Characterization of muscarinic receptors in cultured human iris sphincter and ciliary smooth muscle cells.

Muscarinic receptors present in cultured human iris sphincter and ciliary smooth muscle cells were characterized by both ligand ([3H]QNB binding) and functional (phosphoinositide hydrolysis) studies. Ligand binding studies showed that [3H]QNB represented a single population of binding sites with KD values of 4.02 x 10(-11) M in the ciliary and 5.6 x 10(-11) M in the iris sphincter cells. In competition studies, the selective antagonist, 4-diphenylacetoxy-N-methylpiperidine-methobromide (4-DAMP) was the most potent in displacing [3H]QNB with selectivity of 150-350-fold over pirenzepine (M1) and 450-1700-fold over AF-DX 116 (M2). 4-DAMP recognized one site in the iris sphincter cells (Ki = 0.34 nM) but two sites in the ciliary cells (KH = 0.9 nM and KL = 49 nM). 4-DAMP was also the most potent in inhibiting carbachol-induced hydrolysis of inositol phospholipids (PI) in both cell types. However, the IC50 values for PI hydrolysis were several fold lower than those for [3H]QNB binding. Using these selective antagonists, our data supports the presence of functional muscarinic receptors of M3 subtype in human iris sphincter and ciliary cells. It also shows the presence of a second low affinity site in the ciliary smooth muscle cells that is recognized by 4-DAMP.

Synergistic effects of LTB4 and LTD4 on leukocyte emigration into the guinea pig conjunctiva.

Leukotrienes (LT) B4 and D4, alone and in combination, were topically applied to the eyes of guinea pigs, and their effects on conjunctival leukocyte infiltration studied. LTD4 potentiated the neutrophil response to LTB4, even though no neutrophil emigration was evoked by LTD4 itself over a dose range of 10-1000 ng. LTB4 alone at the 1-ng and 10-ng doses failed to evoke any leukocyte emigration, but significant numbers of neutrophils were observed at these concentrations when LTD4 (1-1000 ng) was present. Although a dose-dependent increase in neutrophil infiltration was observed for the 100-ng and 1000-ng doses of LTB4, cell counts were substantially higher with these doses in the presence of LTD4. Eosinophil numbers increased in a dose-related manner in response to LTB4 and LTD4 alone, with a greater response to LTD4. The addition of either 10 ng or 100 ng of LTB4 to graded doses of LTD4 (10-1000 ng) caused increased eosinophil numbers, the lower dose of LTB4 potentiating the response to LTD4 and the higher LTB4 dose showing no significant effect. The effects on leukocyte infiltration that were evoked by the LT combinations could not be explained simply on the basis of an increase in vascular permeability. Bradykinin (BK), a potent conjunctival microvascular permeability factor that does not elicit any leukocyte infiltration, did not significantly potentiate LTB4-induced eosinophil or neutrophil emigration. The synergistic effects of LTs on leukocyte emigration are also difficult to ascribe to hyperemia (ie, increased blood volume in the conjunctiva), because both LTB4 and LTD4 caused only very modest increases in conjunctival blood content, and BK, which did not potentiate the leukocytic responses to LTB4, caused marked increases in tissue blood content. High-dose LT combinations caused eosinophils, but not neutrophils, to migrate into the conjunctival epithelium and fragment, resulting in overt tissue damage. These results further suggest a synergistic interaction between LTB4 and LTD4 that directly alters leukocyte function. The relevance of these observations to a number of disease and trauma states is discussed.

Topical timolol, at conventional, unilateral doses causes bilateral ocular beta-blockade in rabbits.

The studies described herein reveal that unilateral 0.3% and 1% doses of timolol produce marked beta-adrenoceptor blockade in treated and in contralateral eyes which did not receive timolol. This indicates that unilateral, conventional topical doses of timolol administered to rabbits cause bilateral ocular beta-adrenoceptor blockade. In addition, 0.1% and 1% doses of timolol applied to one eye resulted in blockade of cardiovascular beta-adrenoceptors. Thus, the most likely explanation for bilateral ocular beta-blockade would be systemic absorption of timolol from the treated eye and redistribution to the fellow eye. Since contralateral eyes would not provide an adequate control in circumstances where a large dose of timolol is administered unilaterally, caution must be exercised in interpreting data obtained in laboratory animals with doses of timolol similar to those employed clinically. A 0.01% dose of timolol appears adequate to achieve marked, unilateral ocular beta-adrenoceptor blockade in rabbits.