PACAP activated adenylate cyclase in human sweat glands. An ultracytochemical study

The ultracytochemical localization of adenylate cyclase (AC) was studied after stimulation with pituitary adenylate cyclase activating peptide (PACAP) in human sweat glands. PACAP stimulated AC in both eccrine and apocrine glands. In the secretory cells, enzymatic activity was associated with membranes involved in the secretory mechanism. In both glands, the cells of the excretory duct and myoepithelial cells presented AC activity. These localizations of enzymatic activity suggest a role for PACAP in regulating glandular secretion.     

Evidence for particulate guanylate cyclase in rat kidney after stimulation by atrial natriuretic factor. An ultracytochemical study

Summary Cytochemical localization of particulate guanylate cyclase (GC) in rat kidney, after stimulation with atrial natriuretic factor (ANF), was studied by electron microscopy. In the renal corpuscle GC reaction product was localized on podocytes. Other segments of the nephron that showed ultracytochemical evidence of GC activity were the proximal convoluted tubule, the thick ascending limb of the loop of Henle and the collecting tubule. All GC positivity was associated with plasma membranes. Samples incubated in basal conditions (without ANF) did not reveal any GC reaction product. These results indicate that ANF is a strong activator of particulate GC. Our data also suggests that, through the enzyme, ANF acts directly on epithelial cells of tubules where Na⁺ reabsorption occurs. This is in agreement with the hypothesis that ANF has a direct tubular effect on natriuresis.     

Reversible activation of soluble guanylate cyclase by oxidizing agents.

Soluble guanylate cyclase of human platelets was stimulated by thiol oxidizing compounds like diamide and the reactive disulfide 4, 4'-dithiodipyridine. Activation followed a bell-shaped curve, revealing somewhat different optimum concentrations for each compound, although in both cases, higher concentrations were inhibitory. Diamide at a concentration of 100 microM transiently activated the enzyme. In the presence of moderate concentrations of diamide and 4,4'-dithiodipyridine, causing a two- to fourfold activation by themselves, the stimulatory activity of NO-releasing compounds like sodium nitroprusside was potentiated. In contrast, higher concentrations of thiol oxidizing compounds inhibited the NO-stimulated activation of soluble guanylate cyclase. Activation of guanylate cyclase was accompanied by a reduction in reduced glutathione and a concomitant formation of protein-bound glutathione (protein-SSG). Both compounds showed an activating potency as long as reduced glutathione remained, leading to inhibition of the enzyme just when all reduced glutathione was oxidized. Activation was reversible while reduced glutathione recovered and protein-SSG disappeared. We propose that diamide or reactive disulfides and other thiol oxidizing compounds inducing thiol-disulfide exchange activate soluble guanylate cyclase. In this respect partial oxidation is associated with enzyme activation, whereas massive oxidation results in loss of enzymatic activity. Physiologically, partial disulfide formation may amplify the signal toward NO as the endogenous activator of soluble guanylate cyclase.     

Modifications of adenylate and guanylate cyclase activities during multiplication of KB cells.

Adenylate and guanylate cyclase activities do not vary in concert during the multiplication of KB cells. Adenylate cyclase activity is low and slightly increases at cell confluency, guanylate cyclase activity, great in sparce cells, decreases during cell multiplication period. These variations are not caused by a modification of catalytic sites because the apparent Km for ATP or GTP is not changed, but by a modification of the dependance on Mg⁺⁺ or Mn⁺⁺ ions. Fresh serum increases guanylate cyclase activity but does not affect adenylate cyclase.     

Preparation of renal cortex basal-lateral and bursh border membranes. Localization of adenylate cyclase and guanylate cyclase activities.

Luminal brush border and contraluminal basal-lateral segments of the plasma membrane from the same kidney cortex were prepared. The brush border membrane preparation was enriched in trehalase and gamma-glutamyltranspeptidase, whereas the basal-lateral membrane preparation was enriched in (Na+ + K+1)-ATPase. However, the specific activity of (Na+ + K+)-ATPase in brush border membranes also increased relative to that in the crude plasma membrane fraction, suggesting that (Na+ + K+)-ATPase may be an intrinsic constituent of the renal brush border membrane in addition to being prevalent in the basal-lateral membrane. Adenylate cyclase had the same distribution pattern as (Na+ + K+)-ATPase, i.e. higher specific activity in basal-lateral membranes and present in brush border membranes. Adenylate cyclase in both membrane preparations was stimulated by parathyroid hormone, calcitonin, epinephrine, prostaglandins and 5'-guanylylimidodiphosphate. When the agonists were used in combination enhancements were additive. In contrast to the distribution of adenylate cyclase, guanylate cyclase was found in the cytosol and in basal-lateral membranes with a maximal specific activity (NaN3 plus Triton X-100) 10-fold that in brush border membranes. ATP enhanced guanylate cyclase activity only in basal-lateral membranes. It is proposed that guanylate cyclase, in addition to (Na+ + K+)-ATPase, be used as an enzyme "marker" for the renal basal-lateral membrane.     

Particulate and soluble adenylate cyclase activities of mouse male germ cells

Germ cells from the mouse testis possess both a particulate and a soluble form of adenylate cyclase (EC 4.6.1.1). Germ cell adenylate cyclase activity is Mn⁺⁺ dependent and is not stimulable with either NaF or 5′guanylylimidodiphosphate. Both particulate and soluble adenylate cyclase specific activities increase as germ cells progress through their differentiative stages, but epididymal spermatozoa seem to lack a significant amount of soluble activity. Somatic cells of the seminiferous tubule possess only a membrane bound activity, which is Mg⁺⁺ and Mn⁺⁺ dependent, NaF and 5′guanylylimidodiphosphate stimulable. It is suggested that germ cell adenylate cyclases represent incomplete forms of the enzyme, devoid of regulative subunits.     

Preparation of renal cortex basal-lateral and brush border membranes. Localization of adenylate cyclase and guanylate cyclase activities

Luminal brush border and contraluminal basal-lateral segments of the plasma membrane from the same kidney cortex were prepared. The brush border membrane preparation was enriched in trehalase and γ-glutamyltranspeptidase, whereas the basal-lateral membrane preparation was enriched in (Na⁺ + K⁺)-ATPase. However, the specific activity of (Na⁺ + K⁺)-ATPase in brush border membranes also increased relative to that in the crude plasma membrane fraction, suggesting that (Na⁺ + K⁺)-ATPase may be an intrinsic constituent of the renal brush border membrane in addition to being prevalent in the basal-lateral membrane. Adenylate cyclase had the same distribution pattern as (Na⁺ + K⁺)-ATPase, i.e. higher specific activity in basal-lateral membranes and present in brush border membranes. Adenylate cyclase in both membrane preparations was stimulated by parathyroid hormone, calcitonin, epinephrine, prostaglandins and 5′-guanylylimidodiphosphate. When the agonists were used in combination enhancements were additive. In contrast to the distribution of adenylate cyclase, guanylate cyclase was found in the cytosol and in basal-lateral membranes with a maximal specific activity (NaN₃ plus Triton X-100) 10-fold that in brush border membranes. ATP enhanced guanylate cyclase activity only in basal-lateral membranes. It is proposed that guanylate cyclase, in addition to (Na⁺ + K⁺)-ATPase, be used as an enzyme “marker” for the renal basal-lateral membrane.     

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.     

Differences of adenylate cyclase localization in guinea-pig peritoneal macrophages under different physiological conditions: An ultracytochemical study

Summary The ultrastructural localization of adenylate cyclase activity has been investigated in unfixed guinea-pig peritoneal macrophages in different physiological states (such as suspension, adhesion and phagocytosis) using a medium containing 5-adenylyl-imidodiphosphate (AMP-PNP) as the substrate.Adenylate cyclase activity was observed in cytoplasmic vacuoles of macrophages in suspension; in the perinuclear space, endoplasmic reticulum, Golgi complex and pseudopods of adherent macrophages; and surrounding phagocytosed polystyrene particles. The activity was inhibited by Alloxan added to the incubation medium and no staining was observed when AMP-PNP was omitted from the medium.The segregation of this enzyme to phagocytic vacuoles and pseudopods may have significant implications in understanding cyclic nucleotide function in adhesion and phagocytosis.     

Rhizobium meliloti adenylate cyclase is related to eucaryotic adenylate and guanylate cyclases.

A gene from Rhizobium meliloti coding for an adenylate cyclase was sequenced, and the deduced protein sequence was compared with those of other known adenylate cyclases. No similarity could be detected with the procaryotic counterparts. However, striking similarity was found with the catalytic region of Saccharomyces cerevisiae adenylate cyclase, the cytoplasmic domains of bovine adenylate cyclase, and two mammalian guanylate cyclases. The gene was fused to the enteric beta-galactosidase, and the chimeric protein was purified by affinity chromatography. This fusion protein was found to direct the synthesis of cyclic AMP in vitro. This activity was strongly inhibited by the presence of GTP, but no cyclic GMP synthesis could be detected in conditions permitting cyclic AMP synthesis.     

Growth-dependent changes of guanylate and adenylate cyclase activities in cilia and cell bodies of Tetrahymena pyriformis

The calmodulin-dependent guanylate cyclase of Tetrahymena pyriformis was shown previously to be localized in surface membranes (ciliary and pellicular membranes) (Kudo, S, Nakazawa, K, Nagao, S & Nozawa, Y, Japan j exp med 52 (1952) 193) [21], whereas in a recent report Schultz et al, (Schultz, J E, Schonefeld, U & Klumpp, S, Eur j biochem 137 (1983) 89) [12] demonstrated the localization of this enzyme in ciliary membrane, arguing against its presence in pellicular membrane. To examine the discrepancy, the activities of guanylate and adenylate cyclases were examined in cilia and cell bodies of Tetrahymena pyriformis during transition from early log to stationary growth phase. The guanylate cyclase activity in the cell bodies increased significantly with growth of age, while in cilia the activity was rather consistent. In contrast, adenylate cyclase did not show any growth-dependent activity changes in both cilia and cell bodies. The increase of guanylate cyclase activity was not related to the increase of its activator calmodulin, because the change in enzyme activity could not be negated by addition of a saturating amount of calmodulin. These results suggest that the content of guanylate cyclase itself would be increased in the cell bodies during growth.     

Apparent activation of rabbit lung membrane adenylate cyclase by cytosolic proteins possessing adenylate kinase activity.

Lung cytosolic fraction (23500 x g supernatant) activates cAMP synthesis by lung membrane adenylate cyclase (AC). 23 kDa and 29 kDa proteins were isolated from rabbit lung cytosolic fraction in a homogeneous state, as 'activators' of lung membrane AC. Both of these proteins possess high adenylate kinase (AK) activity and are able to mimic the 'activating' effect of lung cytosol on the lung membrane AC in the standard incubation mixture devoid of adenylate kinase. The activating effect is abolished in the presence of adenylate kinase inhibitor DAPP and after heat- or trypsin-treatment of the cytosolic fraction. Commercial adenylate kinase or nonionic detergent Lubrol PX activate cAMP synthesis by lung membrane AC in a similar manner to that of cytosolic fraction. In the presence of commercial adenylate kinase or Lubrol PX no activating effect of the cytosolic fraction on lung membrane AC is revealed. The ability of cytosolic fraction, commercial adenylate kinase, Lubrol PX or purified 23 kDa and 29 kDa proteins to activate cAMP synthesis by lung membrane AC correlates with their ability to support the constant ATP (AC substrate) concentration in the AC assay mixture. Our data indicate that 'activation' of lung membrane AC in the presence of cytosolic fraction may be produced by cytosolic adenylate kinase activity which regenerates ATP from AMP in the presence of creatine kinase and creatine phosphate providing the substrate for cAMP synthesis by AC.     

From adenylate cyclase to guanylate cyclase. Mutational analysis of a change in substrate specificity.

Adenylate and guanylate cyclases, having different but related substrates, are a paradigm for the study of substrate discrimination. A prokaryotic adenylate cyclase gene, phylogenetically related to eukaryotic counterparts, was screened for mutants remodelling the enzyme's specificity. In a first step, a mutant was selected displaying a significant level of guanylate cyclase activity. This was due to a point mutation destroying most of the adenylate cyclase activity. A second selection step restored most of the original activity. This resulted from an additional mutation in the same region, thus permitting the first identification of a functional domain in adenylate and guanylate cyclases.     

Ultrastructural demonstration of guanylate cyclase in rat lung after activation by ANF.

We studied the cytochemical localization of particulate guanylate cyclase (GC) activity after stimulation with atrial natriuretic factor (ANF) in rat lung, at the electron microscope level. Samples incubated in the absence of ANF did not reveal any GC reaction product. These results indicate that ANF is a strong activator of the enzyme in this organ. In intrapulmonary bronchi, the ANF-activated GC reaction product was localized on mucus secreting goblet cells. GC was seen in bronchioles, alveoli and capillaries. All of the GC reaction product was associated with plasma membranes of Clara cells, of great alveolar cells and of endothelial cells in alveolar capillaries. Our data suggest that, by activation of particulate GC, ANF acts directly on cells where Na+ reabsorption occurs.     

Improved two-step method for the assay of adenylate and guanylate cyclase.

A two-step assay for adenylate and guanylate cyclase is described utilizing α-³²P-labeled ATP or GTP as substrate and involving purification of the resulting ³²P-labeled cAMP or cGMP by sequential chromatography on Dowex 50 and alumina. The Dowex 50 chromatography is performed in acid, 50 mm HCl for cGMP and 10 mm HClO₄ for cAMP, and achieves complete separation from the radiochemical impurities in the substrate which are responsible for blank. The cAMP or cGMP peaks are collected directly onto alumina columns and, under acid conditions, are completely retained by the alumina. After washing the alumina with water, the ³²P-labeled cAMP or cGMP is eluted with 0.2 m imidazole buffer and counted. The method delivers blanks amounting to .0005% of the substrate radioactivity, high recoveries, and excellent reproducibility.     

Specificity for guanine nucleotide activation and stabilization of rabbit cardiac adenylate cyclase

These studies examined the structural specificity for guanine nucleotide-facilitated hormonal activation and guanine nucleotide stabilization of cardiac adenylate cyclase. 1. The phosphonate analogues of GTP, p[CH(2)]ppG (guanosine 5'-[betagamma-methylene]-triphosphate) and pp[CH(2)]pG (guanosine 5'-[alphabeta-methylene]triphosphate), were the most effective activators of adenylate cyclase. Other nucleotides producing significant activation (P<0.01) were, in decreasing order of activation: ITP, GDP, GMP, GTP, XTP, CTP, p[NH]ppG (guanosine 5'-[betagamma-imido]triphosphate), dGTP and 2'-O-methyl-GTP. Guanosine, cyclic GMP, UTP and ppppG (guanosine tetraphosphate) had no effect, and 7-methyl-GTP caused a decrease in the activity. 2. Preincubation of membranes at 37 degrees C for 15min before assay at 24 degrees C produced an 80% decrease in adenylate cyclase activity, and preincubation with p[CH(2)]ppG and pp[CH(2)]pG protected and resulted in a net increase in activity. Other nucleotides that completely or partially preserved activity in decreasing order of effectiveness were p[NH]ppG, GDP, GTP, dGTP, ITP, ppppG, 2'-O-methyl-GTP, GMP, CTP and XTP. Several compounds had no effect, including guanosine, cyclic GMP and UTP, whereas preincubation with 7-methyl-GTP produced a further decrease (P<0.05) in activity. 3. The concentration-dependence for activation and stabilization by the naturally occurring guanine nucleotides was examined in the absence of a regenerating system and revealed GMP to have no stabilizing effect and to be less potent than either GDP or GTP in activating adenylate cyclase. 4. A significant correlation (r=0.90) was found between the properties of activation and stabilization for the compounds examined. These findings are consistent with there being a single nucleotide site through which both the activation and stabilization of adenylate cyclase are mediated.