Intracameral anesthesia: in vitro iris and corneal uptake and washout of 1% lidocaine hydrochloride.

OBJECTIVES: To characterize the uptake, washout, and metabolism of lidocaine hydrochloride in the iris/ciliary body and cornea. METHODS: Iris/ciliary body uptake of lidocaine hydrochloride was measured by incubating human and rabbit irides in radiolabeled carbon 14-1% lidocaine hydrochloride for 2 to 60 minutes. Washout was determined by incubating the iris in 14C-1% lidocaine hydrochloride for 5 minutes and transferring the iris to a series of wells. The wells contained a common intraocular irrigating solution of essential ions, glucose, and glutathione buffered with bicarbonate (an enriched balanced salt solution [BSS PLUS]), which is similar to aqueous humor. Corneal uptake was measured by exposing the endothelial surface to 14C-1% lidocaine hydrochloride for 5 or 15 minutes. Corneal washout was performed after 5-minute exposure to 14C-1% lidocaine hydrochloride using a 2-chambered diffusion apparatus. Samples of the iris, cornea, and BSS PLUS washout solution were analyzed by high-performance liquid chromatography and liquid scintillation spectrometry. RESULTS: In vitro iris/ciliary body uptake of 14C-1% lidocaine hydrochloride follows a logarithmic curve, with 50% to 60% of maximum lidocaine hydrochloride uptake present at 5 minutes. There was no difference in uptake between human, albino rabbit, and pigmented rabbit irides. Washout of lidocaine from the iris occurs with a halflife of 8 to 9 minutes. Corneal uptake of lidocaine was greater after incubation for 15 vs. 5 minutes. The washout of lidocaine from the cornea had a half-life of 5 minutes. Results of high-performance liquid chromatography confirmed that there were no metabolites or breakdown products in the iris, cornea, or washout solution. CONCLUSIONS: Lidocaine is taken up quickly by the iris/ ciliary body and cornea and rapidly removed from these tissues after BSS PLUS washout. Irrigation during phacoemulsification seems to limit lidocaine exposure to the ocular tissues, resulting in a short duration of anesthesia. Lidocaine is not metabolized or broken down by the iris or cornea during this short period.

How might 12 (R) HETE cause the inhibition of Na,K-ATPase?

PURPOSE: 12 (R) hydroxy 5,8,10,14-eicosatetraenoic acid [12 (R) HETE] is a potent inhibitor of Na,K-ATPase. This study was an attempt to determine how the eicosanoid might inhibit the enzyme by using molecular modeling. METHODS: Models were generated using the program HyperChem 2.0 for Windows. Models of 12 (R) HETE, 12 (S) HETE (the "S" isomer of 12 (R) HETE), and 8 (R) hydroxy-hexadecatrienoic acid [8 (R) HHDTrE, a catabolic isomer of 12 (R) HETE] were formed and docked with phosphatidyl choline and the H3-H4 peptide of the alpha-subunit of Na,K-ATPase. In addition, models of 12 (R) HETE, and related compounds, were formed and complexed with calcium, and then docked with phosphatidyl choline. The energies of stabilization were calculated for each optimal docking. RESULTS: Optimal steric fitting and calculated energies of stabilization indicated that 12 (R) HETE and 8 (R) HHDTrE had the best fits when bound to the fatty acid portions of phosphatidyl choline. However, when Ca-HETE complexes were modeled, it was found that they formed even more stable complexes when bound to phosphatidyl choline. Calculated energies of 12 (S) HETE, whether complexed to calcium or not, were less favorable than the other HETE compounds. CONCLUSIONS: The results of the study indicate that plasma membrane lipids rather than Na,K-ATPase itself are more likely to be bound by 12 (R) HETE and its related compounds. Moreover, it was found that the calcium complexes of 12 (R) HETE and 8 (R) HHDTrE are even more likely to dock with plasma membrane lipids. This suggests that such complexes may be able to transport calcium into the cell and make it available for the inhibition of Na, K-ATPase at the enzyme's sodium binding site.

Corneal diffusion and metabolism of 12(R)-hydroxyeicosatetraenoic acid (12(R)HETE).

PURPOSE: To quantify the corneal diffusion and metabolism of tritiated 12(R)-hydroxyeicosatetraenoic acid (12(R)HETE) in the in vitro-mounted rabbit cornea to determine if this compound or one of its metabolites can diffuse across the stroma to the corneal endothelium. METHODS: The studies were performed in a Lucite block perfusion chamber by placing tritiated 12(R)HETE on the tear side of the cornea under the following conditions: (A) cornea completely intact (endothelium and epithelium present); (B) cornea with epithelium removed; and (C) cornea with both epithelium and endothelium removed. Radioactivity of 12(R)HETE and metabolites were measured in the different corneal layers and in the corneal perfusates using scintillation spectroscopy. 12(R)-hydroxyeicosatetraenoic and its metabolites were then quantified in the tissue perfusates using high performance liquid chromatography (HPLC) analysis. RESULTS: 12(R)HETE is rapidly taken up and metabolized by the intact cornea to a number of more polar compounds including a metabolite which has spectral and retention time characteristics of 8(R)-hydroxyhexadecatrienoic acid (8(R)HHDTrE). Both 12(R)HETE and 8(R)HHDTrE can diffuse through the stroma to the endothelium. Corneas having the epithelium removed also allow diffusion of 12(R)HETE across the stroma; however, there is significantly less metabolism. When both the epithelium and endothelium are removed, 12(R)HETE is capable of diffusing across the stroma; however, there is little metabolism, which suggests that the majority of 12(R)HETE metabolism occurs in the epithelium and, to a lesser degree, in the endothelium and stroma. CONCLUSIONS: 12(R)HETE and its metabolites are capable of diffusing from the epithelium through the cornea where they may adversely affect the endothelium.

Swelling in the isolated perfused cornea induced by 12(R)hydroxyeicosatetraenoic acid.

PURPOSE: To evaluate the effect of 12(R)hydroxyeicosatetraenoic acid (12(R)HETE) on corneal swelling when directly perfused to human and rabbit corneal endothelium. METHOD: Excised rabbit and human corneas were mounted in the in vitro specular microscope and the endothelium was perfused with 12(R)HETE at 10(-5), 10(-6), and 10(-7) mol/l. Both 12(R)HETE and 12(S)HETE were compared at equal molar (10(-6) mol/l) concentrations. The reversal of 12(R)HETE and ouabain corneal swelling was also compared. Endothelial permeability to carboxyfluorescein was measured after 12(R)HETE perfusion. High-performance liquid chromatographic analysis confirmed that 12(R)HETE remained in the perfusion media. RESULTS: 12(R)HETE caused a dose-dependent corneal swelling of 25 +/- 2, 24 +/- 1, and 14 +/- 0.5 microns/hr at 10(-5), 10(-6), and 10(-7) mol/l, respectively. Equal molar concentrations (10(-6) mol/l) of 12(S)HETE did not cause corneal swelling. Removal of the 12(R)HETE from the perfusion media resulted in reversal of corneal swelling whereas corneal swelling induced by ouabain did not reverse after ouabain removal. 12(R)HETE (10(-6) mol/l) perfused to the human corneal endothelium inhibited temperature reversal corneal thinning when compared to the paired corneal endothelium perfused with BSS Plus (Alcon Laboratories, Inc., Fort Worth, TX). Na/K adenosine triphosphatase activity was inhibited by 10(-6) mol/l ouabain by 35%, 10(-6) mol/l 12(R)HETE by 54%, and 10(-6) mol/l 12(S)HETE by 0.5%. Endothelial permeability to carboxyfluorescein was unaffected by 12(R)HETE. CONCLUSION: 12(R)HETE causes corneal swelling by inhibiting endothelial pump function. This inhibition of transport appears to be at least partly mediated by inhibition of endothelial Na/K adenosine triphosphatase.

cDNA clones encoding bovine gamma-crystallins.

We have determined the nucleotide sequence of two bovine lens gamma-crystallin cDNA clones, pBL gamma II-1 and pBL gamma III-1. The 644 bp cDNA insert of pBL gamma II-1 contains coding information for the entire amino acid sequence of bovine gamma II-crystallin. The 497 bp cDNA insert of pBL gamma III-1 encodes a homologous but different gamma-crystallin polypeptide, and appears to lack the coding information for the C-terminal 17 amino acid residues. While the nucleotide and predicted amino acid sequences of the coding regions of the clones show a high degree of homology, the untranslated leader sequences are relatively dissimilar. The leader sequence of pBL gamma III-1 is strikingly homologous to a portion of a rabbit immunoglobulin alpha-heavy chain mRNA.

The production and mechanism of ghost cell glaucoma in the cat and primate.

Fresh human ghost blood cells (GBCs) have been shown to cause increased resistance to outflow in enucleated human eyes. In addition, glutaraldehyde-fixed GBCs can cause glaucoma in the rabbit and primate in vivo. The present study shows for the first time that fresh autologous GBCs can cause an acute in vivo rise of intraocular pressure when injected into the anterior chambers of the cat and primate. This rise was of greater magnitude and longer duration than that caused by the injection of a greater number of pliable, fresh red blood cells. It has been theorized that ghost cell glaucoma (GCG) is due to cellular obstruction of the intertrabecular spaces by the nonpliable GBCs. Histologic results from the present study confirm this belief. No evidence of significant trabecular meshwork degeneration or significant GBC phagocytosis was seen.

Effects of prostaglandins and norepinephrine on ocular pressure and pupil size in rabbits following bilateral cervical ganglionectomy.

We wish to report an early (10 to 30 min) 6 to 7 mm Hg increase in IOP above predrug levels following topical norepinephrine (NE) (5mumol) administration in conscious normal rabbits. This early elevation in IOP was significantly more pronounced in bilateral superior cervical ganglionectomized (BG) rabbits. The time course for peak IOP elevation slightly preceded the accompanying peak mydriatic effect in all groups. IOP returned to baseline levels in about 60 min in the BG rabbits, but the mydriasis persisted. Pretreatment with topical indomethacin significantly reversed the early hypertonic NE effect on IOP in the BG animals implying the involvement of prostaglandin synthesis. The IOP continued to fall significantly after 60 min in the BG group with indomethacin pretreatment. This latter result suggests a predominant long-lasting NE effect. The indomethacin pretreatment had no effect on pupil size of any group, but phenoxybenzamine (PBA) reduced the mydriatic effect, implying a direct or a predominant role of NE on iris muscle.