Sensitivity Of Adenylate Cyclase Activity Research Articles

Heterologous sensitization of adenylate cyclase is protein kinase A-dependent in Cath.a differentiated (CAD)-D2L cells.

Persistent activation of Galphai/o-coupled receptors results in a paradoxical enhancement of subsequent drug-stimulated adenylate cyclase activity. The exact mechanism of this up-regulation in the cyclic AMP signaling pathway, known as heterologous sensitization, remains undefined. The present study was designed to investigate the involvement of cyclic AMP-dependent protein kinase in D2L receptor-mediated sensitization in a neuronal cellular environment. The current studies were conducted in the Cath.a differentiated (CAD) cell line transfected stably with the D2L dopamine receptor (CAD-D2L). Long-term 18 h treatment with the D2 receptor agonist, quinpirole, resulted in a two-fold enhancement of forskolin-stimulated cyclic AMP accumulation. Similarly, long-term treatment with the PKA inhibitors, H89 or Rp-8Br-cAMP, also enhanced adenylate cyclase activity. In contrast, long-term activation of protein kinase A (PKA) by forskolin, isobutylmethylxanthine (IBMX), or dibutyryl cyclic AMP caused a significant reduction in subsequent forskolin-stimulated cyclic AMP accumulation and reduced both quinpirole- and H89-induced heterologous sensitization. The effects of PKA inhibitors and activators did not involve changes in PKA subunit expression. RT-PCR analysis of adenylate cyclase isoform expression patterns revealed the expression of mRNA for ACVI and ACIX in CAD-D2L cells. The ability of ACVI to be negatively regulated by PKA is consistent with the observation that inhibition of PKA results in heterologous sensitization of adenylate cyclase activity in CAD-D2L cells.

Adrenergic agonists induce heterologous sensitization of adenylate cyclase in NS20Y-D 2L cells

Adenylate cyclase activity in NS20Y cells expressing D 2L dopamine receptors was examined following chronic treatment with norepinephrine and epinephrine. Initial acute experiments revealed that both norepinephrine and epinephrine inhibited forskolin-stimulated cyclic AMP accumulation via D 2 receptors. Furthermore, chronic (18 h) activation of D 2 dopamine receptors by norepinephrine or epinephrine induced a marked increase (>10-fold) in subsequent forskolin-stimulated cyclic AMP accumulation. This heterologous sensitization of adenylate cyclase activity was blocked by D 2 dopamine receptor antagonists and by pertussis toxin pretreatment. In contrast, concurrent activation of Gα s or adenylate cyclase did not appear to alter noradrenergic agonist-induced sensitization.

Effects of adenosine on cAMP production during early development in the optic tectum of chicks

Accumulation of cyclic adenosine monophosphate (cAMP) elicited by adenosine was studied in slices and membrane preparations of optic tectum from chicks aged 1–13days post-hatch. Accumulation of cAMP promoted by adenosine declined with age, chicks and the lowest in 11-day-old chicks. However, when the slices were incubated with adenosine and the phosphodiesterase inhibitor-Ro 20-1724 the differences between teh two ages were abolished, suggesting a higher phosphodiesterase activity in 11-day-old chicks. In membrane preparations, although basal adenylate cyclase activity was lower in three-day-old chicks, the guanylyl-imidodiphosphate (Gpp(NH)p) concentration curves for stimulation of adenylate cyclase activity indicated a higher sensitivity of G protein to Gpp(NH)p at this age. This hypothesis was reinforced by the observation that the binding of [3H]Gpp(NH)p to the membrane preparation was greater in three-day-old animals. In spite of these differences, the percentage of adenylate cyclase activity stimulation by 2-chloroadenosine (2CADO) + Gpp(NH)p was the same at both ages. These findings suggest that the decreased response evoked by adenosine during development is probably due to increased phosphodiesterase activity and a lower sensitivity of adenylate cyclase activity to Gpp(NH)p.

Heterologous sensitization of adenylate cyclase activity by serotonin in the rat cerebral cortex

In vitro exposure of rat cerebrocortical slices to μM concentrations of serotonin (5HT) results in an increased response of adenylate cyclase to isoproterenol (ISO). No change in the affinity of the β-adrenoceptor toward the agonist was found after 5HT exposure when measuring ISO displacement of [ 3H]CGP 12177 binding. A similar increase of adenylate cyclase response was also found when using VIP as a stimulatory agent. The dose-response curve of adenylate cyclase to the GTP analogue, GppNHp, was modified by 5HT, which promotes a significantly higher maximal response without altering the potency of GppNHp. Forskolin-stimulated adenylate cyclase activity was not affected by 5HT. Serotonergic 5HT 2 receptors are involved in the sensitization of adenylate cyclase to GppNHp, since the selective 5HT 2 antagonist ketanserin inhibits the effect of 5HT, whereas the 5HT 2 agonist DOI mimics 5HT. The involvement of 5HT 2 receptor-coupled activation of protein kinase C is also demonstrated: direct protein kinase C activators such as phorbol esters and s,n-dioctanoylglycerol behave in the same manner as 5HT, while the protein kinase C inhibitor CGP 41251 prevents 5HT from increasing adenylate cyclase responsiveness to GppNHp. Moreover, in vitro exposure of cortical slices to 5HT results in reduced inhibition of adenylate cyclase by somatostatin. Since no change was observed at the receptor level and in the direct stimulation of the catalytic subunit of the enzyme, we propose that 5HT might accomplish the sensitization of adenylate cyclase through protein kinase C by inactivating the inhibitory coupling protein Gi and facilitating the interaction of the exogenous GppNHp with the stimulatory coupling protein Gs.

Regulation of adenylate cyclase in electropermeabilized Dictyostelium discoideum cells

In Dictyostelium discoideum cells the enzyme adenylate cyclase is functionally coupled to cell surface receptors for cAMP. Coupling is known to involve one or more G-proteins. Receptor-mediated activation of adenylate cyclase is subject to adaptation. In this study we employ an electropermeabilized cell system to investigate regulation of D. discoideum adenylate cyclase. Conditions for selective permeabilization of the plasma membrane have been described by C. D. Schoen, J. C. Arents, T. Bruin, and R. Van Driel (1989, Exp. Cell Res. 181, 51–62). Only small pores are created in the membrane, allowing exchange of exclusively low molecular weight substances like nucleotides, and preventing the loss of macromolecules. Under these conditions functional protein-protein interactions are likely to remain intact. Adenylate cyclase in permeabilized cells was activated by the cAMP receptor agonist 2′-deoxy cAMP and by the nonhydrolyzable GTP-analogue GTPγS, which activates G-proteins. The time course of the adenylate cyclase reaction in permeabilized cells was similar to that of intact cells. Maximal adenylate cyclase activity was observed if cAMP receptor agonist or GTP-analogue was added just before cell permeabilization. If these activators were added after permeabilization adenylate cyclase was stimulated in a suboptimal way. The sensitivity of adenylate cyclase activity for receptor occupation was found to decay more rapidly than that for G-protein activation. Importantly, the adenylate cyclase reaction in permeabilized cells was subject to an adaptation-like process that was characterized by a time course similar to adaptation in vivo. In vitro adaptation was not affected by cAMP receptor agonists or by G-protein activation. Evidently electropermeabilized cells constitute an excellent system for investigating the positive and negative regulation of D. discoideum adenylate cyclase.

Increased sensitivity of adenylate cyclase activity in the striatum of the rat to calmodulin and GppNHp after chronic treatment with haloperidol

Chronic treatment of rats with haloperidol causes behavioral supersensitivity to dopaminergic agonists and an increase in the sensitivity of adenylate cyclase activity in the striatum to stimulation by dopamine. In this study the authors examined whether chronic treatment with haloperidol could elicit a change in sensitivity of adenylate cyclase in the striatum of the rat for guanyl nucleotides and the endogenous Ca 2+-binding protein, calmodulin. These agents increase the activation of adenylate cyclase activity by dopamine but act beyond the level of the dopamine receptor. Male, Sprague-Dawley rats were injected subcutaneously with either 0.6 mg/kg haloperidol or vehicle for 14 days. Four days after the last injection, the animals were sacrificed and the activity of adenylate cyclase was measured in a EGTA-washed particulate preparation of the striatum. There was an increase in the activation of adenylate cyclase activity by calmodulin and GppNHp but not by guanosine triphosphate (GTP) in particulate fractions of the striatum from rats treated with haloperidol as compared to controls. The sensitivity of adenylate cyclase to calmodulin was increased 5-fold in particulate fractions from rats treated with haloperidol as opposed to vehicle-treated rats. The lack of change in activation by GTP was not due to an altered activity of GTPase in rats treated with haloperidol. In animals treated for 14 days but not withdrawn from haloperidol there was no statistically significant increase in the sensitivity of adenylate cyclase to calmodulin. There was no change in activation of the enzyme by GppNHp or GTP as compared to control. The activation of adenylate cyclase by calmodulin was not affected when haloperidol was added in vitro to the assay or after the acute injection of rats with haloperidol. These results demonstrate that the development of dopaminergic supersensitivity after withdrawal from chronic blockade of dopamine receptors resulted in increased sensitivity of post-receptor coupling mechanisms, as well as in changes in the receptor-mediated activity. In previous studies, increases in DA-stimulated adenylate cyclase activity were found with this treatment regimen. In this study it was shown that although basal activity was not changed, there was a selective increase in the sensitivity of adenylate cyclase to stimulation by calmodulin and GppNHp after chronic receptor blockade. These findings support previous observations suggesting a regulatory role for calmodulin in dopaminestimulated adenylate cyclase activity.

Enhanced adenylate cyclase activity of turkey erythrocytes following treatment with β-adrenergic receptor antagonists

The turkey erythrocyte contains a β 1-adrenergic receptor-linked adenylate cyclase system. We have examined the effects of pretreatment with receptor antagonists on adenylate cyclase activity and the individual components in the pathway of enzyme activation in this system. Isoproterenol-stimulated adenylate cyclase activity is increased by 30% (P < 0.01) over control in membranes derived from cells preincubated with the antagonist propranolol. The effect is stereospecific and dose-related with a EC 50 of 100 nM for the (−) isomer. The time course of effect is rapid being complete by 90 min. Non-receptor mediated stimulation of adenylate cyclase activity by manganese ion, forskolin and NaF is similarly enhanced following propranolol pretreatment. Sensitization of adenylate cyclase activity also occurs following pretreatment with a number of antagonists but is not seen after preincubation with pindolol or practolol. Quantitation of β-adrenergic receptor (R) density using [ 125I]cyanopindolol indicates no difference between membranes derived from control and antagonist pretreated cells. Coupling of R with the guanine nucleotide regulatory protein (N) as assessed by high affinity agonist binding is unchanged following pretreatment. The efficacy of 5′-guanylylimidodiphosphate Gpp(NH)p in producing a shift of agonist binding curves associated with destabilization of high affinity H-R-N complexes, is also the same (EC 50 = 0.2 μM) in membranes from control and antagonist treated cells. The isoproterenol stimulated rate of release of [ 3H]GDP from membranes preloaded with [ 3H]GTP as an index of formation of an active form of the N protein is similarly unaffected by antagonist preincubation. We conclude that the mechanism of the observed sensitization of turkey erythrocyte adenylate cyclase by β-adrenergic antagonists is receptor mediated and likely involves facilitation of N interaction with the catalytic subunit of the enzyme.

Increased sensitivity to epinephrine stimulated lipolysis during starvation: Tighter coupling of the adenylate cyclase complex

Adipocytes of rats starved for 72 hr showed three-fold increase in sensitivity of the lipolytic response to epinephrine. Adenylate cyclase sensitivity in adipocyte membranes from starved rats was increased almost seven-fold over control rats. Binding studies using [ 3H]-dihydroalprenolol showed that the number of β-adrenergic receptors per fat cell (1.1 × 10 5 and 1.1 × 10 5 receptors/cell, starved vs control) and the dissociation constant (5 vs 4 nM) of the receptor for the antagonist did not change following starvation. Increased sensitivity of adenylate cyclase activity in response to epinephrine without changes in the β-adrenergic binding parameters suggests that tighter coupling of adenylate cyclase with the receptor may mediate the more sensitive lipolytic response of starved rat adipocytes to epinephrine.

378 ONTOGENY OF β-ADRENERGIC RECEPTORS IN MAMMALIAN LUNG

The ontogeny of β-adrenergic receptors (BAR) in lung was described in the rabbit, rat, sheep and guinea pig. Developmental increases in binding capacity were demonstrated in all species, for example, rabbit lung membrane bound (-)-[3H]-dihydroalprenolol, [3H]DHA to a single class of sites, was stereoselective, reversible and saturable; KD 1.78±0.30 nM. Binding at all ages was inhibited by agonists in the order of potency: Iso > Epi=Norepi characteristic of a β1AR subtype. Studies with metoprolol (β1) and zinterol (β2-selective) revealed 60% β1- and 40% β2adrenergic subtypes at all ages. Bmax increased 11.5 fold from day 25 of gestation to adulthood, increasing during the latter days of gestation and the early neonatal period, from 37±10 fmolemg-1 protein to 425±51 in the adult, m±SD. Gpp(NH)p decreased agonist affinity more effectively in adult than in fetal lung and we hypothesize that a “coupling” defect exists in fetal lung BAR. The sensitivity of adenylate cyclase activity to catecholamine was maximal in adult rabbit lung increasing with age from 23% at day 25 gestation to 255% stimulation in the adult. Pulmonary BAR numbers at term gestation were higher in animals which are relatively more mature at birth (guinea pig and sheep) as compared to less mature (rabbit and rat). In conclusion, β-adrenergic receptors appear to be a sensitive indicator of pulmonary maturation and the increase in BAR during development in mammalian lung and supports the role of β-adrenergic receptors in the regulation of pulmonary function.

In vivo and in vitro effects of proteases upon adenylate cyclase activity of liver cells and plasma membranes.

1. 1. Several hours after an intraperitoneal injection of papain (3 mg) the marked stimulation of adenylate cyclase activity was observed as well as the significant increase in cyclic AMP content of rat livers. The fluoride-sensitive adenylate cyclase activity was stimulated more than the basal activity. Trypsin, which is not mitogenic, was much less effective on both events, as compared to papain which is mitogenic to the liver in vivo. 2. 2. Papain stimulated the basal adenylate cyclase activity of isolated liver cells in vitro at concentrations larger than 2.5 μg papain per ml, but the sensitivity of adenylate cyclase activity to fluoride was completely lost upon increasing the papain concentration. Trypsin, on the other hand, gave no effect on the basal adenylate cyclase activity of isolated liver cells at enzyme concentrations from 0 to 500 μg/ml. The fluoride sensitivity was lost upon increasing trypsin concentration similarly to the case of papain. 3. 3. Adenylate cyclase activity (both basal and fluoride-sensitive) of isolated liver plasma membranes was stimulated by papain (but not by trypsin) at concentrations as low as about 0.25 μg papain/ml, similarly to the in vivo effect of proteases. Upon increasing the protease (papain, trypsin) concentration further, both the basal and fluoride-sensitive activities of plasma membranes were markedly reduced. 4. 4. Acid mucopolysaccharides of liver plasma membranes were released almost completely by treatment with papain at concentrations larger than 25 μg/ml, whereas trypsin was less effective in degrading proteoglycans associated with plasma membranes. 5. 5. The results were discussed in relation to the possible involvement of protease in mitogenesis of liver cells through the alteration of cell surface architecture and function.

Inhibition by methylmercuric chloride of prostaglandin E 1-sensitive adenylate cyclase activity in glioma but not in neuroblastoma cells in culture

The responses of prostaglandin E 1 (PGE 1)-sensitive adenylate cyclase activity in glioma (C-6) and neuroblastoma (NBP 2) cells in culture after acute (5 days) and chronic (6–7 weeks) treatment with low concentrations (0.1 μm) of methylmercuric chloride (CH 3HgCl) were investigated. The sensitivity of adenylate cyclase activity to PGE 1, as measured by the rise in the intracellular level of cyclic AMP, was markedly reduced in chronically treated glioma cells, but it was not affected in chronically treated NB cells. Acute treatments of glioma and NB cells with CH 3HgCl did not modify the effect of PGE 1 on the intracellular level of cyclic AMP. The responses of dopamine- and norepinephrine-sensitive adenylate cyclases in actuely treated neuroblastoma (NBA 2(1)) cells and norepinephrine-sensitive adenylate cyclase in acutely or chronically treated glioma cells did not change.

Hormone sensitivity of adenylate cyclase activity in cardiac sarcolemma and sarcoplasmic reticulum preparations

The functional significance of the adenylate cyclase activity found in cardiac microsomal preparations enriched in sarcoplasmic reticulum vesicles was examined by comparing the effects of agents known to modify this enzyme in sarcolemmal and sarcoplasmic reticulum fractions prepared from guinea pig ventricles. The sensitivity of the sarcolemmal adenylate cyclase to adrenergic agonists resembled that of a typical β-receptor. Although the response to these agonists was similar in the case of the sarcoplasmic reticulum enzyme, the extent of stimulation was much less than in the sarcolemma. The pH optimum of the sarcoplasmic reticulum enzyme of 7.5 was slightly lower than that of the sarcolemmal enzyme, which was 8.0; and NaF shifted the pH optimum of the latter, but not the former. The sarcoplasmic reticulum adenylate cyclase was less sentitive to alkali metal salts and GTP than was the sarcolemmal enzyme. While these studies cannot exclude the possibility that the lesser response of the sarcoplasmic reticulum enzyme to catecholamines, alkali metal salts, and GTP arose from contaminating sarcolemmal fragments that were modified during the preparation of these subcellular fractions, they are consistent with the view that the cardiac cell sarcoplasmic reticulum contains an adenylate cyclase that is less regulated, or regulated by different factors, than those which modulate sarcolemmal adenylate cyclase.