Modulation of adenylate cyclase/cyclic AMP response by thyrotropin and prostaglandin E2 in cultured thyroid cells. 2. Positive regulation.

Two different independent processes are operating in cultured thyroid cells to regulate adenylate cyclase/cyclic AMP responsiveness to thyroid stimulators (thyrotropin and prostaglandin E2): firstly, refractoriness or negative regulation [preceding paper], which is specific for each thyroid stimulator, is not mediated by cyclic AMP and is not accompanied by alteration of adenylate cyclase activity; secondly, positive regulation which is characterized by an augmentation of the cyclic AMP response stimulated by thyrotropin and prostaglandin E2. This process is not specific for each thyroid stimulator and is a state of increased susceptibility of cyclic AMP synthesis to stimulation, accompanied by increased activity of the catalytic subunit of adenylate cyclase. Positive regulation is apparently mediated by increased intracellular cyclic AMP levels. It is a time-dependent and dose-dependent process. Very low concentrations (5-50 micronU/ml) of thyrotropin augmented cyclic AMP synthesis stimulated by thyrotropin and prostaglandin E2 whereas higher concentrations (above 0.1 mU/ml) augmented prostaglandin E2 stimulation but induced refractoriness to thyrotropin. Prostaglandin E2 (0.1 to 10 micronM) augmented thyrotropin stimulation and dibutyryl adenosine 3':5'-monophosphate (0.3 to 2 mM) augmented thyrotropin and prostaglandin E2 stimulation. Positive regulation is a slow process which develops within days and increases up to day 5 in culture. Experiments using inhibitors suggested that protein synthesis is required for the full expression of the increase in adenylate cyclase activity induced by the studied thyroid stimulators.     

Cyclic GMP metabolism in relation to the regulation of cell growth in BALB/c3T3 cells.

BALB/c3T3 cell homogenates have guanylate cyclase in both 105000 g paniculate and soluble fraction. The activity in particulate fraction was much higher than that in the soluble fraction. Both enzyme activities were 2- to 3-fold greater in the resting state (G0 phase) than in the logarithmically growing state. In addition, cGMP-phosphodiesterase activity was 2-fold greater in resting than in growing cells. When G0-arrested cells entered into the G1 phase by serum addition, cGMP levels rapidly increased, whereas guanylate cyclase activities did not change within 30 min after serum addition. Four hours after serum addition, these activities had, however, decreased to one third and remained at that low level throughout the G1 phase. The relationship between cell growth and guanylate cyclase activity is discussed.     

Thyrotropin effect on the availability of Ni regulatory protein in FRTL-5 rat thyroid cells to ADP-ribosylation by pertussis toxin

Incubation of FRTL-5 rat thyroid cell membranes with [32P]NAD and pertussis toxin results in the specific ADP-ribosylation of a protein of about 40 kDa. This protein has the same molecular mass of the alpha i subunit of the adenylate cyclase regulatory protein Ni and is distinct from proteins ADP-ribosylated by cholera toxin in the same membranes. Prior treatment of FRTL-5 cells with pertussis toxin results in the ADP-ribosylation of Ni, as indicated by the loss of the toxin substrate in the ADP-ribosylation assay performed with membranes prepared from such cells. Preincubation of FRTL-5 cells with thyrotropin causes the same loss; cholera toxin has no such effect. Pertussis toxin, as do thyrotropin and cholera toxin, increases cAMP levels in FRTL-5 cells. Forskolin together with thyrotropin, cholera toxin or pertussis toxin causes a further increase in cAMP levels. Pertussis toxin and thyrotropin are not additive in their ability to increase adenylate cyclase activity, whereas both substances are additive with cholera toxin. A role of Ni in the thyrotropin regulation of the adenylate cyclase activity in thyroid cells is proposed.     

Oscillating levels of adenylate and guanylate cyclase activities in rat embryo fibroblasts stimulated to divide.

Basal activities of membrane-bound adenylate and guanylate cyclase were determined in confluent rat embryo cells stimulated to proliferate by either the renewal of serum-supplemented growth medium or the addition of a mitogen, the 12-0-tetradecanoyl-phorbol-13-acetate (TPA). A transient increase in guanylate cyclase activity was observed within minutes following either treatment while adenylate cyclase activity either abruptly declined in serum-stimulated cells or remained unaffected in TPA-treated cells. In response to both mitogenic treatment, adenylate and guanylate cyclase activities varied reciprocally throughout the pre-replicative phase up to DNA synthesis. The lower levels of guanylate over adenylate activity ratio occurred prior to the onset of the replicative phase whereas the higher levels were coincident with DNA synthesis. A similar pattern of oscillating levels of sodium-fluoride-stimulated adenylate and lubrol-treated guanylate cyclase activities was observed.     

Differential effects of temperature on cAMP-induced excitation, adaptation, and deadaptation of adenylate and guanylate cyclase in Dictyostelium discoideum

Extracellular cAMP induces excitation of adenylate and guanylate cyclase in Dictyostelium discoideum. Continuous stimulation with cAMP leads to adaptation, while cells deadapt upon removal of the cAMP stimulus. Excitation of guanylate cyclase by cAMP has a lag time of approximately 1 s; excitation of adenylate cyclase is much slower with a lag time of 30 s. Excitation of both enzyme activities is less than twofold slower at 0 degrees C than at 20 degrees C. Adaptation of guanylate cyclase is very fast (t1/2 = 2.4 s at 20 degrees C), and virtually absent at 0 degrees C. Adaptation of adenylate cyclase is much slower (t1/2 = 110 s at 20 degrees C) but not very temperature sensitive (t1/2 = 290 s at 0 degrees C). At 20 degrees C, deadaptation of adenylate cyclase is about twofold slower than deadaptation of guanylate cyclase (t1/2 = 190 and 95 s, respectively). Deadaptation of adenylate cyclase is absent at 0 degrees C, while that of guanylate cyclase proceeds slowly (t1/2 = 975 s). The results show that excitation, adaptation, and deadaptation of guanylate cyclase have different kinetics and temperature sensitivities than those of adenylate cyclase, and therefore are probably independent processes.     

Enzymatic formation of inosine 3',5'-monophosphate and of 2'-deoxyguanosine 3',5'-monophosphate. Inosinate and deoxyguanylate cyclase activity.

Enzymes in particulate fractions from sea urchin sperm and in soluble fractions from rat lung were shown to catalyze the formation of inosine 3',5'-monophosphate (cyclic IMP) and of 2'-deoxyguanosine 3',5'-monophosphate (cyclic dGMP) from ITP and dGTP, respectively. With sea urchin sperm particulate fractions, Mn2+ was an essential metal cofactor for inosinate, deoxyguanylate, guanylate and adenylate cyclase activities. Heat-inactivation studies differentiated inosinate and deoxyguanylate cyclase activities from adenylate cyclase, but indicated an association of these activities with guanylate cyclase. Preincubation of sea urchin sperm particulate fractions with trypsin altered in a very similar manner guanylate, inosinate, and deoxyguanylate cyclase activities, and various metals and metal-nucleotide combinations protected the three cyclase activities to comparable degrees against trypsin. The relative guanylate, deoxyguanylate and inosinate cyclase activities at 0.1 mM nucleoside triphosphate were 1.0, 0.5 and 0.08, respectively. With these three cyclase activities, plots of reciprocal velocities against reciprocal Mn2+-nucleoside triphosphate concentrations were concave upward, suggesting positive homotropic effects. With rat lung soluble preparations, relative guanylate, deoxyguanylate, inosinate and adenylate cyclase activities at 0.09 mM nucleoside triphosphate were 1.0, 1.7, 0.1 and 0, respectively. MnGTP was a competitive inhibitor of deoxyguanylate cyclase activity (Ki equals 12.2 muM) and MndGTP was a competitive inhibitor of guanylate cyclase activity (Ki equals 16.2 muM). Inhibition studies using ITP were not conducted. When soluble fractions from rat lung were applied to Bio-Gel A 1.5 m columns, elution profiles of guanylate, deoxyguanylate and inosinate cyclase activities were similar. These results suggest that deoxyguanylate, guanylate and inosinate cyclase activities reside within the same protein molecule.     

Characterization of adenylate and guanylate cyclases in rat peritoneal mast cells

Adenylate and guanylate cyclase activities were confirmed in crude homogenates from rat peritoneal mast cells. Both enzyme activities were associated with the 105, 000 X g particulate fractions, but not detected in the supernatant fractions. The optimal pH for both cyclase activities was 8.2. Mn⁺⁺ was essentially required for guanylate cylcase activity, while adenylate cyclase activity was observed in the presence of either Mg⁺⁺ or Mn⁺⁺. The apparent Km values of adenylate cyclase for Mn⁺⁺-ATP and Mg⁺⁺-ATP were 160 μM and 340 μM, respectively, whereas the value of guanylate cyclase for Mn⁺⁺-GTP was 100 μM. Adenylate cyclase was activated by 10 mM NaF. However, both adenylate and guanylate cyclase activities were neither stimulated nor inhibited by the addition of various kinds of agents which stimulate or inhibit the release of histamine from mast cells.     

The subcellular distribution of adenylate and guanylate cyclases in murine lymphoid cells.

Membrane vesicles can be prepared from murine lymphoid cells by nitrogen cavitation and fractionated by sedimentation through nonlinear sucrose density gradients. Two subpopulations of membrane vesicles, PMI and PMII, can be distinguished on the basis of sedimentation rate. The subcellular distribution of adenylate and guanylate cyclases in these membrane subpopulations have been compared with the distribution of a number of marker enzymes. Approximately 20-30% of the total adenylate and guanylate cyclase activity is located at the top of the sucrose gradient (soluble enzyme), the remainder of the activity being distributed in the PMI and PMII fractions (membrane-bound enzyme). More than 90% of the 5'-nucleotidase and NADH oxidase activities detected in lymphoid cell homogenates are located in PMI and PMII fractions, whereas succinate cytochrome c reductase activity is detected only in the PMII fractions. In addition, beta-galactosidase activity is distributed in the soluble and PMII fractions of the sucrose density gradients. On the basis of the fractionation patterns of these various enzyme activities, it appears that PMI fractions contain vesicles of plasma membrane and endoplasmic reticulum, whereas PMII fractions contain mitochondria, lysomes, and plasma membrane vesicles. Approximately 30-40% of the adenylate and guanylate cyclase activities in PMII can be converted to a PMI-like form following dialysis and resedimentation through a second nonlinear sucrose gradient. Adenylate and guanulate cyclases can be distinguished on the basis of sensitivity to nonionic detergents.     

Localization of particulate guanylate cyclase in plasma membranes and microsomes of rat liver.

The subcellular localization of guanylate cyclase was examined in rat liver. About 80% of the enzyme activity of homogenates was found in the soluble fraction. Particulate guanylate cyclase was localized in plasma membranes and microsomes. Crude nuclear and microsomal fractions were applied to discontinuous sucrose gradients, and the resulting fractions were examined for guanylate cyclase, various enzyme markers of cell components, and electron microscopy. Purified plasma membrane fractions obtained from either preparation had the highest specific activity of guanylate cyclase, 30 to 80 pmol/min/mg of protein, and the recovery and relative specific activity of guanylate cyclase paralleled that of 5'-nucleotidase and adenylate cyclase in these fractions. Significant amounts of guanylate cyclase, adenylate cyclase, 5'-nucleotidase, and glucose-6-phosphatase were recovered in purified preparation of microsomes. We cannot exclude the presence of guanylate cyclase in other cell components such as Golgi. The electron microscopic studies of fractions supported the biochemical studies with enzyme markers. Soluble guanylate cyclase had typical Michaelis-Menten kinetics with respect to GTP and had an apparent Km for GTP of 35 muM. Ca-2+ stimulated the soluble activity in the presence of low concentrations of Mn-2+. The properties of guanylate cyclase in plasma membranes and microsomes were similar except that Ca-2+ inhibited the activity associated with plasma membranes and had no effect on that of microsomes. Both particulate enzymes were allosteric in nature; double reciprocal plots of velocity versus GTP were not linear, and Hill coefficients for preparations of plasma membranes and microsomes were calculated to be 1.60 and 1.58, respectively. The soluble and particulate enzymes were inhibited by ATP, and inhibition of the soluble enzyme was slightly greater. While Mg-2+ was less effective than Mn-2+ as a sole cation, all enzyme fractions were markedly stimulated with Mg-2+ in the presence of a low concentration of Mn-2+. Triton X-100 increased the activity of particulate fractions about 3- to 10-fold and increased the soluble activity 50 to 100%.     

Postmortem Stability of Dopamine-Sensitive Adenylate Cyclase, Guanylate Cyclase, ATPase, and GTPase in Rat Striatum

The stability of dopamine-sensitive adenylate cyclase, guanylate cyclase, ATPase, and GTPase was measured in homogenates of rat striatal tissue frozen from 0 to 24 h postmortem. ATPase, GTPase, and Mg2+-dependent guanylate cyclase activities showed no significant change over this period. Mn2+-dependent guanylate cyclase activity was stable for 10 h postmortem. Basal and dopamine-stimulated adenylate cyclase activity decreased markedly during the first 5 h. However, when measured in washed membrane preparations, these adenylate cyclase activities remained stable for at least 10 h. Therefore, the postmortem loss of a soluble activator, such as GTP, may decrease the adenylate cyclase activity in homogenates. These results are not consistent with an earlier suggestion that there is a postmortem degradation of the enzyme itself. Other kinetic parameters of dopamine-sensitive adenylate cyclase can also be measured independently of postmortem changes. Thus, it is possible to investigate kinetic parameters of dopamine-sensitive adenylate cyclase, guanylate cyclase, ATPase, and GTPase in human brain obtained postmortem.     

Thyroid membrane ADP ribosyltransferase activity. Stimulation by thyrotropin and activity in functioning and nonfunctioning rat thyroid cells in culture

Bovine thyroid membranes possess both ADP ribosyltransferase and NAD glycohydrolase activities with the same Km values for NAD and the same pH optima. In intact membranes, the ADP ribosyltransferase is limited in its extent by the amount of available membrane acceptor which can be ADP-ribosylated; in membranes solubilized with lithium diiodosalicylate, an artificial acceptor, L-arginine methyl ester, can be substituted to eliminate this limitation. The product of the ADP ribosyltransferase is a mono-ADP-ribosylated acceptor whether the intact or solubilized membrane provides the enzyme activity and whether membrane or exogenous acceptor, L-arginine methyl ester, is utilized. The intact membranes and the solubilized preparation also have an enzyme activity which can release AMP from the mono-ADP-ribosylated acceptor whether formed by the action of the membrane ADP ribosyltransferase or the A promoter of cholera toxin. The NAD glycohydrolase activity appears to represent the half-reaction of the ADP ribosyltransferase, i.e. an activity measurable substituting water for a membrane acceptor or L-arginine methyl ester. Membranes from functional rat thyroid cells in culture, i.e. cells chronically stimulated by thyrotropin and unresponsive to further additions of thyrotropin, have low ADP-ribosylation but high NAD glycohydrolase activities. In contrast, membranes from nonfunctional rat thyroid cells, i.e. cells unresponsive to thyrotropin, have high ADP-ribosylation and low NAD glycohydrolase activities. NAD hydrolysis by the NAD glycohydrolase activity cannot account for the low ADP-ribosylation activity in membranes from the functioning cells, and its low level of ADP-ribosylation can be eliminated by solubilizing the membranes and substituting an artificial acceptor, L-arginine methyl ester. The ADP ribosyltransferase activity of rat thyroid cell membrane preparations can be enhanced by thyrotropin in a dose-dependent manner but not by insulin, glucagon, hydrocortisone, adrenocorticotropin, or its glycoprotein hormone analog, human chorionic gonadotropin. It is thus suggested (i) that, in analogy to cholera toxin, thyrotropin-stimulated ADP-ribosylation may be important in the regulation of the adenylate cyclase response and (ii) that the level of membrane acceptor available for ADP-ribosylation may relate both to a stable "'activated" state of the adenylate cyclase system in cells chronically stimulated with thyrotropin and/or to a desensitized state with regard to a failure of more thyrotropin to elicit additional functional responses.     

Characterization and subcellular localization of nucleotide cyclases in calf thymus lymphocytes

The role of cyclic nucleotides in the regulation of lymphocyte growth and differentiation remains controversial, as an adequate characterization of the key enzymes, adenylate cyclase and guanylate cyclase, in the plasma membrane of lymphocytes is still lacking. In this study, calf thymus lymphocytes were disrupted by nitrogen cavitation and various cellular fractions were isolated by differential centrifugation and subsequent sucrose density ultracentrifugation. As revealed by the chemical composition and the activities of some marker enzymes, the plasma membrane fraction proved to be highly purified. Nucleotide cyclases were present in the plasma membranes in high specific activities, basal activities of adenylate cyclase being 13.7 pmol/mg protein per min and 34.0 pmol/mg protein per min for the guanylate cyclase, respectively. Adenylate cyclase could be stimulated by various effectors added directly to the enzyme assay, including NaF, GTP, 5'-guanylyl imidodiphosphate, Mn2+ and molybdate. Addition of beta-adrenergic agonists only showed small stimulating effects on the enzyme activity in isolated plasma membranes. Basal activity of adenylate cyclase as well as activities stimulated by NaF or 5'-guanylyl imidodiphosphate exhibited regular Michaelis-Menten kinetics. Activation by both agents only marginally affected the Km values, but largely increased Vmax. The activity of the plasma membrane-bound guanylate cyclase was about 10-fold enhanced by the nonionic detergent Triton X-100 and high concentrations of lysophosphatidylcholine, but was slightly decreased upon addition of the alpha-cholinergic agonist carbachol. Basal guanylate cyclase indicated to be an allosteric enzyme, as analyzed by the Hill equation with an apparent Hill coefficient close to 2. In contrast, Triton X-100 solubilized enzyme showed regular substrate kinetics with increasing Vmax but unaffected Km values. Thus the lymphocyte plasma membrane contains both adenylate cyclase and guanylate cyclase at high specific activities, with properties characteristic for hormonally stimulated enzymes.     

Cyclic nucleotides and platelet aggregation. Effect of aggregating agents on the activity of cyclic nucleotide-metabolizing enzymes.

The activities of adenylate and guanylate cyclase and cyclic nucleotide 3':5'-phosphodiesterase were determined during the aggregation of human blood platelets with thrombin, ADP, arachidonic acid and epinephrine. The activity of guanylate cyclase is altered to a much larger degree than adenylate cyclase, while cyclic nucleotide phosphodiesterease activity remains unchanged. During the early phases of thrombin-and ADP-induced platelet aggregation a marked activation of the guanylate cyclase occurs whereas aggregation induced by arachidonic acid or epinephrine results in a rapid diminution of this activity. In all four cases, the adenylate cyclase activity is only slightly decreased when examined under identical conditions. Platelet aggregation induced by a wide variety of aggregating agents including collagen and platelet isoantibodies results in the "release" of only small amounts (1-3%) of guanylate cyclase and cyclic nucleotide phosphodiesterase and no adenylate cyclase. The guanylate cyclase and cyclic nucleotide phosphodiesterase activities are associated almost entirely with the soluble cytoplasmic fraction of the platelet, while the adenylate cyclase if found exclusively in a membrane bound form. ADP and epinephrine moderately inhibit guanylate and adenylate cyclase in subcellular preparations, while arachidonic and other unsaturated fatty acids moderately stimulate (2-4-fold) the former. It is concluded that (1) the activity of platelet guanylate cyclase during aggregation depends on the nature and mode of action of the inducing agent, (2) the activity of the membrnae adenylate cyclase during aggregation is independent of the aggregating agent and is associated with a reduction of activity and (3) cyclic nucleotide phosphodiesterase remains unchanged during the process of platelet aggregation and release. Furthermore, these observations suggest a role for unsaturated fatty acids in the control of intracellular cyclic GMP levels.     

Guanylate cyclase from the rat renal medulla. Physical properties and comparison with adenylate cyclase.

Guanylate cyclase from the rat renal medulla is found in both the soluble and particulate fractions of the cell. Sucrose density gradient centrifugation and gel filtration in H2O and D2O indicate that the enzyme from the soluble cell fraction has the following properties: S20w, 6.3 S; Stokes radius, 54 A; partial specific volume, 0.75 ml/g; mass, 154,000 daltons; f/fo, 1.4; axial ratio (prolate ellipsoid), 7. The addition of 0.1% Lubrol PX to this fraction activates the enzyme and changes thartial specific volume, 0.74 ml/g; mass, 148,000 daltons; f/fo, 1.6; axial ratio (prolate ellipsoid), 11. These findings show that detergent activates the enzyme by changing its conformation and not simply by dispersing nonsedimentable membrane fragments. The dimensions of this guanylate cyclase in detergent are very similar to those of detergent-solubilized adenylate cyclase from the same tissue (Neer, E.J. (1974) J. Biol. Chem. 249, 6527-6531). Guanylate cyclase can be solubilized from the particulate cell fraction with 1% Lubrol PX but has properties quite different from those of the guanylate cyclase in the soluble cell fraction. It is a large aggregate with a value of S20,w of about 10 S, Stokes radius of 65 A, and a mass of approximately 300,000 daltons. However, the peaks of guanylate cyclase activity in column effluents and sucrose density gradients are very broad indicating a mixture of different size proteins. The conditions used to solubilize guanylate cyclase from the particulate fraction also solubilize adenylate cyclase, and the two activities can be separated on the same sucrose gradient. Studies of this sort require a rapid, accurate guanylate cyclase assay. We have developed an assay for guanylate cyclase activity which meets these criteria by adapting the competitive protein binding assay for guanosine cyclic 3':5' monophosphate originally described by Murad et al. (Murad, F., Manganiello, V., and Vaughn, M. (1971) Proc. Natl. Acad. Sci. U.S.A. 68, 736-739).     

Particulate guanylate cyclase and adenylate cyclase activities after activation with various agents in rabbit platelets. An ultracytochemical study

Summary The cytochemical localization of particulate guanylate cyclase and adenylate cyclase activities in rabbit platelets were studied after stimulation with various agents, at the electron microscope level. In the presence of platelet aggregating agents such as thrombin and ADP, the particulate reaction product of guanylate cyclase activity was detectable on plasma membrane and on membranes of the open canalicular system. In contrast, samples incubated with platelet-activating factor showed no activation of the cyclase activity. Atrial natriuretic factor stimulated the particulate guanylate cyclase. The ultracytochemical localization of this activated cyclase was the same as that of thrombin-or ADP-stimulated guanylate cyclase. Adenylate cyclase activity was studied in platelets incubated with prostaglandin E₁ plus or minus insulin. The enzyme reaction product was found at the same sites where guanylate cyclase was detected. Therefore guanylate and adenylate cyclase activities do not seem to be preferentially localised in platelet membranes.     

Guanylate cyclase. Subcellular distribution in cardiac muscle, skeletal muscle, cerebral cortex and liver.

1. Guanylate cyclase of every fraction studied showed an absolute requirement for Mn2+ ions for optimal activity; with Mg2+ or Ca2+ reaction was barely detectable. Triton X-100 stimulated the particulate enzyme much more than the supernatant enzyme and solubilized the particulate-enzyme activity. 2. Substantial amounts of guanylate cyclase were recovered with the washed particulate fractions of cardiac muscle (63-98%), skeletal muscle (77-93%), cerebral cortex (62-88%) and liver (60-75%) of various species. The supernatants of these tissues contained 7-38% of total activities. In frog heart, the bulk of guanylate cyclase was present in the supernatant fluid. 3. Plasma-membrane fractions contained 26, 21, 22 and 40% respectively of the total homogenate guanylate cyclase activities present in skeletal muscle (rabbit), cardiac muscle (guinea pig), liver (rat) and cerebral cortex (rat). In each case, the specific activity of this enzyme in plasma membranes showed a five- to ten-fold enrichment when compared with homogenate specific activity. 4. These results suggest that guanylate cyclase, like adenylate cyclase, and ouabain-sensitive Na+ + K+-dependent ATPase (adenosine triphosphatase), is associated with the surface membranes of cardiac muscle, skeletal muscle, liver and cerebral cortex; however, considerable activities are also present in the supernatant fractions of these tissues which contain very little adenylate cyclase or ouabain-sensitive Na+ + K+-dependent ATPase activities.     

Pivotal role of the RB family proteins in in vitro thyroid cell transformation

Rat thyroid differentiated cells (PC Cl 3) are an excellent model system with which to study the interaction between differentiation and cell transformation. We previously demonstrated that PC Cl 3 cells expressing the adenovirus E1A gene no longer depend on thyrotropin for growth and do not express thyroid differentiation markers. Here we show that an E1A mutant unable to bind the RB protein failed to transform the PC Cl 3 cells. Conversely, mutations in the E1A p300 interacting region did not affect its transforming ability. The pivotal role of RB family proteins in the thyroid cell transformation is supported by the thyrotropin independence induced by the E7 gene of human papilloma virus type 16, but not by a mutated form in the RB-binding region.     

Mn2+-sensitive, soluble adenylate cyclase in rat testis. Differentiation from other testicular nucleotide cyclases.

In subcellular fractions prepared from homogenate of adult rat testis adenylate cyclase (ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1) activity was found in the particulate, primarily 600 X g for 10 min, fractions, as well as in the cytosol. The properties of the adenylate cyclase in the cytosol differs substantially from the adenylate cyclase system associated with the 600 X g for 10 min particulate fraction. The cytosol enzyme, in contrast to the particulate adenylate cyclase, was found to be fluoride- and gonadotropin hormone-insensitive. The cytosol adenylate cyclase appears to be located in the germ cell while the particulate enzyme system in the non-germ cell component of the seminiferous tubules, The cytosol adenylate cyclase was found to be distinct also from the guanylate cyclase present in the rat testis cytosol. The adenylate cyclase appears to be located in the germ cell component while the guanylate cyclase, in the non-germ cell tubular component. Furthermore, it was found that the cytosol guanylate cyclase develops at an earlier stage of spermatogenesis, and precedes the development of the cytosol adenylate cyclase.     

Guanylate cyclase, a cell surface receptor

Guanylate cyclase appears to represent a central member of a diverse family of proteins involved in cell signaling mechanisms including the protein kinases, a low Mr ANP receptor, and possibly adenylate cyclase (based on limited sequence identity with the yeast enzyme). A membrane form of guanylate cyclase represents a new model for cell surface receptors, although such a model was once envisioned for adenylate cyclase (79). In original models for adenylate cyclase, hormone was thought to bind with either the enzyme or with an unknown protein to enhance cyclic AMP production (79). Guanylate cyclase appears to fall into the first adenylate cyclase model where binding of a ligand to an extracellular site on the enzyme transmits a signal to an intracellular catalytic site. The production of cyclic GMP, a second messenger, and of pyrophosphate are then increased. The protein tyrosine kinase family of receptors (80) and possibly another forthcoming family of cell surface receptors containing protein tyrosine phosphatase activity (81-83) contain a single transmembrane domain like guanylate cyclase. Furthermore, the protein tyrosine kinases are activated by ligand binding to the extracellular domain. However, the activation of guanylate cyclase, unlike these cell surface receptors, results in the formation of a low molecular weight second messenger.     

Stimulation of guanylate cyclase activity and reduction of adenylate cyclase activity by granulocyte-macrophage colony-stimulating factor in human blood neutrophils

Human neutrophils were incubated with granulocyte-macrophage (GM)-CSF and examined for changes in second messenger systems. Twofold increases in cGMP but not cAMP were measured after 5 to 20 min with 100 U/ml GM-CSF. Guanylate cyclase activities in membrane and cytosol fractions were increased to the same extent whether measured in the presence of Mg2+ or Mn2+, or in the cytosol with Mg2+ + N-methyl-N'-nitro-N-nitroso-guanidine. Kinetic studies of the cytosol enzyme showed no changes in the Km values for Mg2+ and Mn2+dependent guanylate cyclase activities (0.91 and 0.022 mM, respectively), whereas Vm values were increased after treating intact cells with GM-CSF. Two peaks of guanylate cyclase activity were observed, one at 10 and another at 60 min after adding 100 U/ml GM-CSF, whereas only one peak at 5 min occurred with 1 U/ml. Adenylate cyclase activity was reduced by nearly 50% after adding 100 U/ml GM-CSF for 10 to 30 min. These effects were also seen in the presence of several hormonal and nonhormonal adenylate cyclase stimulators. In contrast, small increases in adenylate cyclase activity occurred after adding 1 U/ml GM-CSF. In experiments to examine the pathway of guanylate cyclase activation by GM-CSF, we observed no changes in inositol phosphates, intracellular calcium ion, or cytosolic protein kinase C. The augmentation of chemotactic peptide-induced superoxide production by GM-CSF concentrations, may be related to the effects of the higher levels of GM-CSF to stimulate late increases in guanylate cyclase or decreases in adenylate cyclase.