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A Sitaramayya

Publications and source records attributed to A Sitaramayya.

At least 19 recordsLinked to original sources
PubMed2001

Nitric oxide attenuates alpha(2)-adrenergic receptors by ADP-ribosylation of G(i)alpha in ciliary epithelium.

PURPOSE: To determine the mechanism by which nitric oxide (NO) regulates alpha(2)-adrenergic receptor coupling to adenylyl cyclase in bovine ciliary epithelium. METHODS: Ciliary epithelial explants were dissected, cultured, and labeled with [(3)H]adenine. [(3)H]Adenosine 3',5'-cyclic monophosphate (cAMP) was measured under basal conditions and after exposure to forskolin, isoproterenol, clonidine, yohimbine, pertussis toxin, and the NO donor spermine-NO. Endogenous and NO-stimulated ADP-ribosylation of ciliary epithelial membrane proteins was determined by [(32)P]nicotinamide adenosine diphosphate (NAD) labeling and autoradiography. The three isoforms of the G(i)alpha protein subunit were evaluated by Western blot analysis. RESULTS: Basal [(3)H]cAMP content was 13.4 +/- 1.3 picomoles/mg protein (SEM). Both isoproterenol and forskolin stimulated [(3)H]cAMP accumulation to 36.0 +/- 3.9 and 73.2 +/- 17.5 picomoles/mg protein, respectively. Clonidine did not affect basal [(3)H]cAMP levels, but attenuated both isoproterenol- and forskolin-mediated [(3)H]cAMP accumulation to 23.2 +/- 4.4 and 31.6 +/- 4.6 picomoles/mg protein, respectively. Yohimbine antagonized the clonidine-mediated adenylyl cyclase inhibition. Pertussis toxin blocked the effect of clonidine. In the presence of the NO donor spermine-NO, the clonidine-mediated inhibition of forskolin- and isoproterenol-stimulated cAMP accumulation was attenuated completely. NO significantly stimulated endogenous [(32)P]ADP-ribosylation of a 40-kDa membrane protein. Western blot analysis with specific antibodies revealed expression of all three G(i) subtypes--G(i1)alpha, G(i2)alpha, and G(i3)alpha--in bovine ciliary epithelium. CONCLUSIONS: NO attenuates alpha(2)-adrenergic receptor-mediated inhibition of adenylyl cyclase in ciliary epithelium through ADP-ribosylation of the G(i)alpha subunit. The findings demonstrate heterologous regulation between the NO and cAMP signaling pathways in ciliary epithelium.

Adenosine Diphosphate Ribose↗
PubMed2000

Rate of deactivation of nitric oxide-stimulated soluble guanylate cyclase: influence of nitric oxide scavengers and calcium.

Soluble guanylate cyclase (sGC) is highly activated by nitric oxide (NO) and is the known mediator of the effects of NO on a variety of physiological processes. The rates at which sGC is activated and deactivated are therefore of wide interest since they determine the duration of a tissue's response to NO. The effect of NO on smooth muscle dissipates in 1-2 min, suggesting that both activation and deactivation are fast. In vitro measurements show that the activation of sGC occurs in less than a second, while the deactivation takes several hours at 20 degrees C. However, recent reports indicate that Mg-GTP, oxyhemoglobin, and reducing and oxidizing agents could deactivate the cyclase in several seconds to minutes, though the effectiveness of each of these agents is in dispute. We investigated the lifetime of NO-sGC in the cytosol of retina by monitoring its enzymatic activity at 20 degrees C. Our results show that Mg-GTP, the substrate of NO-sGC, has no influence on the deactivation. Similarly, reducing agents glutathione and dithiothreitol shortened the half-life of NO-sGC only by about 30%. The greatest effect on the deactivation was caused by scavengers of NO: oxyhemoglobin reduced the half-life of NO-sGC from 106 min to 18 s; another NO scavenger, 2-(4-carboxyphenyl)-4,4,5, 5-tetramethylimidazoline-1-oxyl-3-oxide (CPTIO), reduced it to 42 s (20 degrees C). Similarly rapid deactivation was observed with the enzyme from bovine lung, immunoprecipitated enzyme from bovine retina, and heme-deficient enzyme from bovine retina reconstituted with heme. On the other hand, YC-1, an activator of sGC, stabilized the activated enzyme, preventing NO dissociation, as was evident from the inability of oxyhemoglobin or CPTIO to deactivate NO-sGC. Calcium, which is known to inhibit NO-sGC, also inhibited the effects of oxyhemoglobin and CPTIO, slowing down the deactivation of the enzyme. Lithium, which is also known to inhibit NO-sGC, had no effect on the deactivation rate of the enzyme. These results, taken together, suggest that two factors with major impact on the lifetime of NO-sGC are the proximity to NO scavengers and the calcium concentration in the cell.

Animals↗
PubMed2000

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.

Adrenergic Agonists↗
PubMed1998

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.

Amino Acid Sequence↗
PubMed1998

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.

Animals↗
PubMed1997

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.

Amino Acid Sequence↗
PubMed1997

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.

Adenosine Diphosphate Ribose↗
PubMed1997

Photoreceptor guanylate cyclases: a review.

Almost three decades of research in the field of photoreceptor guanylate cyclases are discussed in this review. Primarily, it focuses on the members of membrane-bound guanylate cyclases found in the outer segments of vertebrate rods. These cyclases represent a new guanylate cyclase subfamily, termed ROS-GC, which distinguishes itself from the peptide receptor guanylate cyclase family that it is not extracellularly regulated. It is regulated, instead, by the intracellularly-generated Ca2+ signals. A remarkable feature of this regulation is that ROS-GC is a transduction switch for both the low and high Ca2+ signals. The low Ca2+ signal transduction pathway is linked to phototransduction, but the physiological relevance of the high Ca2+ signal transduction pathway is not yet clear; it may be linked to neuronal synaptic activity. The review is divided into eight sections. In Section I, the field of guanylate cyclase is introduced and the scope of the review is briefly explained; Section II covers a brief history of the investigations and ideas surrounding the discovery of rod guanylate cyclase. The first five subsections of Section III review the experimental efforts to quantify the guanylate cyclase activity of rods, including in vitro and in situ biochemistry, and also the work done since 1988 in which guanylate cyclase activity has been determined. In the remaining three subsections an analytical evaluation of the Ca2+ modulation of the rod guanylate cyclase activity related to phototransduction is presented. Section IV deals with the issues of a biochemical nature: isolation and purification, subcellular localization and functional properties of rod guanylate cyclase. Section V summarizes work on the cloning of the guanylate cyclases, analysis of their primary structures, and determination of their location with in situ hybridization. Section VI summarizes studies on the regulation of guanylate cyclases, with a focus on guanylate cyclases activating proteins. In Section VII, the evidence about the localization and functional role of guanylate cyclases in other retinal cells, especially in "on-bipolar" cells, in which guanylate cyclase most likely plays a critical role in electrical signaling, is discussed. The review concludes with Section VIII, with remarks about the future directions of research on retinal guanylate cyclases.

Animals↗
PubMed1996

Calcium modulation of bovine photoreceptor guanylate cyclase.

Bovine photoreceptor guanylate cyclase (ROS-GC) consists of a single transmembrane polypeptide chain with extracellular and intracellular domains. In contrast to non-photoreceptor guanylate cyclases (GCs) which are activated by hormone peptides, ROS-GC is modulated in low Ca2+ by calmodulin-like Ca(2+)-binding proteins termed GCAPs (guanylate cyclase-activating proteins). In this communication we show that, like the native system, ROS-GC expressed in COS cells is activated 4-6-fold by recombinant GCAP1 at 10 nM Ca2+ and that the reconstituted system is inhibited at physiological levels of Ca2+ (1 microM). A mutant ROS-GC in which the extracellular domain was deleted was stimulated by GCAP1 indistinguishable from native ROS-GC indicating that this domain is not involved in Ca2+ modulation. Deletion of the intracellular kinase-like domain diminished the stimulation by GCAP1, indicating that this domain is at least in part involved in Ca2+ modulation. Replacement of the catalytic domain in a non-photoreceptor GC by the catalytic domain of ROS-GC yielded a chimeric GC that was sensitive to ANF/ATP and to a lesser extent to GCAP1. The results establish that GCAP1 acts at an intracellular domain, suggesting a mechanism of photoreceptor GC stimulation fundamentally distinct from hormone peptide stimulation of other cyclase receptors.

Animals↗
PubMed1996

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.

Animals↗
PubMed1995

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.

Animals↗
PubMed1995

Regulation of bovine rod outer segment membrane guanylate cyclase by ATP, phosphodiesterase and metal ions.

In vertebrate retina, rod outer segment is the site of visual transduction. The inward cationic current in the dark-adapted outer segment is regulated by cyclic GMP. A light flash on the outer segment activates a cyclic GMP phosphodiesterase resulting in rapid hydrolysis of the cyclic nucleotide which in turn causes a decrease in the dark current. Restoration of the dark current requires inactivation of the phosphodiesterase and synthesis of cyclic GMP. The latter is accomplished by the enzyme guanylate cyclase which catalyzes the formation of cyclic GMP from GTP. Therefore, factors regulating the cyclase activity play a critical role in visual transduction. But regulation of the cyclase by some of these factors--phosphodiesterase, ATP, the soluble proteins and metal cofactors (Mg and Mn)--is controversial. The availability of different types of cyclase preparations, dark-adapted rod outer segments with fully inhibited phosphodiesterase activity, partially purified cyclase without PDE contamination, cloned rod outer segment cyclase free of other rod outer segment proteins, permitted us to address these controversial issues. The results show that ATP inhibits the basal cyclase activity but enhances the stimulation of the enzyme by soluble activator, that cyclase can be activated in the dark at low calcium concentrations under conditions where phosphodiesterase activity is fully suppressed, and that greater activity is observed with manganese as cofactor than magnesium. These results provide a better understanding of the controls on cyclase activity in rod outer segments and suggest how regulation of this cyclase by ATP differs from that of other known membrane guanylate cyclases.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1995

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.

Amino Acid Sequence↗
PubMed1994

Structural and functional characterization of the rod outer segment membrane guanylate cyclase.

In the vertebrate photoreceptor cell, rod outer segment (ROS) is the site of visual signal-transduction process, and a pivotal molecule that regulates this process is cyclic GMP. Cyclic GMP controls the cationic conductance into the ROS, and light causes a decrease in the conductance by activating hydrolysis of the cyclic nucleotide. The identity of the granylate cyclase (ROS-GC) that synthesizes this pool of cyclic GMP is unknown. We now report the cloning, expression and functional characterization of a DNA from bovine retina that encodes ROS-GC.

Adenosine Triphosphate↗
PubMed1993

Structural and biochemical identity of retinal rod outer segment membrane guanylate cyclase.

Recent molecular cloning reports show that there are at least three membrane guanylate cyclases in vertebrate retina: (1) atrial natriuretic factor receptor guanylate cyclase (ANF-RGC), (2) C-type natriuretic peptide receptor guanylate cyclase (CNP-RGC), and (3) "retinal guanylate cyclase" (RetGC). The specific cellular localization of the first two cyclases is unknown, but RetGC is apparently localized in photoreceptor cells, suggesting that it participates in visual transduction. With the overall objective of identifying the guanylate cyclase that is linked to phototransduction, we compared the structural and regulatory properties of the biochemically characterized 112 kDa bovine rod outer segment membrane guanylate cyclase (ROS-GC) with those of RetGC, ANF-RGC and CNP-RGC. The N-terminal and two internal peptide sequences of purified ROS-GC had about 90% similarity with the corresponding sequences of the RetGC; the sequence identity with natriuretic peptide receptor cyclases was about 30%. A 19 amino acid long sequence from a tryptic peptide of ROS-GC had no corresponding sequence in the other three cyclases. ROS-GC was inhibited by ATP but ANF-RGC and CNP-RGC were activated by ATP in the presence of the respective peptide hormones. These results suggest that ROS-GC represents a new subtype of the membrane guanylate cyclase family that is structurally and biochemically distinct from the other retinal cyclases.

Adenosine Triphosphate↗
PubMed1993

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.

Adenosine Diphosphate Ribose↗
PubMed1993

Cloning and expression of an ATP-regulated human retina C-type natriuretic factor receptor guanylate cyclase.

The natriuretic factors are structurally related polypeptide hormones that regulate the hemodynamics of the physiological processes of diuresis, water balance, and blood pressure. Presumably, these hormones act through the activation of guanylate cyclases which are also the specific receptors of these hormones. Two such structurally similar cell surface receptors are known; the ligand for one is atrial natriuretic factor (ANF) and for the other is C-type natriuretic peptide (CNP). Studies with ANF receptor guanylate cyclase (ANF-RGC) have indicated that its ligand binding site is extracellular and the catalytic site is intracellular, but the mere ligand binding to the receptor domain does not activate the cytosolic catalytic domain. An intervening ATP-mediated event is obligatory: ATP binds to a defined ATP-regulated module (ARM) sequence and bridges the events of ligand binding and signal transduction. The mechanism of CNP signaling is not known, although CNP in intact cells transfected with CNP receptor guanylate cyclase (CNP-RGC) stimulates the formation of cyclic GMP. Furthermore, there is no prior evidence of the presence of CNP signal transduction system in retina, although the presence of ANF-RGC has been documented. We now report the molecular cloning and expression of CNP-RGC from human retina and show that ATP is obligatory in CNP signaling also.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗