Stimulation of guanylate cyclase and RNA polymerase II activities in HeLa cells and fibroblasts by biotin

HeLa cells cultured in a biotin-deficient medium showed reduced rates of protein synthesis, DNA synthesis and growth. Continuous synthesis is required for the increase in DNA synthesis observed upon addition of biotin to cells cultured in biotin-deficient medium. The addition of biotin to the biotin-deficient culture medium increased the activity of guanylate cyclase in both HeLa cells and fibroblasts. Both cell types cultured in biotin deficient medium showed reduced activity of RNA Polymerase II. The exogenous addition of biotin to the biotin-deficient cell cultures also resulted in increased activity of RNA Polymerase II in HeLa cells and fibroblasts. The maximal response was observed in 4 hours. Significant increase in enzyme activity was observed at 10^–8 M biotin in the culture medium. The growth promoting effect of biotin seems to involve stimulations of cellular guanylate cyclase and RNA Polymerase II activity.     

Evolution of the membrane guanylate cyclase transduction system

Almost four decades of research in the field of membrane guanylate cyclases is discussed in this review. Primarily, it focuses on the chronological development of the field, recognizes major contributions of the original investigators, corrects certain misplaced facts, and projects its future trend.     

The effect of luteinizing hormone releasing hormone and anti-luteinizing hormone releasing hormone antibodies on chorionic gonadotropin and progesterone secretion by human placental villiin vitro

Gonadotropin releasing hormone has been located and found to be secreted by the human placenta in culture. Addition of the releasing hormone upto 1μg concentration in the placental cultures brings about stimulation of chorionic gonadotropin and progesterone secretion. Higher amounts of the decapeptide has an inhibitory influence on both the gonadotropin and the steroid production. The action of the releasing hormone on the placenta could be blocked by the anti-luteinizing hormone releasing hormone monoclonal antibodies indicating a possible site of action of the antibodies for control of fertility     

Potentiation of NO-dependent activation of soluble guanylate cyclase by polyamines

The influence of polyamines (putrescine, spermidine, and spermine) on the activity of human platelet soluble guanylate cyclase and the stimulation of the enzyme by sodium nitroprusside (SNP), YC-1 and their combination was investigated. All these polyamines stimulated the guanylate cyclase activity and potentiated its activation by sodium nitroprusside. The stimulatory effects of sodium nitroprusside and putrescine (or spermine) were addidive; spermidine produced a synergistic activation and increased the additive effect. All the polyamines inhibited the enzyme activation by YC-1 and decreased the synergistic activation of SNP-stimulated guanylate cyclase activity by YC-1 with nearly the same potency. The ability of the investigated polyamines to potentiate and to increase synergistically (similar to to YC-1, but less effective) NO-dependent activation of soluble guanylate cyclase represents a new biochemical effect of these compounds; this effect should be taken into consideration, especially due to the endogenous nature of polyamines. The data obtained suggest, that specific biological functions of polyamines in the processes of growth and differentiation of cells may be also related to the ability of compounds to activate soluble guanylate cyclase and to increase intracellular cGMP level.     

Cation-dependent prolactin activation of guanylate cyclase

Prolactin enhanced guanylate cyclase [E.C.4.6.1.2] two- to threefold in ovary, testis, mammary gland, liver and kidney. Dose response relationships revealed that maximal activation of this enzyme was at a concentration of one nanomolar and that increasing prolactin's concentration to the millimolar range caused no further increase in activity. There was an absolute cation requirement for prolactin's enhancement of guanylate cyclase. Calcium or manganese allowed prolactin to increase guanylate cyclase activity. Greater enhancement of this enzyme's activity by prolactin was observed when manganese was the co-factor. The data in this investigation suggest that guanylate cyclase may play a role in the mechanism of action of prolactin.     

Moonlighting kinases with guanylate cyclase activity can tune regulatory signal networks

Guanylate cyclase (GC) catalyzes the formation of cGMP and it is only recently that such enzymes have been characterized in plants. One family of plant GCs contains the GC catalytic center encapsulated within the intracellular kinase domain of leucine rich repeat receptor like kinases such as the phytosulfokine and brassinosteroid receptors. In vitro studies show that both the kinase and GC domain have catalytic activity indicating that these kinase-GCs are examples of moonlighting proteins with dual catalytic function. The natural ligands for both receptors increase intracellular cGMP levels in isolated mesophyll protoplast assays suggesting that the GC activity is functionally relevant. cGMP production may have an autoregulatory role on receptor kinase activity and/or contribute to downstream cell expansion responses. We postulate that the receptors are members of a novel class of receptor kinases that contain functional moonlighting GC domains essential for complex signaling roles.     

Peptides from the N-terminus of the atrial natriuretic factor prohormone enhance guanylate cyclase activity and increase cyclic GMP levels in a wide variety of tissues

The 98 amino acid (a. a.) N-terminus of the 126 a. a. atrial natriuretic factor (ANF) prohormone contains three peptides consisting of a. a. 1–30 (proANF 1–30), a. a. 31–67 (proANF 31–67) and a. a. 79–98 (proANF 79–98) with blood pressure lowering, sodium and/or potassium excreting properties similar to atrial natriuretic factor (a. a. 99–126, C-terminus of prohormone). ProANF 1–30 and proANF 31–67 have separate and distinct receptors from ANF in both vasculature and in the kidney to help mediate the above effects. At the cellular level proANFs 1–30, 31–67, and 79–98 as well as ANF's effects are mediated by enhancement of the guanylate cyclase (EC 4.6.1.2) — cyclic GMP system in vasculature and in the kidney. These peptides from the N-terminus of the ANF prohormone circulate normally in man and in all animal species tested. The object of the present investigation was to determine if these peptides have the ability to enhance either guanylate cyclase and/or adenylate cyclase in a variety of other tissues in addition to kidney and vasculature. ProANF 1–30, proANF 31–67, proANF 79–98, and ANF all increased rat lung, liver, heart and testes, but not spleen, particulate guanylate cyclase 2- to 3-fold at their 100 nM concentrations. Dose response curves revealed that maximal stimulation of particulate guanylate cyclase activity by these newly discovered peptides was at their 1 M concentrations, with no further increase in activity above their 1 M concentrations. Half-maximal (EC₅₀) enhancement of particulate guanylate cyclase occurred at 0.15 ± 0.01, 0.3 ± 0.02, 0.5 ± 0.03, and 0.9 ± 0.03 nM for proANF 1–30, proANF 31–67, proANF 79–98 and ANF, respectively. ProANFs 1–30, 31–67, 79–98, and 99–126 (i.e., ANF) each increased cyclic GMP but not cyclic AMP levels in tissue slices of liver, lung, small intestine, heart, and testes. None of these peptides enhanced either adenylate cyclase or the soluble 100,000 G form of guanylate cyclase. The ability of these N-terminal peptides to enhance particulate guanylate cyclase activity in a wide variety of tissues suggests that they may have effects in a much wider variety of tissues than presently thought.     

ATP binding is required for physiological activation of retinal guanylate cyclase

ATP bound to retinal guanylate cyclase (retGC)/membranes prior to the assay (pre-binding effect) and during the assay (direct effect) further enhances retGC activity stimulated by GC-activating proteins (GCAPs). Here we investigate differences between these two effects. We found that the pre-binding effect, but not the direct effect, was absent in membranes pre-washed with Mg(2+)-free hypotonic buffers, that the pre-binding effect, but not the direct effect, was strictly limited to GCAP-stimulated retGC activity, and that these two effects were independent and additive rather than being synergistic. Pre-incubation with amiloride enhanced GCAP2-activated retGC activity in a manner similar to that by ATP pre-binding; however, amiloride did not directly stimulate the retGC activity. These results indicate that these two effects are mechanistically different. Levels of retGC activation by these effects and conditions required for these effects indicate that only the mechanism involving ATP pre-binding is physiologically relevant to retGC activation.     

YC-1-like potentiation of nitric oxide-dependent activation of soluble guanylate cyclase by adrenochrom

The influence of adrenochrome and YC-1 activation of human platelet soluble guanylate cyclase was investigated. Adrenochrome (0.1–10.0 μM) had no effect on the basal activity, but it potentiated in a concentration- dependent manner the spermine NONO-induced activation of this enzyme. Adrenochrome also sensitized guanylate towards nitric oxide (NO) and produced the leftward shift of the spermine NONO concentration response curve. Addition of adrenochrome decreased the YC-1-induced leftward shift of the spermine NONO concentration response curve. Adrenochrome also inhibited enzyme activation byYC-1. Thus, synergistic activation of NO-stimulated guanylate cyclase activity by adrenochrome represents a new biochemical effect of this compound and indicates that adrenochrome may act as an endogenous regulator of the NO-dependent stimulation of soluble guanylate cyclase. This new property of adrenochrome, similar to YC-1 but more effective, should be taken into consideration especially under conditions of adrenochrome overproduction in the body.     

A novel mechanism of soluble guanylate cyclase stimulation: time-dependent activation by bacterial lipopolysaccharide in rat fetal spleen cells

Small amounts of bacterial lipopolysaccharide (LPS) greatly increase cGMP levels in short term cultures of rat fetal liver and spleen cells in a dose and time dependent manner. To determine the role of guanylate cyclase in this response, a series of experiments was undertaken using either intact or broken fetal spleen cells, the most sensitive tissue evaluated to date. The phosphodiesterase inhibitor, 1-methyl-3-isobutylxanthine, potentiated the LPS-cGMP effect in cultures of these cells even at maximal doses of LPS. Moreover, after incubation of intact cells with LPS for 4 h, soluble guanylate cyclase (EC 4.6.1.2) activity was increased 2-fold, whereas particulate activity was unchanged. This increase in soluble activity was proportional to the dose of LPS, was synchronous with the elevation of cGMP levels, and was not associated with any change in cGMP-phosphodiesterase (EC 3.1.4.17) activity. In contrast to intact cells, neither total nor soluble guanylate cyclase activity was increased by the addition of LPS to spleen cells, neither total cytosol for various times from 10 min to 3.5 h. These results suggest that the LPS-cGMP response is due to a persistent indirect stimulation of soluble guanylate cyclase activity that is both dose and time dependent.     

Third calcium-modulated rod outer segment membrane guanylate cyclase transduction mechanism

Ca²⁺-modulated rod outer segment membrane guanylate cyclase (ROS-GC1) has been cloned and reconstituted to show that it is regulated by two processes: one inhibitory, the other stimulatory. The inhibitory process is consistent with its linkage to phototransduction; the physiology of the stimulatory process is probably linked to neuronal transmission. In both regulatory processes, calcium modulation of the cyclase takes place through the calcium binding proteins; guanylate cyclase activating proteins (GCAP1 and GCAP2) in the case of the phototransduction process and calcium-dependent GCAP (CD-GCAP) in the case of the stimulatory process. The cyclase domains involved in the two processes are located at two different sites on the ROS-GC1 intracellular region. The GCAP1-modulated domain resides within the aa 447-730 segment of ROS-GC1 and the CD-GCAP-modulated domain resides within the aa 731-1054 segment. In the present study the GCAP2-dependent Ca²⁺ modulation of the cyclase activity has been reconstituted using recombinant forms of GCAP2 and ROS-GC1, and its mutants. The results indicate that consistent to phototransduction, GCAP2 at low Ca²⁺ concentration (10 nM) maximally stimulates the cyclase activity of the wild-type and its mutants: ext- (deleted aa 8-408); kin- (deleted aa 447-730) and hybrid consisting of the ext, transmembrane and kin domains of ANF-RGC and the C-terminal domain, aa 731-1054, of ROS-GC1. In all cases, it inhibits the cyclase activity with an IC₅₀ of about 140 nM. A previous study has shown that under identical conditions the kin- and the hybrid mutant are at best only minimally stimulated. Thus, the GCAP1 and GCAP2 signal transduction mechanisms are different, occurring through different modules of ROS-GC1. These findings also demonstrate that the intracellular region of ROS-GC1 is composed of multiple modules, each designed to mediate a particular calcium-specific signalling pathway.     

Acidic triazoles as soluble guanylate cyclase stimulators

A series of acidic triazoles with activity as soluble guanylate cyclase stimulators is described. Incorporation of the CF(3) triazole improved the overall physicochemical and drug-like properties of the molecule and is exemplified by compound 25.     

Novel frequenin-modulated Ca2+-signaling membrane guanylate cyclase (ROS-GC) transduction pathway in bovine hippocampus

Frequenin is a member of the neuronal Ca²⁺ sensor protein family, implicated in being the modulator of the neurotransmitter release, potassium channels, phosphatidylinositol signaling pathway and the Ca²⁺-dependent exocytosis of dense-core granules in the PC12 cells. Frequenin exhibits these biological activities through its Ca²⁺ myristoyl switch, yet the switch is functionally inactive. These structural and functional traits of frequenin have been derived through the use of recombinant frequenin. In the present study, frequenin (BovFrq) native to the bovine hippocampus has been purified, sequenced for its 9 internal fragments, cloned, and studied. The findings show that structure of the BovFrq is identical to its form present in chicken, rat, mouse and human, indicating its evolutionary conservation. Its Ca²⁺ myristoyl switch is active in the hippocampus. And, BovFrq physically interacts and turns on yet undisclosed ONE-GC-like ROS-GC membrane guanylate cyclase transduction machinery in the hippocampal neurons. This makes BovFrq a new Ca²⁺-sensor modulator of a novel ROS-GC transduction machinery. The study demonstrates the presence and mechanistic features of this cyclic GMP signaling pathway in the hippocampal neurons, and also provides one more support for the evolving concept where the Ca²⁺-modulated membrane guanylate cyclase transduction machinery in its variant forms is a central operational component of all neurons.     

Cation-dependent substance P activation of the enzyme guanylate cyclase

Substance P enhanced guanylate cyclase (E.C.4.6.1.2) two- to fourfold in pancreas, small intestine, cerebellum, liver, kidney, and lung. Dose response relationship revealed that substance P caused a maximal augmentation of guanylate cyclase activity at concentration of 1 micromolar. Increasing substance P's concentration to the millimolar range caused no further increase in activity. There was an absolute cation requirement for substance P's enhancement of guanylate cyclase activity. Substance P could increase guanylate cyclase activity with either calcium or manganese in the incubation medium but more augmentation was observed with manganese. The data in this investigation suggest that guanylate cyclase may play a role in the mechanism of action of substance P.     

MsGC-II,a receptor guanylyl cyclase isolated from the CNS of Manduca sexta that is inhibited by calcium

We describe the cloning of a receptor guanylyl cyclase, MsGC-II, from the CNS of the insect Manduca sexta. Sequence comparisons with other receptor guanylyl cyclases show that MsGC-II is most similar to a predicted guanylyl cyclase in the Drosophila genome and to vertebrate retinal guanylyl cyclases. When expressed in COS-7 cells, MsGC-II exhibited a low level of basal activity that was nearly abolished in the presence of 10 micro m calcium. Incubation with either a mammalian guanylyl cyclase-activating protein or Drosophila frequenin resulted in only mild stimulation of activity, whereas incubation of COS-7 cells expressing MsGC-II with a variety of Manduca tissue extracts failed to stimulate enzyme activity above basal levels. Analysis of the tissue distribution of MsGC-II revealed that it is nervous system specific. In the adult, MsGC-II is present in neurons in the optic lobes, antennal lobes and cellular cortex, but it is most highly expressed in subsets of intrinsic mushroom body neurons. Thus, MsGC-II appears to be a neural-specific receptor guanylyl cyclase whose activity may be regulated either directly or indirectly by calcium.     

Identification of residues crucially involved in soluble guanylate cyclase activation

The ubiquitous heterodimeric nitric oxide (NO) receptor soluble guanylate cyclase (sGC) plays a key role in various signal transduction pathways. Binding of NO takes place at the prosthetic heme moiety at the N-terminus of the beta(1)-subunit of sGC. The induced structural changes lead to an activation of the catalytic C-terminal domain of the enzyme and to an increased conversion of GTP into the second messenger cyclic GMP (cGMP). In the present work we selected and substituted different residues of the sGC heme-binding pocket based on a sGC homology model. The generated sGC variants were tested in a cGMP reporter cell for their effect on the enzyme activation by heme-dependent (NO, BAY 41-2272) stimulators and heme-independent (BAY 58-2667) activators. The use of these experimental tools allows the enzyme's heme content to be explored in a non-invasive manner. Asp(44), Asp(45) and Phe(74) of the beta(1)-subunit were identified as being crucially important for functional enzyme activation. beta(1)Asp(45) may serve as a switch between different conformational states of sGC and point to a possible mechanism of action of the heme dependent sGC stimulator BAY 41-2272. Furthermore, our data shows that the activation profile of beta(1)IIe(145) Tyr is unchanged compared to the native enzyme, suggesting that Tyr(145) does not confer the ability to distinguish between NO and O(2). In summary, the present work further elucidated intramolecular mechanisms underlying the NO- and BAY 41-2272-mediated sGC activation and raises questions regarding the postulated role of Tyr(145) for ligand discrimination.     

Plasma levels of gonadotropin releasing hormone during menstrual cycle ofMacaca radiata

A sensitive radioimmunoassay for gonadotropin releasing hormone has been developed. The assay has been validated for its specificity by testing various analogues of gonadotropin releasing hormone. Analysis of plasma samples during the menstrual cycle of 4 female bonnet monkeys showed a significant increase in the immunoreactive gonadotropin releasing hormone levels during preovulatory period of the menstrual cycle.     

Heat shock protein 90 regulates stabilization rather than activation of soluble guanylate cyclase

Endothelium-derived nitric oxide (NO) activates the heterodimeric heme protein soluble guanylate cyclase (sGC) to form cGMP. In different disease states, sGC levels and activity are diminished possibly involving the sGC binding chaperone, heat shock protein 90 (hsp90). Here we show that prolonged hsp90 inhibition in different cell types reduces protein levels of both sGC subunits by about half, an effect that was prevented by the proteasome inhibitor MG132. Conversely, acute hsp90 inhibition affected neither basal nor NO-stimulated sGC activity. Thus, hsp90 is a molecular stabilizer for sGC tonically preventing proteasomal degradation rather than having a role in short-term activity regulation.     

Requirement of thiols for activation of coronary arterial guanylate cyclase by glyceryl trinitrate and sodium nitrite possible involvement of S-nitrosothiols

Glyceryl trinitrate specifically required cysteine, whereas NaNO₂ at concentrations less than 10 mM required one of several thiols or ascorbate, to activate soluble guanylate cyclase from bovine coronary artery. However, guanylate cyclase activation by nitroprusside or nitric oxide did not require the addition of thiols or ascorbate. Whereas various thiols enhanced activation by nitropruside, none of the thiols tested enhanced activation by nitric oxide. S-Nitrosocysteine, which is formed when cysteine reacts with either NO₂^? or nitric oxide, was a potent activator of guanylate cyclase. Similarly, micromolar concentrations of the S-nitroso derivatives of penicillamine, GSH and dithiothreitol, prepared by reacting the thiol with nitric oxide, activated guanylate cyclase. Guanylate cyclase activation by S-nitrosothiols resembled that by nitric oxide and nitroprusside in that activation was inhibited by methemoglobin, ferricyanide and methylene blue. Similarly, guanylate cyclase activation by glyceryl trinitrate plus cysteine, and by NaNO₂ plus either a thiol or ascorbate, was inhibited by methemoglobin, ferricyanide and methylene blue. These data suggest that the activation of guanylate cyclase by each of the compounds tested may occur through a common mechanism, perhaps involving nitric oxide. Moreover, these findings suggest that S-nitrosothiols could act as intermediates in the activation of guanylate cyclase by glyceryl trinitrate, NaNO₂ and possibly     

Synthesis of adenosine 3′,5′-monophosphate by guanylate cyclase,a new pathway for its formation

The 105 000 × g supernatant fractions from homogenates of various rat tissues catalyzed the formation of both cyclic GMP and cyclic AMP from GTP and ATP, respectively. Generally cyclic AMP formation with crude or purified preparations of soluble guanylate cyclase was only observed when enzyme activity was increased with sodium azide, sodium nitroprusside, N-methyl-N′-nitro-N-nitrosoguanidine, sodium nitrite, nitric oxide gas, hydroxyl radical and sodium arachidonate. Sodium fluoride did not alter the formation of either cyclic nucleotide. After chromatography of supernatant preparations on Sephadex G-200 columns or polyacrylamide gel electrophoresis, the formation of cyclic AMP and clycic GMP was catalyzed by similar fractions. These studies indicate that the properties of guanylate cyclase are altered with activation. Since the synthesis of cyclic AMP and cyclic GMP reported in this study appears to be catalyzed by the same protein, one of the properties of activated guanylate cyclase is its ability to catalyze the formation of cyclic AMP from ATP. The properties of this newly described pathway for cyclic AMP formation are quite different from those previously described for adenylate cyclase preparations. The physiological significance of this pathway for cyclic AMP formation is not known. However, these studies suggest that the effects of some agents and processes to increase cyclic AMP accumulation in tissue could result from the activation of either adenylate cyclase or guanylate cyclase.