Adenylate cyclase activity in lymphocyte subcellular fractions. Characterization of a nuclear adenylate cyclase.

Nuclei from purified human peripheral lymphocytes were prepared by incubations with Triton X-100 to disrupt the cells, followed by sucrose-density gradient centrifugation. The nuclei were pure as judged by phase-contrast microscopy and had low contents of non-nuclear marker enzymes. In addition, nuclei prepared from lymphocytes surface-labelled with 125I had only 2-7% of the radioactivity bound to intact lymphocytes. At 3.3 mM-Ca2+ and 100 micronM-ATP a fluoride-sensitive adenylate cyclase was demonstrated in nuclei prepared in 0.2% Triton X-100 or 0.33% Triton X-100. There was linear accumulation of cyclic AMP for 10 min in both preparations. The apparent Km for ATP was 90 micronM. Adenylate cyclase activity was augmented by 1.0 mM-Mn2+ and inhibited at higher concentrations. Ca2+ showed two peaks of stimulation, at 1.0-2.5 mM- and above 10 mM-Ca2+. Mg2+ was inhibitory at all concentrations. EDTA OR EGTA only slightly decreased adenylate cyclase activity, suggesting that another metal ion may be necessary for activity. Adenylate cyclase activity was stimulated by 10mM-isoproterenol and 10 micronM-adrenaline in the presence of a phosphodiesterase inhibitor. Phytohaemagglutinin and prostaglandin E1 alone or in combination with isoproterenol had no effect on nuclear adenylate cyclase activity in either nuclei preparation. These results indicate that human lymphocyte nuclei contain one or several adenylate cyclases which differ from adenylate cyclases found in other subcellular fractions of these cells with regard to their bivalentcation requirements and responsiveness to pharmacological agents.     

Biological activities of catfish glucagon and glucagon-like peptide

The ability of catfish glucagon and glucagon-like peptide to bind and activate mammalian glucagon receptors was investigated. Neither catfish peptide binds to glucagon receptors of rat liver, hypothalamus or pituitary. Neither stimulates adenylate cyclase activity in liver membranes. Catfish glucagon fails to activate adenylate cyclase in hypothalamic or pituitary membranes in contrast to mammalian glucagon. However, catfish glucagon-like peptide does stimulate hypothalamic and pituitary adenylate cyclase (EC50 approximately 1 pM) possibly through mammalian glucagon-like peptide receptors.     

Plasma hyperosmolality increases G protein and 3',5'-cyclic adenosine monophosphate synthesis in the paraventricular and supraoptic nuclei

Hyperosmotic stimuli produce profound changes in cellular morphology and biosynthetic activities within the hypothalamic paraventricular and supraoptic nuclei (SON) of the rat. The mechanisms by which osmoreceptive signals are transduced within these nuclei are poorly understood. We examined several components of the cAMP-associated second messenger system after giving rats 2% saline to drink for one week, a strong hyperosmotic stimulus. We found that mRNA levels for both the stimulatory and inhibitory guanine-nucleotide binding protein alpha-subunits were increased in the paraventricular nucleus and SON. In the SON, these changes were accompanied by increased basal cAMP levels, cholera toxin-stimulated adenylate cyclase, and Gs alpha. Our results suggest that Gs alpha levels are not saturated with respect to adenylate cyclase coupling and that osmoreception activates the cAMP second messenger system.     

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.     

The purification and characterization of plasma membranes and the subcellular distribution of adenylate cyclase in mouse parotid gland.

1. Plasma membranes have been purified 17-fold from mouse parotid gland homogenates prepared in hypertonic sucrose media using differential centrifugation. The method is fast and simple. The membranes were characterised by electron microscopy, enzyme composition and chemical composition. Further purification was achieved by isopycnic centrifugation in discontinuous sucrose gradients. 2. The purified membranes contain an adenylate cyclase activity which is stimulated by isoproterenol and fluoride. Only 50% of the total adenylate cyclase activity sedimented in the plasma membrane fraction. The rest of the activity resided in the crude nuclear and mitochondrial pellets. However, this adenylate cyclase activity was not associated with these organelles but with membrane fragments in the pellets. Purified nuclei did not contain adenylate cyclase activity. 3. Adenylate cyclase activity was also localised by electron microscopic cytochemistry. Besides being found at the plasma membrane, large amounts of adenylate cyclase were found in a small proportion of the vesicles within the acinar cells, which appeared to be secondary lysosomes. 4. Adenylate cyclase activities, under standard assay conditions, are proportional to the time of incubation and the concentration of enzyme. The enzyme requires both Mg-2+ and CA-2+ for activity. Isoproterenol increased activity 2-fold and this increase is abolished by beta-adrenergic blocking agents.     

Ultracytochemical localizations of adenylate cyclase, guanylate cyclase and cyclic 3',5'-nucleotide phosphodiesterase activity on the trophoblast in the human placenta. Direct histochemical evidence

Ultracytochemical localizations of cyclic nucleotide-metabolizing enzymes, namely adenylate cyclase (AC), guanylate cyclase (GC) and cyclic 3',5'-nucleotide phosphodiesterase (PDE), have been demonstrated in the human term placenta. AC activity was found positive on the basal plasma membrane of the syncytiotrophoblast and on the pinocytotic vesicle of the fetal capillary endothelial cell. GC activity was observed to be strong on the plasma membrane of the microvilli of the syncytiotrophoblast. The cAMP PDE activity was shown positive both on the basal plasma membrane and on the microvillous membrane, while cGMP PDE activity was exclusively confined to the microvilli of the syncytiotrophoblast. These observations suggest that the syncytiotrophoblast plays an important role in the cyclic nucleotide metabolism in the human term placenta and that there might be significant functional differences between its basal plasma membrane and its microvillous membrane.     

Desensitization of beta-adrenergic stimulated adenylate cyclase in turkey erythrocytes.

Desensitization of catecholamine stimulated adenylate cyclase (AC) activity is demonstrated in membranes derived from turkey erythrocytes pre-treated with isoproterenol. Membranes from desensitized cells had a loss in maximal catecholamine stimulated adenylate cyclase activity of 104 +/- 13 (pmols/mg protein/10', p less than .001) compared with controls. When adenylate cyclase was maximally stimulated with NaF or Gpp(NH)p, the decrements were 84 +/- 19 (p less than .005) and 92 +/- 32 (p less than .05) pmol/mg protein/10' respectively. There was no change in beta-adrenergic receptor number in membranes derived from treated cells. While the molecular mechanism accounting for the desensitization is uncertain, the data is consistent with the hypothesis that there is a lesion distal to the beta-adrenergic receptor, possibly involving the nucleotide site or the catalytic subunit of adenylate cyclase, causing the desensitization in the isoproterenol treated cells.     

Activation of fluoride-stimulated adenylate cyclase by phospholipase A2 in the caudate nucleus of the rat brain

Phospholipase A₂ (PLA₂) increases adenylate cyclase (AC) activity in the rat caudate nucleus in a dose-dependent manner. After maximal stimulation by fluoride, PLA₂ treatment further increases AC activity 2.4 fold. Adenylate cyclase activity is maximal after 45% hydrolysis of the phospholipids. Of the products of PLA₂ treatment only lysophosphatidylcholine (LPC) produces such an increase in AC activity. In contrast to PLA₂ treatment, LPC solubilizes the enzyme, decreases the Km value for ATP, and requires much larger amounts of LPC than that produced by lipase treatment. After maximal stimulation with fluoride and PLA₂, removal of most of the LPC does not reduce the activity of adenylate cyclase. These findings suggest that removal of membrane lipid rather than generation of LPC is responsible for the activation of brain adenylate cyclase by phospholipase A₂.     

An impaired response of adenylate cyclase to stimulation by epinephrine in adipocyte plasma membranes from genetically obese mice (ob/ob).

The present studies have established that there is an impaired response to epinephrine of the adenylate system in adipocyte preparations from obese hyperglycemic mice as compared to their thin littermates. In contrast, membrane preparations from both groups of animals were found to exhibit a similar response to fluoride ion. The response of adenylate cyclase to epinephrine was enhanced to a similar extent by increasing the ATP concentration in adipocyte plasma membranes from the two groups of animals. While GTP (0.1 muM) elicited an ATP-like response of similar magnitude in adenylate cyclase activity in both membrane preparations, it did not therefore abolish the impaired response to epinephrine of adenylate cyclase activity in membranes of obese mice. The response of adenylate cyclase activity to (--)-epinephrine in membrane preparations from obese mice progressively diminished with the age of these animals. In contrast, the concentration of (--)-epinephrine required for half-maximal stimulation of adenylate cyclase was similar and remained unchanged with the age for both membrane preparations. These data suggest that a perturbation may occur in the coupling step between the hormone receptor and the catalytic site of the adenylate cyclase system in obese mice. While a 15-day restrictive diet or a 72-h period of fasting was found to normalize the hyperinsulinemia of obese animals, neither affected the response of adenylate cyclase to epinephrine in preparations of adipocyte membranes from these mice. These results suggest that the observed defect in the response of plasma membrane adenylate cyclase activity to epinephrine in obese mice does not result from their hyperinsulinism.     

Increase in the amount of adenylate cyclase in rat gastrocnemius muscle after denervation

After section of the sciatic nerve, the basal adenylate cyclase (AC) activity in rat gastrocnemius muscle increased 6-7 times per membrane protein and about 2 times per whole muscle in the following 30 or 40 days. The AC activity in the muscle 30 days after denervation was increased about 4 times by forskolin. Calcitonin gene-related peptide (CGRP) also increased the adenylate cyclase activity in the denervated muscle. The binding of [3H]-forskolin (10nM) to cells isolated from gastrocnemius muscle was examined to determine the amount of AC molecules. Inhibition of [3H]-forskolin binding by increasing amounts of unlabeled forskolin gave a sigmoid curve with a IC50 value of 3 x 10(-7) M. Results showed that the number of [3H]-forskolin binding sites per cell was higher on the denervated side than on the control side, like the basal AC activity. The IC50 values for inhibition by unlabeled forskolin of binding of [3H]-forskolin were similar to muscles on the control and denervated sides. These results suggest that an increase in the AC activity induced by denervation was due to an increase in the numbers of AC molecules in the muscle.     

Isolation of a neuropeptide corresponding to the N-terminal 27 residues of the pituitary adenylate cyclase activating polypeptide with 38 residues (PACAP38)

A novel neuropeptide with 38 residues (PACAP38) was isolated from ovine hypothalamic tissues using the pituitary adenylate cyclase activation in rat pituitary cell cultures as a parameter of the biological activity (Miyata et al, Biochem. Biophys. Res. Commun. 164, 567-574, 1989). From the side fractions obtained during the purification of PACAP38, a shorter form peptide with 27 residues corresponding to the N-terminal 27 amino acids of PACAP38 and amidated C-terminus was isolated and named as PACAP27. Synthetic PACAP27 showed a biological activity of adenylate cyclase stimulation comparable to PACAP38. Moreover PACAP27 which shows a considerable homology with vasoactive intestinal polypeptide (VIP) demonstrated a similar vasodepressor activity as VIP, but the adenylate cyclase stimulating activity was about 1000 times greater than VIP.     

Calcium-Dependent Increases in Protein Kinase-A Activity in Mouse Retinal Ganglion Cells Are Mediated by Multiple Adenylate Cyclases

Neurons undergo long term, activity dependent changes that are mediated by activation of second messenger cascades. In particular, calcium-dependent activation of the cyclic-AMP/Protein kinase A signaling cascade has been implicated in several developmental processes including cell survival, axonal outgrowth, and axonal refinement. The biochemical link between calcium influx and the activation of the cAMP/PKA pathway is primarily mediated through adenylate cyclases. Here, dual imaging of intracellular calcium concentration and PKA activity was used to assay the role of different classes of calcium-dependent adenylate cyclases (ACs) in the activation of the cAMP/PKA pathway in retinal ganglion cells (RGCs). Surprisingly, depolarization-induced calcium-dependent PKA transients persist in barrelless mice lacking AC1, the predominant calcium-dependent adenylate cyclase in RGCs, as well as in double knockout mice lacking both AC1 and AC8. Furthermore, in a subset of RGCs, depolarization-induced PKA transients persist during the inhibition of all transmembrane adenylate cyclases. These results are consistent with the existence of a soluble adenylate cyclase that plays a role in calcium-dependent activation of the cAMP/PKA cascade in neurons.     

Isolation, purification and characterization of regulatory properties of adenylate cyclase from rabbit heart]

Rabbit heart membranes possessing the adenylate cyclase activity were isolated and purified by extraction with high ionic strength solutions and centrifugation in the sucrose density gradient. It was shown that the membranes are characterized by a high percentage of cholesterol (molar ratio cholesterol/phospholipids is 0.24) and an increased activity of Na, K-ATPase, which suggests the localization of adenylate cyclase in the sarcolemma. During centrifugation in the sucrose density gradient the activities of andenylate cyclase and Na,K-ATPase are not separated. Treatment of heart sarcolemma with a 0.3% solution of lubrol WX results in 10--20% solubilization of adenylate cyclase. Purification of the enzyme in the membrane fraction is accompanied by a decrease in the activity of phosphodiesterase; however, about 2% of the heart diesterase total activity cannot be removed from the sarcolemma even after its treatment with 0.3% lubrol WX. Epinephrine and NaF activate adenylate cyclase without changing the pH dependence of the enzyme. The alpha-adrenergic antagonist phentolamine has no effect on the adenylate cyclase activation by catecholamines, glucagon and histamine; the beta-adrenergic antagonist alprenolol competitively inhibits the effects of isoproterenol, epinephrine and norepinephrine, having no effect on the enzyme activation by glucagon and histamine. There is no competition between epinephrine, glucagon and histamine for the binding site of the hormone; however, there may occur a competition between the hormone receptors for the binding to the enzyme. A combined action of several hormones on the membranes results in the averaging of their individual activating effects. When the hormones were added one after another, the extent of adenylate cyclase activation corresponded to that induced by the first hormone; the activation was insensitive to the effect of the second hormone added. It is assumed that the outer membrane of myocardium cells contains a adenylate cyclase and three types of receptors, each being capable to interact with the same form of enzyme. The activity of adenylate cyclase is determined by the type of the receptor, to which it is bound and by the amount of the enzyme-receptor complex.     

Cerebral endothelial cell culture. I. The presence of beta 2 and alpha 2-adrenergic receptors linked to adenylate cyclase activity

Cultured endothelial cells derived from cerebral microvessels separated from 2-day-old rat brain contain a specific beta 2 and alpha 2-adrenergic sensitive adenylate cyclase (AC). Among the various tested hormones, PGE1 and PGE2 were found to be the most potent activators, while adenosine, angiotensin I and II, gamma-aminobutyric acid and vasoactive intestinal peptide inhibited the enzyme activity. However, acetylcholine, histamine, serotonin, glycine, glutamine, bradykinin, neurotensin and vasopressin (Lysine and Arginine) had no effect on the adenylate cyclase activity in this model. The susceptibility of the cerebrovascular endothelial AC system to the vasoactive substances as well as presence of beta 2 and alpha 2-type adrenergic receptors in the cultured endothelium provides additional support for the proposed endothelial involvement in the regulation of cerebrovascular permeability and blood flow.     

Solubilization and chromatographic separation of gonadotropin receptor from adenylate cyclase in ovarian preparations

The distribution of gonadotropin receptor and adenylate cyclase activities has been monitored in Sepharose 6B chromatographic eluates of a Lubrol solubilized fraction of 25 day old rat ovarian tissue. The receptor was resolved from the adenylate cyclase activity. Chromatography of a gonadotropin desensitized rat ovarian preparation showed that the desensitized preparation contained normal amounts of the adenylate cyclase activity when compared with the control ovarian preparation, but there was a marked reduction in the receptor activity in the desensitized ovaries. This study demonstrates that in ovarian tissue adenylate cyclase is a separate entity distinct from the receptor molecule and that the desensitization causes a reduction in the receptor activity with no detectable change in the catalytic and chromatographic properties of the adenylate cyclase.     

Ethanol Does Not Modify Opiate-Mediated Inhibition of Striatal Adenylate Cyclase

Ethanol increases the activity of "basal," guanine nucleotide- and dopamine-stimulated adenylate cyclase in mouse striatum. In contrast, ethanol, in vitro, did not modify the inhibition of striatal adenylate cyclase activity by opiates (morphine or [D-Ala2,D-Leu5] enkephalin). Following chronic in vivo ethanol treatment of mice, there was also no change in the character of opiate inhibition of striatal adenylate cyclase activity. Since ethanol, in vitro, does decrease striatal opiate receptor binding, the results suggest that the changes in affinity detected by ligand binding studies are not relevant for receptor-coupled adenylate cyclase activity, or that opiate receptor binding and opiate regulation of adenylate cyclase can be modulated independently. The selective effects of ethanol on systems that modulate adenylate cyclase activity may produce imbalances in neuronal function during in vivo ethanol exposure.     

The role of a guanine nucleotide-binding protein in the activation of rat liver plasma-membrane adenylate cyclase by forskolin.

The effects of the photoreactive GTP analogue GTP-gamma-azidoanilide on rat liver plasma-membrane adenylate cyclase are described. U.v. irradiation in the presence of the analogue abolished activation by any effector or combination of effectors that function via the activatory G protein. Partial protection against this inhibition was given by F- and guanosine 5'-[gamma-thio]triphosphate. It is concluded that GTP-gamma-azidoanilide acts by a light-induced covalent reaction with the G protein. In the dark the effects of the analogue were similar to those of GTP. Irradiation in the presence of GTP-gamma-azidoanilide was found to reduce but not to abolish activation of rat liver plasma membrane adenylate cyclase by forskolin. The activation by forskolin and GTP together were greater than the sum of the individual activations. Forskolin doubled adenylate cyclase activity in the presence of glucagon and guanosine 5'-[beta, gamma-imido]triphosphate, which might be expected to activate to the maximum possible extent via the G protein. It is concluded that there are two components to the forskolin activation, a guanine nucleotide-dependent and a guanine nucleotide-independent component.     

Adenylate cyclase activity of the plasma membranes of lung tissue at lengthy periods following non-lethal gamma-irradiation of rats

Basal and stimulated activity of adenylate cyclase (cyclizing ATP-pyrophosphate lyase, E.C. 4.6.1.1., AC) in plasma membranes of pulmonary tissue was being studied during a year after fractionated irradiation of rats (2 GyX3). Basal and hormone-stimulated activity of AC was shown to vary significantly from normal 6 and 12 months after irradiation. The exposed membranes responded differently to AC activation by isoproterenol and F-.     

Species and organ differences in the activity and regulation of adenylate and guanylate cyclases

The activity of adenylate and guanylate cyclases was determined in adrenal, heart, liver and fat tissues of guinea pigs, mice, rabbits and monkeys. The enzymes activities varied markedly depending both on the species and organs. The highest basal activities of adenylate cyclase was observed in all organs of guinea pigs. It was found that organs with low basal level of adenylate cyclase possess high guanylate cyclase. Species variations of the basal and stimulated adenylate cyclase activity may determine the functional activity of an organ: the higher the adenylate cyclase activity, the more intensive steroidogenesis in adrenals, lipolysis in the fat tissue, muscle contraction and nerve impulse conduction in heart.