Cell signaling in bovine ciliary epithelial organ culture.

The ciliary epithelium secretes aqueous humor, an intraocular fluid whose production is regulated in part by transmembrane signaling pathways including those mediated by G protein-coupled receptors. Many drugs, such as beta-adrenergic receptor (AR) antagonists and alpha2-AR agonists, are used to lower intraocular pressure by presumably decreasing fluid transport across this epithelium. Hence, our purpose was to establish a ciliary epithelial organ culture system suitable for the study of cell signaling pathways. A trypsin-mediated dissection method was established to isolate bovine ciliary epithelial sheets. These sheets were cultured in a 5% CO2 incubator. The quality was assessed by light microscopy, by protein analysis, and by the evaluation of epinephrine-mediated phosphoinositide turnover. The cultured epithelial explants were viable as evidenced by minimal trypan blue staining. The explants were composed primarily of nonpigmented cells and some pigmented cells, but no other ciliary body tissues were present on histology. Membrane preparations showed proteins with a distribution from 31 to 116 kDa. Epinephrine caused a dose-dependent increase in [3H]inositol phosphates (InsPs) accumulation with a maximal increase of two- to three-fold over basal levels. This epinephrine-mediated increase was inhibited by prazosin. We established an organ culture system of isolated bovine ciliary epithelium suitable for the study of transmembrane signaling pathways.

Soluble guanylate cyclase and nitric oxide synthase in synaptosomal fractions of bovine retina.

Cyclic GMP has been shown in recent years to directly activate ion channels in bipolar and ganglion cells, and to indirectly regulate coupling between horizontal cells, and between bipolar and amacrine cells. In all of these cases, the effects of cyclic GMP are mimicked by nitric oxide. An increase in calcium concentration stimulates the production of nitric oxide by neuronal and endothelial forms of nitric oxide synthase, which in turn activates soluble guanylate cyclases, enhancing the synthesis of cyclic GMP. Though some effects of nitric oxide do not involve cyclic GMP, the nitric oxide-cyclic GMP cascade is well recognized as a signaling mechanism in brain and other tissues. The widespread occurrence of nitric oxide/cyclic GMP-regulated ion channel activity in retinal neurons raises the possibility that nitric-oxide-sensitive soluble guanylate cyclases play an important role in cell-cell communication, and possibly, synaptic transmission. Immunohistochemical studies have indicated the presence of soluble guanylate cyclase in retinal synaptic layers, but such studies are not suitable for determination of the density or quantitative subcellular distribution of the enzyme. Microanalytical methods involving microdissection of frozen retina also showed the presence of cyclase activity in retinal plexiform layers but these methods did not permit distinction between nitric oxide-sensitive and insensitive cyclases. In this study, we fractionated retinal homogenate into the cytosolic and synaptosomal fractions and investigated the specific activity and distribution of soluble guanylate cyclase and nitric oxide synthase. The results show that both enzymes are present in the synaptosomal fractions derived from inner and outer plexiform layers. The synaptosomal fraction derived from inner retina was highly enriched in cyclase activity. Nitric oxide synthase activity was also higher in the inner than outer retinal synaptosomal fraction. The results suggest that the nitric oxide-cyclic GMP system is operational in both synaptic layers of retina and that it may play a more significant role in the inner retina.

Identification of effector binding sites on S100 beta: studies with guanylate cyclase and p80, a retinal phosphoprotein.

S100 beta is a calcium-binding protein, which regulates the activities of several enzymes and inhibits the phosphorylation of a variety of protein kinase C substrates in a calcium-dependent manner. The protein was recently found to activate a retinal membrane guanylate cyclase, and in this paper, we report that it inhibits the phosphorylation of an 80 kDa retinal protein (p80). Structurally, S100 beta consists of two EF-hands connected by a hinge region. In view of its small size, wide distribution in a variety of tissues, and regulation of many different proteins, it is of interest to identify the sites on the protein that interact with the effectors, and to determine if the same sites are responsible for interaction with different effectors. We addressed these questions with the use of synthetic peptides with sequences corresponding to different regions of S100 beta and testing their effects on the protein's activation of guanylate cyclase, and inhibition of p80 phosphorylation. Peptides with sequences corresponding to effector interaction sites were anticipated to either block or simulate the effects of S100 beta. The results show that two regions of S100 beta interact with effectors: the C-terminal region of Thr81-Glu91 and the hinge region of Leu32-Leu40. The synthetic peptide containing the latter sequence blocked the S100 beta activation of guanylate cyclase and inhibition of p80 phosphorylation, while the peptide containing the former sequence blocked cyclase activation and simulated S100 beta in inhibiting p80 phosphorylation. By determining the effects of including or excluding dithiothreitol in the assays, we observed that the cysteine residue in the C-terminal region of S100 beta (Cys84) participates in the regulation of guanylate cyclase but not of p80 phosphorylation. We conclude from these results that the C-terminal and hinge regions of S100 beta are important in the regulation of effector proteins and that Cys84 is essential for interaction with only specific effectors.

Differential activation of rod outer segment membrane guanylate cyclases, ROS-GC1 and ROS-GC2, by CD-GCAP and identification of the signaling domain.

The ROS-GC is one of the two subfamilies of membrane guanylate cyclases. It distinguishes itself from the other surface receptor subfamily in that its members are not regulated by extracellular peptides; instead, they are modulated by intracellular Ca2+ signals. There are two members of the subfamily, ROS-GC1 and ROS-GC2. An intriguing feature of ROS-GC1 is that it has two Ca2+ switches. One switch inhibits the enzyme at micromolar concentrations of Ca2+, and the other stimulates. The inhibitory switch is linked to phototransduction, and it is likely that the stimulatory switch is linked to retinal synaptic activity. Ca2+ acts indirectly via Ca(2+)-binding proteins, GCAPs and CD-GCAP. GCAPs modulate the inhibitory switching component of the cyclase and CD-GCAP turns on the activation signaling switch. The activating switch of ROS-GC2 has not so far been scrutinized. The present study shows that CD-GCAP is linked to the activation signaling switch of ROS-GC2, but the linkage is about 10-fold weaker than that of the ROS-GC1. Thus, CD-GCAP is a specific ROS-GC1 activator. Furthermore, through a series of expression studies on the mutants involving deletion, building of hybrids, and reconstruction of a heterologous cyclase, the study confirms that the CD-GCAP regulated switch resides within the amino acid segment 736-1053 of the cyclase.

Structural and functional characterization of retinal calcium-dependent guanylate cyclase activator protein (CD-GCAP): identity with S100beta protein.

Calcium-dependent guanylate cyclase activator protein (CD-GCAP) is a low-molecular-weight retinal calcium-binding protein which activates rod outer segment guanylate cyclase (ROS-GC) in a calcium-dependent manner. This investigation was undertaken to determine the protein's structure and identity. Partial amino acid sequencing (72% of the protein), mass spectral analysis, cloning, and immunological studies revealed that CD-GCAP is identical to S100beta, another low-molecular-weight calcium-binding protein whose structure was known. We had shown earlier that the latter protein, which is usually called S100b (S100betabeta or dimer of S100beta), also activates ROS-GC but that the Vmax of activated cyclase was about 50% lower than when stimulated by CD-GCAP. S100b also required about 15 times more calcium (3.2 x 10(-)5 vs 1.5 x 10(-)6 M) for half-maximal stimulation of cyclase. To investigate the possibility that CD-GCAP is a post-translationally modified form of S100b, both proteins were treated with 1 M hydroxylamine which is known to deacylate proteins. After the treatment, CD-GCAP did not activate cyclase while S100b activation remained unaffected suggesting that CD-GCAP could not be a modified form of S100b. Hydroxylamine also broke down CD-GCAP into smaller fragments while leaving S100b intact. It therefore appeared that in spite of identical primary structures, the conformations of the two proteins were different. We then investigated the possibility that the purification procedures of the two proteins, which were quite different, could have contributed to such conformational differences: CD-GCAP purification included a step of heating at 75 degrees C in 5 mM Ca, while S100b purification included zinc affinity chromatography. To test the influence of these treatments on the properties of the proteins, CD-GCAP was subjected to zinc affinity chromatography and purified as S100b (CD-GCAP-->S100b) and S100b was heated in Ca and purified as CD-GCAP (S100b-->CD-GCAP). Cyclase activation, calcium-sensitivity, and hydroxylamine-lability measurements revealed that CD-GCAP-->S100b is identical to S100b and that S100b-->CD-GCAP is identical to CD-GCAP. Taken together the results demonstrate that CD-GCAP and S100b are one and the same protein and that their functional differences are due to different interconvertible conformational states.

Endogenous ADP-ribosylation of a G(alpha i) protein in bovine ciliary body is stimulated by nitric oxide.

Five ciliary body membrane proteins were labeled when incubated with (adenylate-32P)NAD. Nitric oxide donors stimulated the labeling of 64, 40, and 29-30 kDa proteins and inhibited that of 58 and 56 kDa proteins. The greatest influence of nitric oxide was on the 40 kDa protein: a 17-fold stimulation. Western blotting and immunoprecipitation with specific antibodies identified this protein as the alpha-subunit of G(i-1). Studies with inhibitors showed that the protein was mono-ADP-ribosylated. Treatment of (32P)NAD-labeled G(i-1) with Hg and analysis of the released radioactive material showed that the protein was ADP-ribosylated on a cysteine residue.

Activation of bovine photoreceptor guanylate cyclase by S100 proteins.

S100 proteins are acidic calcium-binding proteins present in high concentration in the brain and at lower concentrations in peripheral tissues. Their function remains to be elucidated in many tissues. In this report we show for the first time that S100 proteins stimulate the bovine photoreceptor membrane guanylate cyclase. The extent of stimulation varied between the different S100 proteins with the S100b exhibiting the highest level of influence. The stimulation by all S100 proteins was calcium dependent, with the half-maximal stimulation occurring at about 35-40 microM calcium. These results suggest that in some cells S100 proteins can mediate calcium signals via cyclic GMP.

A novel calcium-dependent activator of retinal rod outer segment membrane guanylate cyclase.

The membrane guanylate cyclase in retinal rod outer segments (ROS-GC) is known to be negatively regulated by calcium; when the calcium concentration is reduced below the dark-adapted level of about 500 nM, the enzyme is activated by a soluble protein. We now report that the enzyme is also positively regulated by calcium; a novel soluble protein is identified and purified from bovine retina which activates ROS-GC, with half-maximal activation occurring at 2-5 microM calcium. The activation is dose-dependent, and at its maximum, cyclase is stimulated up to 25-fold. The activator has a molecular mass of about 40 kDa and is a multimer of a 6-7 kDa peptide.

Nitric oxide synthesis in retinal photoreceptor cells.

Nitric oxide (NO) is known to be synthesized in several tissues and to increase the formation of cyclic GMP through the activation of soluble guanylate cyclases. Since cyclic GMP plays an important role in visual transduction, we investigated the presence of nitric oxide synthesizing activity in retinal rod outer segments. Bovine rod outer segments were isolated intact and separated into membrane and cytosolic fractions. Nitric oxide synthase activity was assayed by measuring the conversion of L-arginine to L-citrulline. Both membrane and cytosolic fractions were active in the presence of calcium and calmodulin. The activity in both fractions was stimulated by the nitric oxide synthase cofactors FAD, FMN, and tetrahydrobiopterin and inhibited by the L-arginine analog, L-monomethyl arginine. The Km for L-arginine was similar, about 5 microM for the enzyme in both fractions. However, the two fractions differed in their calcium/calmodulin dependence: the membrane fraction exhibited basal activity even in the absence of added calcium and calmodulin while the cytosolic fraction was inactive. But the activity increased in both fractions when supplemented with calcium/calmodulin: in membranes from about 40 to 110 fmol/min/mg of protein and in the cytosol from near zero to about 350 fmol/min/mg of protein in assays carried out at 0.3 microM L-arginine. The two enzymes also responded differently to detergent: the activity of the membrane enzyme was doubled by Triton X-100 while that of the cytosolic enzyme was unaffected. These results show that NO is produced by cytosolic and membrane-associated enzymes with distinguishable properties.(ABSTRACT TRUNCATED AT 250 WORDS)

The bovine rod outer segment guanylate cyclase, ROS-GC, is present in both outer segment and synaptic layers of the retina.

Cyclic-GMP, which plays a pivotal role in visual transduction in the vertebrate retina, is synthesized by guanylate cyclase. The purpose of this study was to localize a rod outer segment-derived particulate guanylate cyclase (ROS-GC) to the retina of several species that have different populations of rods and cones. A rabbit antibody was raised against a synthetic peptide, corresponding to the sequence A107-L125 of bovine ROS-GC. Western blot analysis showed a single immunoreactive band at about 115 kDa with bovine rod outer segments but not with human rod outer segments. Light microscopic immunocytochemistry of tissue sections revealed immunoreactivity in the outer segment layer and in the outer and inner plexiform layers. The rod-rich rat retina showed uniform immunolabeling of outer segments; the cone-containing cat retina showed heavily labeled cone outer segments and lighter labeling of rod outer segments; the cone-rich chicken retina showed a uniformly and intensely labeled outer segment layer. Preincubation of the primary antibody with the peptide completely blocked antibody binding. Electron microscopic immunocytochemistry of the cat retina confirmed the presence of guanylate cyclase in photoreceptor outer segments and demonstrated its association with disk and plasma membranes. These data support a concept in which guanylate cyclase is much more concentrated in the outer segments of cones than rods. The immunolabeling of the plexiform layers suggests that the particulate guanylate cyclase is not unique to the photoreceptor outer segments, and may also play a role in transduction processes of retinal synapses.

Nitric oxide-regulated endogenous ADP-ribosylation of rod outer segment proteins.

Both membrane and cytosolic fractions of retinal rod outer segments contain ADP-ribosylases that modify proteins in the respective fractions. Nitroprusside and endogenously produced NO regulate the activities of these ADP-ribosylases. The ADP-ribosylation of the membrane proteins of molecular weight 116K, 66K and 46K is inhibited by NO and nitroprusside, while that of the 38K cytosolic protein and the 39K membrane-associated protein is activated. The 39K protein is identified as the alpha-subunit of G-protein. The ADP-ribosylation of this protein is activated 6 to 11-fold by NO suggesting that NO may play a significant role in modulating the activity of G-protein in visual transduction.