A3 adenosine receptors regulate Cl- channels of nonpigmented ciliary epithelial cells.

Adenosine stimulates Cl- channels of the nonpigmented (NPE) cells of the ciliary epithelium. We sought to identify the specific adenosine receptors mediating this action. Cl- channel activity in immortalized human (HCE) NPE cells was determined by monitoring cell volume in isotonic suspensions with the cationic ionophore gramicidin present. The A3-selective agonist N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (IB-MECA) triggered shrinkage (apparent Kd = 55 +/- 10 nM). A3-selective antagonists blocked IB-MECA-triggered shrinkage, and A3-antagonists (MRS-1097, MRS-1191, and MRS-1523) also abolished shrinkage produced by 10 microM adenosine when all four known receptor subtypes are occupied. The A1-selective agonist N6-cyclopentyladenosine exerted a small effect at 100 nM but not at higher or lower concentrations. The A2A agonist CGS-21680 triggered shrinkage only at high concentration (3 microM), an effect blocked by MRS-1191. IB-MECA increased intracellular Ca2+ in HCE cells and also stimulated short-circuit current across rabbit ciliary epithelium. A3 message was detected in both HCE cells and rabbit ciliary processes using RT-PCR. We conclude that human HCE cells and rabbit ciliary processes possess A3 receptors and that adenosine can activate Cl- channels in NPE cells by stimulating these A3 receptors.

A release mechanism for stored ATP in ocular ciliary epithelial cells.

Purines can modify ciliary epithelial secretion of aqueous humor into the eye. The source of the purinergic agonists acting in the ciliary epithelium, as in many epithelial tissues, is unknown. We found that the fluorescent ATP marker quinacrine stained rabbit and bovine ciliary epithelia but not the nerve fibers in the ciliary bodies. Cultured bovine pigmented and nonpigmented ciliary epithelial cells also stained intensely when incubated with quinacrine. Hypotonic stimulation of cultured epithelial cells increased the extracellular ATP concentration by 3-fold; this measurement underestimates actual release as the cells also displayed ecto-ATPase activity. The hypotonically triggered increase in ATP was inhibited by the Cl--channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) in both cell types. In contrast, the P-glycoprotein inhibitors tamoxifen and verapamil and the cystic fibrosis transmembrane conductance regulator (CFTR) blockers glybenclamide and diphenylamine-2-carboxylate did not affect ATP release from either cell type. This pharmacological profile suggests that ATP release is not restricted to P-glycoprotein or the cystic fibrosis transmembrane conductance regulator, but can proceed through a route sensitive to NPPB. ATP release also was triggered by ionomycin through a different NPPB-insensitive mechanism, inhibitable by the calcium/calmodulin-activated kinase II inhibitor KN-62. Thus, both layers of the ciliary epithelium store and release ATP, and purines likely modulate aqueous humor flow by paracrine and/or autocrine mechanisms within the two cell layers of this epithelium.

Galanin immunoreactivity in autonomic innervation of the cat eye.

In an immunohistochemical study, we find that galanin is much more widely distributed in the peripheral innervation of the cat eye than in other animals so far examined. Previous studies of rat and pig eyes have revealed sparse galanin-positive nerves that presumably originate in the trigeminal ganglion. In contrast, the cat has a rich supply of galanin-containing nerve fibers throughout the uvea. Galanin-positive varicose nerves concentrate densely in iris muscles and distribute more sparsely in the ciliary muscle. The ciliary processes have a plexus of galanin-positive nerves underlying the ciliary epithelium at their base and positive nerve fibers coursing within their stroma. The ciliary artery and its branch vessels in the uvea are invested with a dense plexus of galanin-positive nerves. All autonomic ganglia supplying the eye contain cells that express galanin. It is present in 97% of superior cervical ganglion cells, coexisting with both tyrosine hydroxylase and neuropeptide Y; in 80% of pterygopalatine ganglion cells, most of which also contain vasoactive intestinal peptide; and in approximately 25% of ciliary ganglion cells. After unilateral superior cervical ganglionectomy, galanin-positive nerves almost totally disappear from the iris muscles, demonstrating that they are predominantly of sympathetic origin. Galanin-positive nerves investing the ciliary artery and choroidal blood vessels are not detectably reduced by sympathectomy, indicating that perivascular parasympathetic nerves from the pterygopalatine ganglion also express galanin. Other galanin-containing nerves in the eye can originate from the trigeminal and ciliary ganglia. The prominence of galanin in the ocular autonomic innervation of the cat provides an opportunity to explore the physiological effects of this neuropeptide in the eye.

Brain natriuretic peptide-immunoreactive nerves in the porcine eye.

Using immunohistochemistry, brain natriuretic peptide (BNP)-like immunoreactive (-LI) nerves localize to the anterior segment of the porcine eye. BNP-LI nerve fibers are visualized mainly in the aqueous outflow pathway, ciliary processes and anterior ciliary muscle. A small number of immunoreactive nerve fibers occur in the anterior iris stroma and in relation to the iris muscles and in the cornea. Given the known ocular effects of atrial natriuretic peptide (ANP) and that BNP and ANP seem to share common receptors, these results suggest an ocular role for peptides of this class independent of delivery by the systemic circulation.

An immunohistochemically distinct population of cat ciliary ganglion cells.

In the cat ciliary ganglion, 16% and 23% respectively of the neurons are surrounded by nerve fibers immunoreactive to calcitonin gene-related peptide (CGRP) and somatostatin (SOM). One-third of the ganglion cell perikarya are immunoreactive to neuropeptide Y (NPY). Analysis of the coincidence of immunoreactivities shows a striking correlation. Practically all of the ganglion cells surrounded by CGRP-like immunoreactive (LI) nerve fibers also are surrounded by SOM-LI nerve processes and demonstrate NPY-LI perikarya. These observations define a subset of NPY-LI ciliary ganglion cells with a particular peptidergic input and perhaps a specific physiologic function.

Peptide immunoreactivity of the ciliary ganglion and its accessory cells in the rat.

By means of immunohistochemistry, calcitonin gene-related peptide, Leu-enkephalin and neuropeptide Y localize to rat ciliary and accessory ganglion cells. The proportion of ciliary and accessory neurons immunoreactive to each peptide is provided and compared to previous data for vasoactive intestinal peptide. These findings indicate considerable neurochemical complexity for a parasympathetic ganglion with a small cell population.

Calcitonin gene-related peptide immunoreactive nerves in human and rhesus monkey eyes.

Using immunohistochemical methods, calcitonin gene-related peptide localizes to peripheral nerve fibers of the human and rhesus monkey eye. Immunoreactive nerve fibers are found in the cornea, about limbal blood vessels and in the trabecular meshwork. Many immunoreactive iris nerve fibers are present, mostly within the stroma. A particularly dense network occurs just anterior to the iris sphincter muscle, but only a small number of immunoreactive nerve fibers are visualized within it. The ciliary muscle and ciliary processes also are innervated. Immunoreactive nerve fibers are associated with uveal blood vessels, most prominently in the choroid and ciliary body. Apposition of immunoreactive nerve fibers to uveal melanocytes is seen. In lower mammalian species, calcitonin gene-related peptide co-localizes with substance P in many ocular nerve fibers. The comparative distribution in human and monkey eyes of nerve fibers immunoreactive to these two peptides is discussed.

Increase of basal cell membrane area of the retinal pigment epithelium in experimental diabetes.

Stereological analysis of electron micrographs of the pigment epithelium of rats with drug-induced diabetes demonstrated an increase of plasma membrane surface area at the basal aspect of the cells. In none of the diabetic animals examined was there any evidence of breakdown of the blood-retinal barrier to the protein tracer, horseradish peroxidase. Statistically significant increases in basal plasma membrane length and surface density (surface area per unit cell volume) were measured in both streptozotocin and alloxan-injected rats after four weeks of diabetes. When hyperglycemia in streptozotocin-injected rats was promptly reversed by transplantation of normal pancreatic islets, the increase of membrane surface area did not occur. We conclude, therefore, that increased basal surface area of pigment epithelial cells is related to the diabetic condition rather than to a toxic action of the diabetogenic agents. Furthermore, increased membrane surface area was present in streptozotocin-diabetic rats killed after six months of diabetes indicating that the structural change is relatively stable. Relation of basal membrane alteration in the pigment epithelium to any functional disturbance of the barrier cell layer or of the retina in diabetes remains to be established.

Hypertensive breakdown of cerebral but not of retinal blood vessels in rhesus monkey.

Acute hypertension, induced either by intravenous injection of metaraminol bitartrate (Aramine), infusion of isotonic saline into the common carotid artery, or a combination of both procedures did not in the rhesus monkey lead to breakdown of the blood-retinal barrier. Whereas the cerebral vasculature was made permeable to blood-borne dye at carotid pressure above 160 mm Hg, the retinal blood vessels were intact even at pressures as high as 310 mm Hg. Hypertensive blood-brain barrier opening was associated with neurologic defects and brain edema. The results indicate that the retina is more resistant to acute hypertension than is the brain. The greater resistance in the retina may be due to the high number of contractile, perivascular mural cells counteracting increased intravascular hydrostatic pressure. An alternative or supplementary explanation is that choroidal and retinal blood vessels are better protected from surges in blood pressure than are brain blood vessels. Differences between the innervation of brain and ocular blood vessels could account for this.