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.     

Characterization of rat testicular guanylate cyclase during development.

The biochemical characteristics of rat testicular guanylate cyclase were investigated and the activity and subcellular distribution of the enzyme was determined during testicular development. Examination of the effects of metal ions, nucleotides, detergents and other in vitro activators on the activity of guanylate cyclase revealed that the testicular enzyme is similar in most respects to guanylate cyclase isolated from other mammalian tissues. Changes in the total activity of guanylate cyclase during testicular development paralleled changes in the tissue concentration of cyclic GMP; i.e. guanylate cyclase activity and tissue cyclic GMP were highest during the early stages of development. Subcellular fractionation revealed that the activity of the soluble form of guanylate cyclase was best correlated with tissue cyclic GMP. Biochemical analysis of the soluble enzyme prepared from testes of neonatal and adult rats did not reveal any significant differences in the characteristics of the enzyme during ontogeny with the exception of a 2.5 fold increase in V noted in the neonatal testis. The results of this study are consistent with a molecular mechanism that allows independent regulation of the different forms of guanylate cyclase.     

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.     

Separation of soluble adenylate and guanylate cyclases from the mature rat testis

The mature rat testis contains both a soluble guanylate cyclase and a soluble adenylate cyclase. Both these soluble enzymes prefer manganous ion for activity. It is known that guanylate cyclase can, when activated by a variety of agents, catalyze the formation of cyclic AMP. The following experiments were performed to determine whether the testicular soluble adenylate and guanylate cyclase activities were carried on the same molecule. Analysis of supernatants from homogenized rat testis by gel filtration and sucrose density gradient centrifugation showed that the two activities were clearly separable. The molecular weight of guanylate cyclase is 143 000, while that of adenylate cyclase is 58 000.Treatment of the column fractions with 0.1 mM sodium nitroprusside allowed guanylate cyclase activity to be expressed with Mg²⁺ as well as with Mn²⁺. Sodium nitroprusside did not affect the metal ion or substrate specificity of adenylate cyclase.These experiments show that adenylate and guanylate cyclase activities are physically separable.     

Somatostatin binding capacity, guanylate cyclase and tyrosine phosphatase activities during pancreatic proliferation in the rat induced by gastrectomy

Gastrectomy increased pancreatic growth and this effect was associated with an increase in the number of somatostatin-14 (SS) receptors (146% of control) without altering their affinity. SS increased guanylate cyclase activity twofold in pancreatic acinar membranes from gastrectomized rats. The gastrectomy decreased pancreatic SS-like immunoreactivity (SS-LI) content (55% of control levels) and tyrosine phosphatase activity (74% of control levels). Administration of proglumide (20 mg/kg, IP), a gastrin/cholecystokinin (CCK) receptor antagonist, suppressed the inhibitory effect of gastrectomy on basal tyrosine phosphatase activity and SS-LI content, which returned to control levels. Furthermore, proglumide suppressed the increase of the number of SS receptors and of SS-stimulated guanylate cyclase activity induced by gastrectomy. All this suggests that pancreatic acinar cell growth is associated with upregulation of SS receptors, which could represent a mechanism promoted by the cell to negatively regulate the mitogenic activity of pancreatic growth factors such as CCK. In addition, the results also suggest that the negative regulation of tyrosine phosphatase activity may be important in the events involved in the pancreatic hyperplasia observed after gastrectomy.     

Assay of soluble guanylate cyclase activity by isocratic high-performance liquid chromatography

A HPLC method alternative to labelled or unlabelled procedures was developed for the assay of guanylate cyclase (GC) activity. The substrate (GTP) and the product (cGMP) of the enzymatic reaction were separated in the isocratic mode on a μBondapak C₁₈ column. The activity of GC was linearly dependent on the amount of cGMP produced in the presence of sodium nitroprusside. This approach was applied to follow the purification of GC from bovine lung and to evaluate its stability in different storage conditions.     

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     

Regulation of rat lung adenylate cyclase by cytoplasmic factor(s) during development

Basal adenylate cyclase activity in rat lung homogenate was low prenatally but increased several-fold after birth and remained elevated to maturity. The results also demostrate the appearance of some factors(s) in the lung cytoplasm at a certain age which markedly activated adenylate cyclase. During late gestation and early neonatal life, when the cytoplasmic factor(s) was low or absent, basal adenylate cyclase activity was low and norepinephrine and NaF produced maximum activation of the enzyme. However, when the cytoplasmic factor(s) appeared in the adult lungs, basal adenylate cyclase activity was elevated and both norepinephrine and NaF produced little or no activation of the enzyme. These data suggest a role for the cytoplasmic factor(s) in regulating rat lung adenylate cyclase.The cytoplasmic factor(s) appeared to be a protein since it was inactivated by trypsin digestion and by heating to 75°C. Activation of adenylate cyclase was not due to small ions or other low molecular weight components of the cytoplasm as dialysis of the supernatant did not alter its activation of adenylate cyclase. The cytoplasmic factor(s) did not appear to be either GTP or calcium-dependent regulator of cyclic AMP phosphodiesterase as these did not activate the rat lung adenylate cyclase.     

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.     

鸟苷酸环化酶C激动剂对便秘小鼠肠道菌群的影响

通过动物模型和16S rDNA高通量测序,探究鸟苷酸环化酶C（guanylate cyclase C,GC-C）激动剂（利那洛肽）对便秘的治疗作用和肠道菌群的影响,以期为便秘的治疗提供理论依据。

随机将30只雄性C57BL/6小鼠平均分为对照组、便秘模型组及治疗组,每组10只。其中对照组小鼠给予生理盐水灌胃,便秘模型组和治疗组小鼠给予洛哌丁胺灌胃构建便秘模型,造模成功后治疗组小鼠给予GC-C激动剂灌胃。干预结束后检测各组小鼠粪便含水率、首粒黑便排出时间、小肠推进率以及血清中P物质（Substance P,SP）、血管活性肠肽（vasoactive intestinal peptide,VIP）水平;使用16S rDNA高通量测序分析3组小鼠肠道菌群特点和差异。

与便秘模型组相比,治疗组小鼠粪便含水量、小肠推进率显著提升（t = 3.418,P = 0.003 1;t = 3.141,P = 0.005 6）,首粒黑便排出时间缩短（t = 4.756,P = 0.000 2）;血清VIP水平降低（t = 4.894,P = 0.000 5）,SP水平有升高趋势。与对照组相比,便秘模型组小鼠肠道菌群多样性显著降低,门水平上,Firmicutes丰度升高和Bacteroidetes丰度降低;属水平上,便秘模型组小鼠Muribaculaceae、Lachnospiraceae、Akkermansia丰度降低（均P＜0.050 0）,Allobaculum、Turicibacter、Clostriclium丰度升高（均P＜0.001 0）,通过GC-C激动剂干预可以改善便秘引起的菌群改变,并显著提高了Lactobacillus丰度（P = 0.030 5）。

GC-C激动剂可能通过影响血清中SP、VIP水平,并调节便秘小鼠的肠道菌群趋于正常,增加Lactobacillus、Muribaculaceae等产短链脂肪酸菌群丰度,提高肠道动力,从而起到改善便秘的作用。

     

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.     

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.     

Adenylyl cyclase isoforms in rat testis and spermatozoa from the cauda epididymidis

Expression of adenylyl cyclase genes in rat testis and spermatozoa from the cauda epididymidis was investigated using RT-PCR analysis. Genes encoding the transmembrane adenylyl cyclases (tmAC) II, III, IV, V, VI, VII, and VIII were expressed in the testis, whereas only the gene for tmAC III was expressed in caudal spermatozoa. Immunocytochemistry was used to investigate which tmAC were translated into putative, functional proteins in spermatozoa. Indirect immunofluorescence localized the tmAC II enzyme to a region on the head occupied by the acrosome. The tmAC III enzyme was localized to the posterior margin of the head and to the flagellum, whereas tmAC V and/or VI was localized to the region where the ventral surface of the acrosomal equatorial segment is located. The tmAC VII and VIII enzymes were localized to the convex margin of the head, covering the dorsal region of the acrosomal crescent. To our knowledge, this is the first demonstration that five apparently different tmAC enzymes are localized to discrete subcellular regions of mammalian spermatozoa. These findings provide a fundamental basis for future studies, to determine the physiological roles of tmAC in testis and mature spermatozoa.This work was supported by a grant from the Australian Research Council/Department of Education, Training and Youth Affairs (ARC A40001141)     

Cation-dependent gonadotropin releasing hormone activation of guanylate cyclase

Summary Gonadotropin releasing hormone enhanced guanylate cyclase [E.C.4.6.1.2] two- to threefold in pituitary, testis, liver and kidney. Dose response relationships revealed that at a concentration of 1 nanomolar, gonadotropin releasing hormone caused a maximal augmentation of guanylate cyclase activity and that increasing its concentration to the millimolar range caused no further enhancement of this enzyme. There was an absolute cation requirement for gonadotropin releasing hormone's enhancement of guanylate cyclase activity as there was no increase without any cation present. Gonadotropin releasing hormone 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 gonadotropin releasing hormone.     

Hepatic guanylate cyclase activity is decreased in a model of cirrhosis: a quantitative cytochemistry study

The production of nitric oxide (NO) in liver disease and its role in vascular control has been a subject of much interest in recent years. However, the activity of guanylate cyclase (GC), the enzyme activated by NO has received little attention with regard to liver disease. In this study we have utilised a quantitative cytochemical technique to examine the activity of GC on a per cell basis in a rat model of cirrhosis. Our results show a significant reduction in GC activity, indicating that vascular regulation is likely to be substantially affected irrespective of NO generation in this disease model.     

Plasma membrane guanylate cyclase is a multimodule transduction system

Summary This minireview highlights the studies which suggest that guanylate cyclase is a single-component transducing system, containing distinct signaling modules in a single membrane-spanning protein. A guanylate cyclase signaling model is proposed which envisions the following sequential events: (1) a signal is initiated by the binding of the hormone to the ligand binding module; (2) the signal is potentiated by ATP at ARM; and (3) the amplified signal is finally transduced at the catalytic site. All of these signaling steps together constitute a switch, which when turned on, generates the second messenger cyclic GMP.