Mechanism of adenylate cyclase activation by the rat lung cytoplasmic factors

Epinephrine, histamine and prostaglandin E₁ stimulated adenylate cyclase activity in lung membranes and their stimulation of the enzyme activity was completely blocked by propranolol, metiamide and indomethacin, respectively. A partially-purified activator from the adult rat lung also enhanced adenylate cyclase activity in membranes. However, stimulation of adenylate cyclase by the rat lung activator was not abolished by the above receptor antagonists. Further, epinephrine, NaF and Gpp(NH)p stimulated adenylate cyclase activity rather readily, whereas stimulation of the enzyme activity by the lung activator was evident after an initial lag phase of 10 min. Also, the lung activator produced additive activation of adenylate cyclase with epinephrine, NaF and Gpp(NH)p. These results indicate that the lung activator potentiates adenylate cyclase activity in membranes by a mechanism independent from those known for epinephrine, NaF and Gpp(NH)p. Incubation of lung membranes for 30 min at 40°C resulted in a loss of adenylate cyclase activation by NaF and Gpp(NH)p. Addition of the released proteins to the heat-treated membranes did not restore the enzyme response to these agonists. However, heat treatment of lung membranes in the presence of 2-mercaptoethanol or dithiothreitol prevented the loss of adenylate cyclase response to NaF and Gpp (NH)p. N-ethylmaleimide abolished adenylate cyclase activation by epinephrine, NaF, Gpp(NH)p and the lung activator. These results indicate that the sulfhydryl groups are important for adenylate cyclase function in rat lung membranes.Abbreviations Gpp(NH)p 5-Guanylimidodiphosphate     

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.     

Partial purification and properties of the cytoplasmic factors modulating adenylate cyclase activity in rat lung plasma membranes

The adult rat lung supernatant contains some factors which markedly enhance adenylate cyclase activity in membranes (Nijjar, M.S. (1979) Biochim. Biophys. Acta 584, 43–50). These factors were separated into two less active components (peaks 1 and 2) by DEAE-cellulose chromatography. However, their recombination restored the full activation of adenylate cyclase. Further purification and characterization of these factors revealed that the activation in peak 1 contained two proteins of low (14 500) and high (65 000) molecular weight whereas the activator in peak 2 contained only one protein of 65 000. The kinetics of adenylate cyclase activation revealed that both the K_m and V values were affected. The data also demonstrate that calmodulin was not involved in the cytoplasmic activation of adenylate cyclase in rat lungs.     

Calmodulin activation of rat lung adenylate cyclase is independent of the cytoplasmic factors modulating the enzyme.

Adenylate cyclase activity in the rat lung membranes washed with 150 microM-EGTA was stimulated by calmodulin in the presence of 100 microM-Ca2+. The calmodulin activation of the enzyme was concentration-dependent; however, at high concentrations the activation was diminished. Activation of adenylate cyclase by calmodulin was immediate, reversible and due to an increase in the Vmax. without apparent effect on the affinity of the enzyme for ATP. The rat lung supernatant produced additive activation of the adenylate cyclase that was already maximally stimulated by calmodulin, indicating that either calmodulin and cytoplasmic factors act at different sites on adenylate cyclase or different adenylate cyclases may be involved. The data further support our previous conclusion that calmodulin is not involved in the activation of adenylate cyclase by cytoplasmic factors in rat lungs.     

Further purification and partial characterization of the rat lung cytoplasmic factors modulating adenylate cyclase activity in plasma membrane

Summary The adult rat lung cytoplasm contains some factors which markedly stimulate adenylate cyclase activity in plasma membranes (Nijjar, M. S. Biochim. Biophys. Acta 584:43–50, 1979). Adenylate cyclase activator (ACA) was purified from rat lungs by DEAE-cellulose chromatography, preparative isoelectric focusing and by repeated high-performance liquid chromatography on a Sepharogel TSK 2000SW column. The final preparation showed about 200 fold purification in ACA activity over the original lung supernatant, and appeared to be homogeneous on the basis of its migration into a single band on SDS-polyacrylamide gel electrophoresis, and co-elution of ACA activity with protein from a gel exclusion column. ACA is an acidic (pl 4.8 ± 0.1), heat labile, monomeric protein of 40000 ± 2000 dalton molecular weight, and does not resemble calmodulin.     

Dopamine-inhibited adenylate cyclase in female rat adenohypophysis

A dopamine-inhibited adenylate cyclase has been demonstrated in anterior pituitary gland of adult female rats, lactating and not lactating. This inhibitory effect was completely GTP dependent. In contrast, in the adenohypophysis of male rats, dopamine had no detectable effect on adenylate cyclase activity. In female rats the inhibition of the enzyme appears mediated by specific dopaminergic receptors: the effect of dopamine was mimicked by the dopaminergic agonists apomorphine and the ergot derivative CH 29–717, while norepinephrine was much less potent. On the other hand, the dopaminergic antagonists trifluoperazine and sulpiride competitively antagonized the dopamine inhibition of the adenylate cyclase. The possibility that the dopamine-inhibited enzyme is located in mammotrophs appears supported 1) by its observation in the female rat pituitary, which contains this type of cells in much larger proportion than the male gland (33–38% vs. < 5%); 2) by the pharmacological similarity between the dopaminergic receptors mediating the adenylate cyclase inhibition (this work) and those regulating prolactin release (which have been characterized in previous studies). The well known inhibition of prolactin release brought about by dopamine could therefore be mediated, at least in part, by a decrease in the intracellular level of cAMP.     

Does glucocorticoid deprivation promote the expression of adenosine receptor-sites stimulating adenylate cyclase in rat adipocyte membranes?

The influence of N⁶-phenylisopropyladenosine (PIA) on adenylate cyclase was compared in adipocyte membranes from adrenalectomized and sham operated rats. In the presence of 100 mM sodium, 10 μM GTP and adenosine deaminase, PIA inhibited basal adenylate cyclase activity in sham rats, but elicited biphasic effects in adrenalectomized rats: at concentrations up to 10 nM, PIA first stimulated the enzyme, after which higher concentrations produced inhibition. In the presence of theophylline, these biphasic effects could not be observed. When isoproterenol maximally-stimulated adenylate cyclase was studied, the same biphasic effects of PIA were also observed in adrenalectomized rats, provided that no sodium was added in the assay, since with 100 mM sodium, only inhibition was seen. Finally, the stimulatory but not the inhibitory effect of PIA was prevented by glucocorticoid administration, a phenomenon which suggests that glucocorticoid deprivation may promote the expression of adenosine receptorsites which activate adenylate cyclase and which are normally absent, cryptic or unfunctional in normal adipocytes.     

Relationship between the cytoplasmic activator of adenylate cyclase and glycogen metabolism in rat lung

Summary The role of cytoplasmic activator of adenylate cyclase in rat lung metabolism was investigated. Mouse adrenal tumor (MAT) cells undergo differentiation in response to choleratoxin which acts through cyclic AMP. The activator of adenylate cyclase from rat lung also produced cyclic AMP in a disrupted MAT cell preparation. However, unlike choleratoxin, it did not induce MAT cell differentiation in whole cells. These results suggest impermeability of MAT cells, and possibly other cells, to the activator. Thus, means of altering activator activity in lung cytoplasm were sought, and changes in activator activity were related to lung glycogen. Adrenalectomy (ADX) in rats led to a reduction in activator activity that was accompanied by an elevation in lung glycogen. Dexamethasone treatment of adrenalectomized rats reversed both of these effects. Streptozotocin-induced diabetes in rats elevated activator activity and lowered lung glycogen. Insulin treatment of the diabetic rats restored activator activity to the normal control values. Preweaning of rats on day 16 instead of day 22 increased activator activity on the 19th day over the controls and there was a concomitant decrease in lung glycogen. Feeding the separated pups with homogenized milk restored glycogen and activator activity to the control values. These results indicate that activator activity in rat lung cytoplasm was dependent on the circulating levels of cortisol and insulin, and that there appeared to be an inverse relationship between activator activity and glycogen level in rat lungs.     

Presence of vasoactive intestinal peptide receptors coupled to adenylate cyclase in rat lung membranes

(1) The binding of 125I-labelled vasoactive intestinal peptide (VIP) to a particulate fraction from rat lung was rapid, temperature dependent, saturable and specific. This process was also reversible and 125I-labelled VIP dissociation was accelerated by guanine triphosphate nucleotides. The curves describing the inhibition of tracer binding by peptides of the VIP-secretin family suggested the presence of at least two classes of VIP receptor: a "high-affinity' type with decreasing affinity for VIP in the order: VIP = [Val5]secretin greater than [Ala4, Val5]secretin; and a "low-affinity type' with decreasing affinity for VIP in the order: VIP greater than [Val5]secretin greater than [Ala4, Val5]secretin = secretin greater than [Ala4]secretin. (2) VIP and related peptides stimulated the adenylate cyclase activity of the same lung membrane preparation more efficiently than beta-adrenergic agonists and prostaglandins E1 and E2. The dose-effect curves of stimulation of adenylate cyclase by VIP and parent peptides were also compatible with the existence of two classes of VIP receptor, the relative peptide potencies being identical with their ability to compete with 125I-labelled VIP for binding.     

Multiple localizations of adenylate cyclase in rat hippocampus

Summary A new method for the histochemical demonstration of adenylate cyclase activity, introduced and biochemically tested by Poeggel et al. (1981a), was employed in nervous tissue. Using this method a multiple pattern of activity was detectable. Activity occurs in nervous as well as glial elements. Biochemical results and physiological conclusions could be confirmed by ultrahistochemical visualization of adenylate cyclase activity in nervous tissue. The specificity of the reaction is controlled by a number of variations of the incubation methods.     

II. Beta-adrenergic receptors and catecholamine sensitive adenylate cyclase in the developing rat lung

β-Adrenergic receptors were identified in membrane fractions of fetal and postnatal rat lung with the β-adrenergic antagonist (?)?[³H] dihydroalprenolol, (?)?[³H] DHA. β-Receptor number (Bmax) increased 11-fold from day 18 of gestation to day 28 of postnatal life, 46±7 to 491±69 femtomole·mg^?1 protein. Neither the K_D, approximately 0.8nM for [³H]DHA, nor the β-adrenergic subtype changed with age. Classical agonists competed for the β-receptor with properties characteristic of β₂-adrenergic binding. Analysis of the inhibition of receptor binding by selective β-adrenergic agents demonstrated approximately 75% β₂ and 25% β₁ β-adrenergic subtypes in fetal rat lung membranes. The increase in β-adrenergic receptor during development was associated with adenylate cyclase activity which was sensitive to catecholamines at all ages studied, supporting the possible role of the β-adrenergic receptor system in the postnatal regulation of pulmonary function.     

Hormonal control of FAT cells adenylate cyclase

Summary Fat cells were preincubated for 2 h in the presence and absence of growth hormone (GH) and Dexamethasone (Dex) before the addition of increasing concentrations of either epinephrine, theophylline or glucagon and final incubation of the cells for an additional 5 minutes. GH and Dex increased by 85%, 28% and 72%, respectively, the cAMP levels reached in the sole presence of 10^–5 m epinephrine, 10^–2 m theophylline or 5 × 10^–5 m glucagon. An adenylate cyclase particulate preparation shows that epinephrine decreases Km from 2mm to 0.6mm and increases V_max and the strength of interaction value (n) from 0.91 to 1.75.     

Beta-adrenergic receptor-linked adenylate cyclase in rat posterior pituitary

A specific β-adrenergic-stimulated cyclic AMP generating system has been evidenced in rat posterior pituitary. This is clearly demonstrated by: 1) the adenylate cyclase (AC) affinity for stimulants was in the order ISO > NA > DA, and 2) propranolol, a specific β-adrenergic receptor blocker, was the only antagonist of the system. Clonidine and apomorphine were completely inactive, thus excluding an α-adrenergic and/or dopaminergic component in this AC system. Our data also indicate that dopaminergic receptors present in both pituitary lobes are not coupled to an AC.     

Failure to demonstrate rat hippocampus adenylate cyclase

Summary The fate of ATP exposed to rat hippocampal extracts was investigated after their separation by a microdisc electrophoresis technique. It could be demonstrated that the histochemical adenylate cyclase procedure using ATP as substrate is not suitable for specific localization of the enzyme, since other ATP hydrolysing enzymes were also able to convert ATP unless the concentrations of inhibitors reached 1 mM (ouabain) and 40 mM (NaF). With a prolonged incubation time of 18 h further substrate splitting protein zones could be revealed, possibly reflecting activities of enzymes involved in the hydrolysis of degradation products of ATP.Supported by Ministerium für Hoch- und Fachschulwesen der DDR     

Effects of premature weaning and diet on lung growth and appearance of adenylate cyclase activator in rat lung

Early weaning of rat pups on day 16 to semi-ground Purina chow food and drinking water, ad libitum, delayed growth of body and lungs, and the appearance of adenylate cyclase activator (ACA) in lung after day 22. However, early weaning of pups to either milk or a gel diet containing semi-ground Purina chow food, agarose gel, water (30:1:69, w/w), and drinking water, restored lung and body growth and the appearance of ACA to control values. Early weaning of pups to dry semi-ground Purina chow food and drinking water also induced a transient rise in ACA on day 19. This early rise in ACA was completely absent in pups weaned on day 16 to milk, whereas it persisted in pups weaned similarly to a gel diet. Interestingly, lung glycogen decreased on day 19 in pups weaned early to dry semi-ground Purina chow food without (group 2) or with triiodothyronin administration (group 3), and on day 25 after normal weaning on day 22 (Nijjar, M.S. Biochim. Biophys. Acta 586: 464–472,1979). These data indicate 1) that reduced food intake (starvation) in pups weaned on day 16 to dry semi-ground Purina chow food was responsible for the delayed growth of body and lung, and the delayed appearance of ACA in lung after day 22, and 2) that a change in diet from milk to Purina chow food and associated alterations in hormones, possibly cortisol and insulin, were responsible for the appearance of ACA in rat lung. It would appear that reduced intake of food (starvation) and associated changes in hormones in rat pups at weaning induce ACA in lungs, stimulating adenylate cyclase to produce more cyclic AMP. A rise in cyclic AMP may initiate a cascade of enzymic reactions resulting in enhanced metabolism of glycogen to intermediates which are utilized for the production of energy during the time that the exogenous source of energy i.e. food, is restricted.     

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.     

Ultracytochemical localization of adenylate cyclase activity in rat thymocytes

Summary Ultrastructural localization of adenylate cyclase (AC) activity was investigated in suspensions of unfixed isolated rat thymocytes using a medium containing 0.6 mM 5-adenylylimidodiphosphate (AMP-PNP) as a substrate, 10 mM MgSO₄ as an activator, 5 mM theophylline as an inhibitor of 3,5-AMP-phosphodiesterase and 2 mM lead nitrate as a capturing agent. AC activity was demonstrated in plasma membrane, perinuclear space, endoplasmic reticulum, Golgi complex, centriole microtubules and mitochondria. AC was activated with 10^–4 M adrenalin in the presence of 5-guanylylimido-diphosphate (GMP-PNP) as well as with 10^–2 M NaF. In the cells incubated in a medium devoid of theophylline and containing 5-AMP instead of AMP-PNP, 5-nucleotidase activity was observed in the same cell structures as AC activity. Hydrolysis of 5-AMP in the nucleus was much stronger than that of AMP-PNP. 10 mM NaF markedly inhibited hydrolysis of 5-AMP in all cell structures. No staining was observed with 2 mM -glycerophosphate as a substrate. Incubation of unfixed thymocytes in media containing AMP-PNP, 5-AMP or p-nitrophenyl phosphate, but not -glycerophosphate, induced both in the nucleus and in the cytoplasm in some cells an appearance of a transitory reticular formation consisting of about 30 nm thick strands which could penetrate the nuclear envelope and plasma membrane and form connections with adjacent cells. The transitory reticular formation seems to belong to the cytoskeleton and to be involved in cell aggregation.     

Subcellular location of adenylate cyclase in rat cardiac muscle

Crude homogenates of rat cardiac muscle were fractionated in order to examine the subcellular location of adenylate cyclase in this tissue. The fractionation procedure employed differential centrifugation of homonized material, followed by collagenase treatment, centrifugation on a discontinuous sucrose density gradient and extraction with 1 M KCl. The particulate fraction obtained by this procedure contained a high specific activity and yield of adenylate cyclase, moderate levels of mitochondria and low levels of sarcoplasmic reticulum and contractile protein as judged by marker enzyme activities. Adenylate cyclase was purified 20-fold with a 33% yield from the crude homogenate, while mitochondrial, sarcoplasmic reticulum and contractile protein yields were 5, 0.4 and 0.7% respectively. The membrane fractions prepared in this manner were examined by sodium dodecyl sulfate · gel electrophoresis.Adenylate cyclase copurified with ouabain-sensitive (Na⁺ + K⁺)-ATPase, a plasma membrane marker enzyme, and not with Ca²⁺-accumulating activity, which is associated with the sarcoplasmic reticulum. The distribution of marker enzyme activities indicates that heart adenylate cyclase is not located in the sarcoplasmic reticulum but is localized predominantly, if not exclusively, in the plasma membrane.