Cytochemistry of protein kinase C and Na-K-ATPase in rabbit ciliary processes treated with phorbol ester.

Immunocytochemical localization of protein kinase C (PKC) in rabbit ciliary processes was investigated using anti-PKC monoclonal antibodies (MAbs) against rabbit Types 1, 2, and 3 PKC. Specific immunolabeling was observed in nonpigmented epithelial (NPE) cells and in the capillaries of the ciliary processes with anti-Types 2 and 3 MAbs. No apparent staining was seen with anti-Type 1 MAbs. Immunoelectron microscopy of Types 2 and 3 MAbs revealed a diffuse distribution of immunoreactive PKC in the cytoplasm, in the nucleus, and on the plasma membrane in the NPE cells. When incubated with phorbol 12-myristate 13-acetate (PMA), the distribution of PKC was basically similar to that of the untreated group. However, the labelling density on the plasma membrane at basolateral interdigitation increased considerably for anti-Types 2 and 3 PKC MAbs. In addition, the enzyme cytochemical activity of Na-K-ATPase (ouabain-sensitive K-NPPase) and its change after PMA administration in the ciliary processes were observed. An intense reaction was seen on the basolateral plasma membrane of the NPE cells. In the PMA-treated group, the enzyme activity of Na-K-ATPase apparently was decreased. These findings provide evidence that PKC plays a crucial role in the function of the NPE cells of the ciliary processes, possibly in aqueous humor production.     

Marijuana-derived material-induced changes in monkey ciliary processes differ from those in rabbit ciliary processes

The morphologic changes in ciliary processes and the associated intraocular pressure (IOP) were observed in owl and squirrel monkeys after intravitreal (IVT) and intravenous (IV) injections of water soluble marijuana-derived material (MDM). The response in monkeys differed from that reported in rabbits wherein IV injection induced severe ciliary swelling and a significant decrease in IOP. Only moderate swelling occurs in monkey processes after IV injection of relatively high dose of MDM, and this change, which includes disruption of the basal lamina of the pigment epithelium, is not associated with a change in IOP. Severe swelling occurs in the crests of monkey ciliary processes after IVT injection, which is accompanied by a fall in IOP. The difference in the response in monkey versus rabbit ciliary processes after IV injection of MDM may be due to a more compact stroma in the monkey processes.     

Beta-adrenergic receptors in ciliary processes of the rabbit

Identification and characterization of beta-adrenergic receptors were attempted in particulate membrane fractions derived from isolated ciliary processes (CP) of rabbit eyes. High-affinity binding sites for 125I-hydroxybenzylpindolol (125I-HYP), a beta-adrenergic antagonist, were identified in particulate membrane fractions of homogenized CP that were recovered from discontinuous sucrose density gradients. Adenylate cyclase activity was recovered in the same fraction as the 125I-HYP binding sites. The dissociation constant of 125I-HYP for the high affinity site is 0.25 nM, with a minimum capacity of about 35 fmol/mg of protein. Adrenergic agonists and antagonists, including timolol, 1-alprenolol, d,1-propranolol, 1-isoproterenol, 1-epinephrine, and phentolamine, were examined for their ability to displace 125I-HYP from its binding site. The results were consistent with the identification of the high-affinity 125I-HYP binding sites as beta-adrenergic receptors. This is the first report which identifies by ligand binding techniques beta-adrenergic receptors in CP exclusive of iridial or other uveal tissue and supports the possibility of direct action of beta-adrenergic agents on the formation of aqueous humor.     

Calcium-dependent phosphorylation of proteins in rabbit ciliary processes

Calcium-dependent phosphorylation of endogenous substrate proteins in albino rabbit ciliary processes was studied by SDS-polyacrylamide gel electrophoresis and autoradiography. In the soluble fraction, a modest augmentation of phosphorylation was observed by Ca2+ alone and together with the additional activators, calmodulin (CAM) or phorbol myristate acetate (PMA). However, there was a greater enhancement of protein phosphorylation by Ca2+ and activators in the particulate fraction. The degree of Ca2+-CAM-dependent protein phosphorylation was greater than that of Ca2+-PMA-dependent phosphorylation. Endogenous substrate proteins for Ca2+-CAM-dependent protein kinases had apparent molecular sizes of 205,170,150,130,77,58,40,32 and 18 kDa. Phosphorylation of the 58 kDa protein band was strongest. This protein was identified as vimentin on the basis of its behavior with Triton-X100 treatment, and by Western blotting using anti-vimentin antibody. Endogenous substrates of protein kinase C (Ca2+-PMA-dependent) were located at 87 kDa and possibly in the 56 and 54 kDa protein bands. A 50 kDa protein was found to be phosphorylated in the presence of Ca2+ alone, and was not affected by the presence of other activators (CAM or PMA). A Ca2+-dependent dephosphorylation of a 43 kDa protein was observed, and some proteins rapidly phosphorylated by Ca2+-CAM kinase were also relatively quickly dephosphorylated at incubation times greater than 1 min.     

Functional morphology of rabbit iridial ciliary processes

Using scanning electron microscopy of vascular resin casts, it was found that the capillary network of the iridial ciliary processes is supplied by arterioles of its own and drained posteriorly by long isolated venules. These venules pass the ciliary body without greater connection to the venules of the major ciliary processes (shunt-system). Transmission electron microscopy showed that in contrast to the pigment epithelium (PE) of the major ciliary processes, the PE of the iridial processes develops elongated finger-like cytoplasmic processes which protrude into the stroma, often in the direction of the vessels, forming a 'basolateral labyrinth', like the kidney tubule epithelium. After paracentesis, an enlargement and leakage of the iridial ciliary process capillaries was found, resulting in resin leakage at various sites; no leakage was found in the vasculature of the major ciliary processes. Electron microscopically, the basolateral labyrinth of the PE appeared enormously enlarged, forming wide protein-containing intercellular spaces. These findings suggest that the function of the iridial ciliary processes in rabbits has a dual nature. Under normal circumstances, the main function of the iridial processes may be fluid reabsorption rather than aqueous humor secretion. In cases of paracentesis, for example, the very sensitive blood aqueous barrier of this area breaks down with resulting protein exudation.     

Cyclic nucleotide-dependent phosphorylation of proteins in rabbit ciliary processes

Cyclic nucleotide-dependent protein phosphorylation in albino rabbit ciliary processes was studied in particulate and soluble fractions of the tissue by the technique of SDS-polyacrylamide gel electrophoresis and autoradiography. In the presence of gamma-32P-ATP, the soluble fraction showed increased phosphorylation of proteins of 200, 32 and 16 kDa molecular weight when 10 microM cAMP was added. Protein phosphorylation increased with time up to 5 min. No significant augmentation of phosphorylation was observed in the presence of 10 microM cGMP compared to control. In the particulate fraction, proteins with molecular weights of 200, 160, 105, 72, 58, 32 and 16 kDa showed increased phosphorylation in the presence of 10 microM cAMP. Phosphorylation caused by the addition of cAMP was maximal between 30 sec and 1 min for the particulate membrane fraction, but with longer incubation times the incorporation of phosphate residues decreased. The same molecular weight proteins of the membrane fraction that were phosphorylated in a cAMP-dependent manner were phosphorylated in the absence of exogenous cAMP by addition of either the catalytic subunit of cAMP-dependent protein kinase or activators of membrane-bound adenylate cyclase such as l-isoproterenol, vasoactive intestinal peptide, aluminum fluoride or forskolin. A cAMP-dependent dephosphorylation of a 56 kDa protein was observed in the membrane fraction. Cyclic GMP did not cause observable changes in the pattern of protein phosphorylation in the particulate fraction of rabbit ciliary processes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Circadian change of adenylate cyclase activity in rabbit ciliary processes

PURPOSE: To characterize in vivo changes of adenylate cyclase activity in rabbit ciliary processes during the circadian cycle. METHODS: After removal of vitreous, lens, retina and choroid from freshly enucleated rabbit eyes, the anterior segment and attached sclera was chilled in cold buffer containing the non-selective phosphodiesterase (PDE) inhibitor, 3-isobutyl-1-methylxanthine (IBMX). Then ciliary processes were excised and homogenized in ice cold trichloro-acetic acid (TCA) 2.5 min after IBMX treatment. Increased cyclic AMP in response to PDE inhibition was measured in ciliary processes at six times during the circadian cycle, after preganglionic section of the cervical sympathetic trunk (decentralization, DX), and after topical instillation of hydroxyamphetamine, timolol, brimonidine, rauwolscine or a soluble derivative of forskolin. RESULTS: The increase of rabbit ciliary process cyclic AMP levels in response to PDE inhibition with IBMX was enhanced at night. Much of the enhanced nocturnal increase persisted in constant dark and was blunted by DX. Topical instillation of hydroxyamphetamine enhanced the increase during the day; whereas, timolol, a beta-adrenergic antagonist, or brimonidine, an alpha2-adrenergic agonist, blunted the IBMX induced increase. Neither instillation of rauwolscine, an alpha2-adrenergic antagonist, nor the soluble forskolin derivative enhanced the increase of cyclic AMP after IBMX. CONCLUSIONS: The technique reported here can be used to estimate ciliary process adenylate cyclase activity in vivo. There is a circadian rhythm of adenylate cyclase activity in rabbit ciliary processes that is driven in part by ocular sympathetic input and stimulation of beta-adrenergic receptors.     

Steroid potentiation of beta-adrenergic-sensitive adenylate cyclase in rabbit ciliary processes

Dexamethasone (3.3 X 10(-8) M) potentiated 1-isoproterenol-stimulated adenylated cyclase activity in vitro in rabbit ciliary process epithelial membrane preparations. The ka for isoproterenol stimulation of the cyclase was 5 X 10(-7) M in control membranes, 2.5 X 10(-9) M in membranes preincubated with dexamethasone, and 6 X 10(-8) M with corticosterone. No change was observed in basal adenylate cyclase activity. Changes in enzyme activity at maximally stimulating levels of isoproterenol were not consistently different. Dose response to corticosterone in the presence of 10(-7) M 1-isoproterenol shows maximum effect at 6.6 X 10(-8) M corticosterone.     

Alpha 2-adrenergic and VIP receptors in rabbit ciliary processes interact

The interaction between alpha 2-adrenergic and VIP receptors has been studied by examining inhibition of VIP-stimulated cyclic AMP production by adrenergic agonists in intact, excised rabbit ciliary processes. Epinephrine, norepinephrine, isoproterenol, dopamine, and the specific alpha 2-adrenergic agonists clonidine and p-aminoclonidine exhibit dose-dependent inhibition of VIP-stimulated cyclic AMP production. I50s, clonidine (0.05 microM) = p-aminoclonidine (0.05 microM) congruent to epinephrine (0.1 microM) less than norepinephrine (2.0 microM) less than isoproterenol (15 microM) = dopamine (15 microM), are consistent with the characteristic binding affinities of these adrenergic agonists for alpha 2-adrenergic receptors. Inhibition of VIP-stimulated cyclic AMP production by clonidine, epinephrine, isoproterenol, and dopamine is blocked by yohimbine but not by prazosin. These data establish the alpha 2-adrenergic specificity of the inhibitory effects observed. We have previously shown that beta 2-adrenergic receptor-mediated stimulation of cyclic AMP production in rabbit ciliary processes is also inhibited by postjunctional alpha 2-adrenergic receptors. These studies support the idea that the catecholamines may regulate aqueous humor formation by inhibiting stimulation of cyclic AMP production via postjunctional alpha 2-adrenergic receptors in ciliary processes.     

Effects of Rho-associated protein kinase inhibitors Y-27632 and Y-39983 on isolated rabbit ciliary arteries

Purpose

In normotensive eyes, reduced ocular blood flow can lead to glaucoma pathogenesis. Drugs that reduce intraocular pressure (IOP) often cause vasodilation of the ciliary arteries and improve blood flow to the eye. A novel class of drugs called Rho-associated coiled coil-forming protein kinase (ROCK) inhibitors can lower IOP. Therefore, we tested the ability of two ROCK inhibitors, Y-27632 and Y39983, to relax rabbit ciliary arteries.

Methods

We measured in vitro ciliary artery smooth muscle contractions by isometric tension recordings and changes of intracellular free calcium concentration ([Ca²⁺]_i) by fluorescence photometry.

Results

Both Y-27632 and Y-39983 induced a concentration-dependent relaxation in rabbit ciliary arteries precontracted with a high-potassium (high-K) solution. The amplitude of relaxation induced by Y-27632 and Y-39983 was not affected by either 100 ??M N ^G-nitro-l-arginine methyl ester (l-NAME) or 10 ??M indomethacin. In Ca²⁺-free solution, Y-27632 and Y-39983 significantly inhibited the transient contraction of ciliary arteries induced by 10 ??M histamine. However, neither Y-27632 nor Y-39983 affected the elevation of [Ca²⁺]_i induced by high-K solution and histamine.

Conclusions

We concluded that Y-27632 and Y-39983 relaxed isolated rabbit ciliary artery segments in vitro. The mechanism of relaxation was not dependent on endothelial-derived factors such as nitric oxide (NO) or prostacyclin, nor was it dependent on changes in intracellular Ca²⁺ concentration.     

Microendoscopic observations of the hemodynamics in the rabbit ciliary processes.

The in vivo hemodynamics of the ciliary process vasculature in the albino rabbit were studied with an improved technique of intraocular microendoscopy. The spreading pattern and velocity of the dye front has been observed in defined vessel segments following intracarotid injection of Evans Blue. In the anterior portion of the ciliary processes a "thoroughfare channel" (bypass) was found leading from the arteriolar tree directly into the marginal venule bypassing the capillary network of the ciliary processes. This bypass is characterized by a higher blood flow velocity (1.5-3mm/sec) than was found for the capillary network (0.8-1mm/sec). A stoppage of blood flow was observed in the capillary network at 45-50mmHg after step-wise elevation of intraocular pressure (IOP): blood flow was not stopped in the thoroughfare channel until IOP values of 55-60mmHg were reached. Intraarterial administration of vasoconstrictive agents could lead to a complete stoppage of blood flow in the capillary net whereas the marginal route often remained patent.     

Carbonic anhydrase in the developing ciliary processes of the rabbit eye

Carbonic anhydrase activity in the developing ciliary processes of rabbit eyes has been investigated by a histochemical method.     

Ontogeny and localization of the IgG Fc receptor of rabbit ciliary processes

We have previously demonstrated the presence of IgG Fc binding sites in adult rabbit ciliary processes. The present study of 22 perinatal rabbit eyes defines the age-related expression of the IgG receptor activity and the selective localization of this activity on the posterior portions of the ciliary processes where it appears to be associated with the superficial non-pigmented epithelium. Our findings indicate that the rabbit ciliary processes IgG binding activity is only slightly expressed at birth but develops rapidly after birth, reaching adult levels within 2 weeks. Moreover, our results support the concept of important regional specialization within the ciliary body and suggest that the posterior ciliary processes, like other tissues similarly endowed with IgG binding sites, may have specialized immune functions.     

Neuropeptide Y and somatostatin inhibit stimulated cyclic AMP production in rabbit ciliary processes

The interaction of adrenergic and peptide receptors linked to adenylate cyclase and the inhibition by bioactive peptides of stimulated cyclic AMP production has been investigated in intact, excised rabbit ciliary processes. Cyclic AMP production stimulated by isoproterenol, vasoactive intestinal peptide, or forskolin was inhibited by the biologically active peptides neuropeptide Y, somatostatin, and the synthetic somatostatin analogue SMS 201-995. IC50s determined from dose-response curves of inhibition are consistent with the known abilities of these ligands to modulate cyclic AMP and physiological responses in other tissues. Inhibition by neuropeptide Y or SMS 201-995 was unaffected by the specific alpha 2-adrenergic antagonist yohimbine, which shows that peptide inhibition is not occurring via peptide binding to the inhibitory alpha 2-adrenergic receptor. These results suggest that endogenous peptides may participate in modulation of cyclic AMP production and subsequent physiological events influenced by cyclic AMP levels in rabbit ciliary processes by inhibiting stimulated cyclic AMP synthesis.     

Inhibitory effects of neuropeptide Y on adenylate cyclase of rabbit ciliary processes

The inhibitory effect of neuropeptide Y (NPY) was studied on the adenylate cyclase (AC) activity in homogenates of rabbit ciliary processes and compared with that of the alpha 2-adrenergic agonist clonidine (CLN). NPY inhibited basal AC activity as well as AC activity stimulated by isoproterenol (ISO), vasoactive intestinal polypeptide (VIP) or forskolin (FSK). The extent of this inhibition corresponded well to the inhibition elicited by CLN. The inhibitory effects of NPY and CLN appeared to be nonadditive. AC activity stimulated by ISO was considerably more sensitive to the effects of either NPY or CLN than basal, VIP- or FSK-stimulated AC activity. It was inferred that NPY inhibitory effects were mediated by the activation of NPY receptors coupled negatively to the catalytic unit of AC via the inhibitory Gi protein. Moreover, involvement of NPY in physiological modulation of AC activity in ciliary processes and in the regulation of aqueous humor formation and intraocular pressure is suggested.     

Stimulatory and inhibitory cyclic AMP responses in rabbit ciliary processes after cervical ganglionectomy.

Cyclic AMP production in response to agonists which act at a variety of receptors to either stimulate or inhibit cyclic AMP production has been studied in intact, dissected ciliary processes from rabbit eyes after unilateral surgical removal of the cervical ganglion. Cyclic AMP responses to stimulatory ligands vasoactive intestinal peptide (VIP), isoproterenol, and forskolin and inhibitory agonists neuropeptide Y (NPY), the synthetic somatostatin analogue SMS 201-995, and alpha-adrenergic agents were investigated in tissues from normal eyes and compared to the same responses in tissues from sympathetically denervated eyes. Neither stimulated cyclic AMP production nor inhibition of stimulated cyclic AMP production was significantly different in tissues from denervated vs. normal eyes. Inhibition of VIP-stimulated cyclic AMP production by epinephrine and paraaminoclonidine in tissues from both normal and denervated eyes was blocked by the alpha 2-adrenergic antagonist yohimbine but not by the alpha 1-adrenergic antagonist prazosin. These data indicate that the VIP, NPY, somatostatin, and alpha 2- and beta 2-adrenergic receptors which regulate cyclic AMP production in rabbit ciliary processes are postjunctional and suggest that ligands known to modulate cyclic AMP levels in this tissue may exert effects on aqueous humor formation independently of adrenergic innervation.     

Interaction between alpha 2- and beta 2-adrenergic receptors in rabbit ciliary processes

The interaction between the alpha 2- and beta 2-adrenergic receptors of ciliary processes has been studied by examining dose-response curves for adrenergic agonist stimulation of cyclic AMP production by intact, excised rabbit ciliary processes. Stimulation of cyclic AMP production by 1-isoproterenol is maximum from 0.1 to 1.0 microM; at higher concentrations stimulation decreases and approaches basal levels. Decreased cyclic AMP production at high concentrations of isoproterenol is blocked by the specific alpha 2-adrenergic antagonist, yohimbine, but not by the alpha 1-adrenergic antagonist, prazosin. Ciliary processes from animals after bilateral cervical ganglionectomy also show reduced cyclic AMP production at high concentrations of isoproterenol and this reduction is blocked by yohimbine, but not prazosin. This experiment suggests that the inhibition at high concentrations of isoproterenol is mediated by postsynaptic alpha 2-adrenergic receptors. Cyclic AMP production is relatively insensitive to epinephrine and norepinephrine, but their responses are potentiated by yohimbine. Catecholamines and clonidine, a specific alpha 2-adrenergic agonist, exhibit dose-dependent inhibition of forskolin-stimulated cyclic AMP production by ciliary processes. I50s from the dose-response curves are consistent with the characteristic binding affinities of these adrenergic agonists for alpha 2-adrenergic receptors: clonidine = epinephrine greater than norepinephrine greater than isoproterenol. Inhibition of forskolin-stimulated cyclic AMP production by clonidine is blocked by yohimbine but not by prazosin.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effects of adrenergic agonists and antagonists on the adenylate cyclase in albino rabbit ciliary processes

An adenylate cyclase from rabbit ciliary processes was shown to be activated by norepinephrine, epinephrine, isoproterenol, salbutamol, guanylylimidodiphosphate and sodium fluoride. Activation of the adenylate cyclase by isoproterenol was inhibited by propranolol, timolol, metripranolol and practolol, while phentolamine was without clear effect on the activation of the adenylate cyclase by epinephrine. From the comparison of the effects of used agents, it has been concluded that ciliary processes of rabbit may contain stimulatory beta₂-adrenoceptors coupled to the adenylate cyclase. The presence of α-adrenoceptors inhibiting the adenylate cyclase was questioned. Importance of the β-adrenoceptors for ocular hypotensive effects of the adrenergic agonists and antagonists was briefly discussed.     

Evidence for catecholamine-stimulated adenylate cyclase activity in frog and rabbit corneal epithelium and cyclic AMP-dependent protein kinase and its protein substrates in frog corneal epithelium

Evidence was obtained for catecholamine-stimulated adenylate cyclase activity in particulate fractions of frog and rabbit corneal epithelium. Epinephrine (10(-5)M) stimulated adenylate cyclase by 22 and 53% in the frog and rabbit, respectively. The corresponding changes were statistically significant (P less than 0.01) when the data was analyzed using paired variates. Preincubation with 10(-4)M propranolol eliminated any stimulatory effect by 10(-5)M isoproterenol. Adenylate cyclase activity derived from either source was activated several fold by either 10 mM NaF or 10(-5)MGpp (NH)p. Soluble fractions of homogenized frog corneal epithelium contained cyclic AMP-dependent protein kinase activity which was half-maximally stimulated by about 6 nM cyclic AMP. Evidence was also obtained for the presence of protein substrates of cyclic AMP dependent protein kinase in frog corneal epithelium. With exogenous cyclic AMP and protein kinase, a rapid 32P labelling of proteins having approximate molecular weights of 56, 46, 23 and 21 K was obtained with sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. A less marked and slower increase in phosphoprotein formation was observed when corneal membranes were incubated with cyclic AMP in the absence of added protein kinase.