3, 4-thiadiazole-5-sulfonamides as carbonic anhydrase inhibitors: Their effects on the transepithelial potential difference of the isolated rabbit ciliary body and on the intraocular pressure of the living rabbit eye

In order to understand the pharmacology of carbonic anhydrase inhibitors in reduction of aqueous secretion, three sorts of studies were conducted using five 2-substituted 1, 3, 4-thiadiazole-5-sulfonamides: an inhibition study of carbonic anhydrase II, electrical measurements of the isolated ciliary body, and pharmacological study on intraocular pressure of living animals. The inhibitors of carbonic anhydrase employed here were 2-amino-1, 3, 4-thiadiazole-5-sulfonamide; 2-methylamino-1, 3, 4-thiadiazole-5-sulfonamide; 2-formylamino-1, 3, 4-thiadiazole-5-sulfonamide; 2-acetylamino-1, 3, 4-thiadiazole-5-sulfonamide (acetazolamide); and 2-propionylamino-1, 3, 4-thiadiazole-5-sulfonamide. All of these compounds showed significant inhibitory activity to carbonic anhydrase II which exists in the ciliary epithelium, but their potencies of inhibition varied relative to one another (I50S were 1.91 X 10(-7) to 3.3 X 10(-8) M). The effects of the five compounds on electrical phenomena were observed using isolated rabbit ciliary body mounted on an Ussings chamber. Each compound decreased the negative electrical potential of the tissue (-0.70 mV as the average of the initial values) by 10- to 33%, and this effect was proportional to its inhibitory activity to carbonic anhydrase II. The effects of the five compounds on intraocular pressure were determined, and each compound decreased the intraocular pressure (18 mmHg as the average of the initial values) by 7- to 32%. This effect was also proportional to the inhibitory activity to the enzyme. Correlation between the two effects was studied, and good correlation was observed. This implies that both effects have a common basis which relates to the physiological role of carbonic anhydrase. The present study, therefore, shows the importance of the bicarbonate ion in the aqueous humor formation since it is both substrate and product of carbonic anhydrase II.

Cysteine conjugate of methazolamide is metabolized by β‐lyase

Abstract Bovine kidney and liver homogenates degraded a cysteine conjugate of methazolamide, S ‐(5‐acetylimino‐4‐methyl‐Δ 2 ‐1,3,4‐thiadiazolin‐2‐yl)cysteine. We isolated the degradation product following incubation with kidney homogenate by high‐performance liquid chromatography on reversed‐phase columns. The chemical structure was confirmed by proton and carbon‐13 nuclear magnetic resonance spectroscopy ( 1 H NMR and 13 C NMR, respectively), and elemental analysis by high‐resolution mass spectrometry to be N ‐(3‐methyl‐5‐mercapto‐Δ 4 ‐1,3,4‐thiadiazol‐2‐yl)acetamide, a thiol compound. The reaction is thought to be catalyzed by a pyridoxal‐dependent enzyme(s) as indicated by an inhibition study using aminooxyacetic acid. Possible involvement of the thiol compound in the development of an adverse effect is discussed.

An Application of Mass Fragmentography to the Determination of Acetazolamide in Body Fluids

Abstract A reliable mass fragmentographic determination method for acetazolamide levels in body fluids was developed to favor the pharmacokinetic study on the patients receiving multiple doses. The use of 2-(N-propionamido)-1,3,4-thiadiazole-5-sulfonamide as the internal standard permitted the internal standard to be added to the sample before the extraction, and enabled the single ion measurement at m/e 249. Single ion measurement at m/e 249 detected 25 pg of acetazolamide. Multiple ion detection at m/e 249, 264 and 278 favored more accurate identification of each compound on the chromatogram. The assay results of the samples from volunteers were consistent with the ones reported elswhere.

Effects of Carbonic Anhydrase Inhibitors on Electrical Phenomenon of the Ciliary Body of the Rabbit Eyea

The objective of the present work was to investigate the role of carbonic anhydrase in the aqueous humor secretion in the eye, especially in relation to anion transport(s) that brings about negative value of transepithelial electrical potential difference across the ciliary body. We studied the effects of five carbonic anhydrase inhibitors on the electrical potential difference of the isolated rabbit ciliary body, and then correlated the findings with the results of enzymological and aqueous humor dynamics studies.