Effects of norepinephrine and 5-hydroxytryptamine on phosphoinositide-PO4 turnover in rabbit cornea.

We have investigated: (a) Phospholipid composition and phosphoinositide-PO4 turnover in rabbit cornea tissues; and (b) the effects of adrenergic and serotonergic agonists on breakdown of phosphoinositides in the rabbit cornea. The data obtained from these studies can be summarized as follows: (1) in the cornea phosphatidylcholine and phosphatidylethanolamine constitute about 55%, phosphatidylinositol (PI) 10%, and phosphatidylinositol 4-phosphate (PIP), phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidic acid (PA) comprise about 1% each of the total phospholipids; (2) incubation of cornea in 32Pi-containing medium resulted in incorporation of radioactivity in tissue phospholipids. The radioactivity was highest in PIP2 (39%), followed by PI (19%), PIP (16%) and PA (5% of the total radioactivity). When compared with stroma and endothelium, the cornea epithelium was most active in phosphoinositide metabolism; (3) addition of norepinephrine (NE) or 5-hydroxytryptamine (5-HT), 200 microM each, to 32P-labeled cornea resulted in a loss of radioactivity in PIP and PIP2 by about 12- and 20%, respectively. Concomitantly, the radioactivity in PA and PI was increased by 44- and 66%, respectively. The effects of the neurotransmitters were time- and concentration-dependent. When added to the cornea labeled with myo [3H] inositol, NE and 5-HT increased the production of labeled myo-inositol phosphates; (4) prazosin (20 microM), but not yohimbine or propranolol, blocked the effects of NE. Similarly, the effects of 5-HT were antagonized by methysergide (20 microM) and ketanserin (10 microM) but not by prazosin. These data demonstrate that NE and 5-HT stimulate phospholipase C-mediated hydrolysis of PIP2 into diacylglycerol (DG) and myo-inositol trisphosphate (IP3). Furthermore, the effects of NE and 5-HT are mediated by alpha 1-adrenergic and 5-HT2 receptors, respectively. It is suggested that IP3, by releasing Ca2+ from ER, and DG, by activating protein kinase C, may function as second-messenger molecules which may participate in agonist-induced functional responses, including chloride transport, in the cornea epithelium.

Energy dispersive x-ray analysis of the cornea. Application to paraffin sections of normal and diseased corneas.

The distribution of chemical elements in the normal human cornea was studied by energy dispersive x-ray analysis and scanning electron microscopy of routinely prepared paraffin sections. Calcium, phosphorus, and sulfur were consistently present in quantities above background and varied in concentration regionally. Analysis of fresh-frozen tissue, an approximation of the in vivo state, gave a similar elemental profile to paraffin sections, except for the loss of diffusable electrolytes in the latter. After fixation, S was the most abundant element and was highest in Descemet's membrane. Corneas with granular, lattice, macular, and Fuchs' endothelial dystrophies, band keratopathy, and spheroidal degeneration were also examined. Characteristic patterns of abnormal S and Ca distribution were found in each of the dystrophies. The relative proportions of Ca, P, and S gave diagnostic profiles for distinguishing band keratopathy and spheroidal degeneration.

Phosphatic metabolites of the intact cornea by phosphorus-31 nuclear magnetic resonance.

The principal low molecular weight phosphatic metabolites of the intact cornea were identified and quantitated nondestructively by phosphorus-31 nuclear magnetic resonance (P-31 NMR) spectroscopy. As part of this analytical procedure, the intracorneal pH was approximated from the resonance shift position of inorganic orthophosphate. In addition the metabolic and pH stability of incubated corneas at 37 C in MK medium was evaluated during an 8-hr time course and compared to similar dynamic analyses performed on corneas with denuded endothelium. Perchloric acid extracts prepared from these same corneas were analyzed by P-31 NMR to verify the metabolite peak assignments and to quantitate the concentrations of minor corneal metabolites. The concentrations of phosphatic metabolites of the cornea, including three previously unidentified phosphorus-containing substances, were determined for freshly excised corneas. The initial corneal spectroscopic profile was not altered by removal of the endothelium. At 37 C the MK media-incubated intact whole corneas experienced a time-dependent decline in ATP levels with a concomitant rise in inorganic orthophosphate; however, the tissue levels of the other principal phosphatic metabolites were not altered by prolonged incubation. In contrast, removal of the endothelial layer of the cornea-induced progressive metabolic deterioration of intact corneas characterized, most prominantly, by time-dependent declines in ATP and glycerol 3-phosphorylcholine levels and concomitant increases in ADP and inorganic orthophosphate levels relative to intact whole corneas. This study has established the feasibility of monitoring the metabolic status of intact rabbit corneas nondestructively and noninvasively. As such, P-31 NMR spectroscopy offers a promising method that may enable analysis of the metabolic viability of intact human donor corneas to provide a basis for selecting donor corneas for transplantation.

Haemosiderin deposits in the human cornea.

A siderotic cornea from a case of post-traumatic hyphaemia showed large numbers of siderosomes in the corneal fibroblasts and a few in the corneal epithelial and endothelial cells. Electron-probe x-ray analysis revealed iron, phosphorous and sulphur in the siderosomes.

The significance of the arginine and arginase of tears in experimentally-induced herpes simplex corneae.

Experimental corneal herpes is always accompanied by the accumulation of arginine, the substrate of arginase, in tears, ensuring the multiplication of the herpes hominis virus. The main source of the large amount of arginine is the desquamating corneal epithelium, since after the epithelium of the cornea is abraded the arginine content of the tears again equals that of healthy tears. The low arginase content of rabbit tears can be supplemented by arginase applied as eyedrops, and this results in the cure of the herpetic process.

Antibiotic therapy of experimental Pseudomonas keratitis in guinea pigs.

Antibiotic therapy of experimental Pseudomonas keratitis was evaluated quantitatively by determining numbers of viable bacteria in the cornea of guinea pigs. Topically applied carbenicillin disodium, gentamicin sulfate, and tobramycin sulfate were often significantly more effective than topically applied polymyxin B sulfate. Intramuscular therapy with tobramycin was as effective as topical therapy, and the results exhibited less variability. Topical tobramycin every 30 minutes was significantly more effective than topical therapy every 60 minutes. No combination of antibiotics was significantly better than a single effective drug. The concentration of tobramycin in the aqueous correlated more closely to therapeutic efficacy than did the concentration in the cornea. Although all antibiotics reduced numbers of bacteria in the cornea by more than 99% in the first 24 hours of therapy, none was able to sterilize the cornea in four additional days of continuous therapy. Persistence of organisms despite apparently adequate topical therapy may explain some reported cases of relapse in humans.