Corneal function after storage in dexsol or optisol.

Rabbit corneas were stored in Dexsol or Optisol (Chiron, Irvine, CA) for up to 2 wk at 4 degrees C. The thickness of corneas placed in Dexsol decreased 10 microns after they were placed initially in Dexsol, then increased approximately 8 microns/d for 7 d and 3 microns/d thereafter. Corneas placed in Optisol decreased 35 microns in thickness initially, then increased 2 microns/d thereafter. Human corneas showed similar thickness changes to those of the rabbit when stored in these media. After 5.5, 10, and 14 d in storage, rabbit corneas from each medium were cultured to assess their net deturgescence ability. Identical groups were cultured in media containing 20 microM ouabain to monitor the corneas' passive swelling characteristics. Corneas stored in either medium showed similar net deturgescence and passive swelling patterns after each storage period. Deturgescence rates decreased with increasing storage time, primarily because the rates of passive corneal swelling increased with storage time. Knowledge of the net deturgescence and passive swelling rates allowed an estimation of the total deturgescence activity of corneas after removal from Dexsol or Optisol. The total deturgescence activity of corneas stored in Dexsol for 5.5, 10, and 14 d was 85%, 68%, and 63% of control corneas, which were processed identically but not stored before culture. Corneas stored in Optisol exhibited 87%, 71%, and 69% of control deturgescence activity, respectively. These experiments show that Optisol was not significantly better than Dexsol in retaining poststorage corneal deturgescence activity but was superior to Dexsol in preventing corneal swelling during storage.

Alpha 1-adrenoceptors in human corneal epithelium.

Specific binding of the potent, selective alpha 1-adrenoceptor antagonist 3H-prazosin was demonstrated in cultured human corneal epithelial cells. Specific binding of the radioligand was concentration-dependent between 0.5 and 6 nM, with apparent saturation of receptor sites seen at higher concentrations. The cells exhibited a maximum binding capacity for 3H-prazosin of 225 fmol/mg of cellular protein and a dissociation constant of 2 nM. The binding of 3H-prazosin was competitive with known alpha 1-adrenoceptor ligands and was reversible. Epithelium of intact human corneas also exhibited specific 3H-prazosin binding, as did cultures of bovine and rabbit corneal epithelium. The alpha-adrenergic agonist methoxamine significantly stimulated phosphatidylinositol 4,5-bisphosphate hydrolysis, measured as myoinositol trisphosphate accumulation in cultures of human corneal epithelium. This stimulation was inhibited by the presence of prazosin during the assays. These findings indicate the existence of specific, reversible, high-affinity receptors for alpha 1-adrenoceptors that regulate inositol phosphate turnover in human, rabbit, and bovine corneal epithelial cells.

Atrial natriuretic peptide receptors on the corneal endothelium.

PURPOSE: To ascertain whether the corneal endothelium exhibits specific receptors for atrial natriuretic peptide (ANP) and to characterize partially any binding activity. METHODS: Most experiments employed radioligand-binding techniques to characterize 125I-ANP binding activity. Guanylate cyclase and corneal deturgescence assays were used in an attempt to correlate 125I-ANP binding activity with physiologic processes. RESULTS: Corneal endothelial cells reversibly bound 125I-ANP with high affinity and exhibited a finite number of 125I-ANP binding sites. These binding sites also bound several ANP fragments but did not show binding affinity for the peptide hormone, vasopressin. Autoradiograms of affinity labeled ANP receptors separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed specifically radiolabeled proteins of several molecular weights in nonreduced samples, but only one major radiolabeled protein of 65 kd when samples were chemically reduced before separation. CONCLUSIONS: Corneal endothelial cells from several species exhibit specific binding of 125I-ANP. The binding characteristics of these receptors are similar to physiologic ANP receptors identified in other tissues. Several lines of evidence indicate that corneal endothelial ANP receptors are the "clearance" (ANP-C) type.

The effects of UW solution and its components on corneal thickness during and after storage.

The ability of rabbit corneas to undergo energy-dependent deturgescence was examined after the corneas were stored at 4 degrees C in UW solution, M-K media, or selected modifications of these media. All corneas slowly increased in thickness during storage, despite the presence of colloidal osmotic agents. Corneas stored for 2.5 days in M-K became slightly thinner when cultured over a 24-hour period. Corneas stored in UW swelled quickly in culture and became too opaque to measure within three hours. Corneas stored in UW with 1.8 mM CaCl2 swelled transiently, then maintained their thickness and exhibited deturgescence in the latter stages of the culture period. Deturgescence of corneas stored for 7 days in M-K was only slightly worse than those stored for 2.5 days. Corneas stored for 7 days in UW, however, became opaque almost immediately in culture and those stored in calcium-supplemented UW became opaque within 4.5 hours. Replacement of the dextran in M-K with hydroxyethyl starch produced a slower rate of corneal swelling during storage and a substantially better corneal deturgescence profile during culture. Use of high concentrations of potassium ion in the M-K formulation had no significant effect on post-storage deturgescence. Replacement of glucose in M-K with the impermeable sugar, raffinose had a slight deleterious effect on corneal deturgescence in subsequent culture. Use of the impermeable anions gluconate or lactobionate to replace chloride ion caused profound corneal swelling during culture, compared with those stored in M-K. These experiments show that UW solution is inferior to M-K at preserving post-storage corneal function.(ABSTRACT TRUNCATED AT 250 WORDS)

Muscarinic cholinoceptors in native and cultured human corneal endothelium.

Specific and high affinity binding of the potent muscarinic cholinergic antagonist, [3H]quinuclidinylbenzilate ([3H]QNB) was observed using intact native and cultured adult human corneal endothelium (HCE). Specific binding was proportional to radioligand concentration between 0.03 and 5 nM, indicating a maximal binding capacity (Bmax) of 130 fmol of [3H]QNB/mg protein and a dissociation constant (Kd) of 0.3 nM. Atropine competed effectively with [3H]QNB for binding sites; requiring 3 nM to inhibit 50% of the binding of 1 nM [3H]QNB. Carbachol also competed with [3H]QNB at higher concentrations, but nicotine did not affect [3H]QNB binding at levels up to 1 nM. [3H]QNB binding was also observed in cultured cells of adult human, rabbit, and bovine corneal endothelium. Native and cultured HCE were affinity labelled using tritium-labelled propylbenzilylcholine mustard (PBCM). Separation of the proteins in affinity labelled native and cultured tissue by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) showed that only one protein in each preparation, of 60 and 55 kilodaltons (kDa), respectively, was specifically radiolabelled. These data indicate that the corneal endothelium of human and several animal species exhibit muscarinic cholinoceptors.

Electroacupuncture alters catecholamines in brain regions of rats.

Electroacupuncture (EA) stimulation mediated the release of [3H]norepinephrine (NE) from synaptosomes prelabeled with [3H]NE. The pulse release of [3H]NE by EA stimulation was dependent on the presence of Ca2+. Treatment of rats with EA for 30 min at 4 Hz did not significantly alter the dopamine (DA) content in hypothalamus, cerebellum, pons, midbrain, and cerebral cortex regions, but the DA level was decreased by 20% in caudate nucleus. The NE level was found to increase by 43% in caudate nucleus and 38% in hypothalamus. The results indicate that only certain neuronal pathways are affected by the EA treatment, and that NE and DA may respond differently to such stimulation.

Inhibition of Na+ reabsorption in the rat parotid gland by prostaglandin E1 and kallidin: implications for cystic fibrosis.

Prostaglandin E1 caused a dose-related inhibition of sodium reabsorption in the rat parotid gland when injected by retrograde perfusion into the glandular ducts. The extent of inhibition ranged from 11.7 +/- 2.4% at a dose of 2.5 micrograms to 63.8 +/- 8.9% at a dose of 31.2 micrograms. Both phospholipase A2, an enzyme involved in prostaglandin synthesis, and arachidonic acid, a precursor of prostaglandins, also increased the Na+ concentration of parotid saliva in a dose-dependent fashion. With phospholipase A2 the inhibition ranged from 21.6 +/- 4.4% at a dose of 3 micrograms to 73.5 +/- 8.2% at a dose of 30 micrograms. With arachidonic acid, the degree of inhibition was 5.1 +/- 3.0% at a 10(-5) M dose and 57.7 +/- 10.2% at a dose of 10(-3) M. Lysine bradykinin (kallidin), a peptide present in salivary and other exocrine glands and their secretions, also caused a 30% inhibition of Na+ reabsorption when retroperfused at a concentration of 12.5 micrograms, as did kallikrein (176 micrograms) and trypsin (33.3 micrograms). These results indicate that prostaglandins and kinins can inhibit Na+ reabsorption in the rat parotid duct when present in the luminal side of the cells. Since they are normally present in exocrine glands and can presumably be secreted, they may have a role as luminal factors in the regulation of transductal transport of Na+. The possibility that they may be increased in the exocrine secretions of patients with cystic fibrosis and that they may act as the so-called cystic fibrosis "factors" is also raised by the findings of this study.

Alpha 1-adrenoceptors in the corneal endothelium.

The potent alpha 1-adrenoceptor antagonist, [3H]prazosin, exhibited high affinity, specific and reversible binding to intact rabbit, bovine and human corneal endothelial cells in culture. The binding of 1 nM [3H]prazosin to rabbit cells reached a steady-state level within 10 min at 37 degrees C. Under these conditions, approximately 50% of the [3H]prazosin bound was specific. The level of specific [3H]prazosin binding was concentration-dependent, but Rosenthal analysis indicated that [3H]prazosin bound to at least two sites. One site exhibited a high affinity for [3H]prazosin (Kd = 0.2 nM), but a relatively low binding capacity (Bmax = 175 fmol bound mg-1 protein); the other site showed a relatively low affinity for the radioligand (Kd = 85 nM), but a much higher binding capacity (1280 fmol mg-1). Several known alpha 1-adrenoceptor antagonists and agonists competitively inhibited [3H]prazosin binding at the high affinity site when incubated with the radioligand. The relative potencies of these competing ligands were generally consistent with their binding affinities for alpha 1-adrenoceptors in other tissues. Phenylephrine stimulated the rate of hydrolysis of phosphatidylinositol 4,5-bisphosphate to inositol 1,4,5-trisphosphate by 63% in these cells. This stimulation was inhibited by 52% if phentolamine was also present during the incubation. These data indicate that corneal endothelial cells have alpha 1-adrenoceptors which can modulate polyphosphoinositide turnover in this tissue.