Expression of rat mRNA coding for hormone-stimulated adenylate cyclase in Xenopus oocytes

Beta-Adrenergic agonist-stimulated hyperpolarization, whole-cell cAMP accumulation, and activity of isoproterenol-stimulated membrane-bound adenylate cyclase (EC 4.6.1.1) in Xenopus laevis ovarian oocytes are entirely dependent on the presence of nascent follicle cells. A method was developed to remove rapidly and completely all extra-oocyte cell types to yield defolliculated oocytes that exhibited normal viability and resting membrane potentials yet lacked beta-adrenergic receptor (beta AR)-stimulated responses. Purified follicle membranes contained beta AR-stimulated adenylate cyclase activity, whereas oocyte cell membranes did not. Purified oocyte membrane preparations from X. laevis oocytes previously microinjected with C6-2B rat astrocytoma mRNA, and subsequently defolliculated, exhibited novel beta AR and forskolin-stimulated adenylate cyclase activity. These experiments demonstrate that oocytes expressed rat C6-2B mRNA coding for the beta-adrenergic receptor and the components necessary for forskolin-stimulated adenylate cyclase activity.     

Differential amplification of antagonistic receptor pathways in neutrophils.

In human neutrophils approximately 500 ligand-occupied beta-adrenergic receptors almost completely inhibit the superoxide production generated by at least 50,000 formyl peptide receptors, suggesting a massive amplification of the inhibitory receptor signals. We estimated two stages of amplification. In the first stage, we quantitated the ligand-dependent GTPase activities. For the formyl peptide receptor, the number of phosphates released from GTP in the presence of the saturating ligand is relatively modest, i.e. approximately 1/min/receptor, even though there are approximately 200 Gn (Gi type II) proteins/formyl peptide receptor in neutrophil membranes. In contrast, the number of GTPs cleaved in the presence of a beta-adrenergic agonist is approximately 100/min/beta-adrenergic receptor, and there are about 700 Gs/beta-adrenergic receptor in membranes. Thus the signal of the beta-adrenergic receptor is already massively amplified at the G protein, whereas the signal of the formyl peptide receptor is likely to be amplified at subsequent steps. New kinetic evidence from intact cells and biochemical evidence from permeabilized cells is provided that the second messenger of the inhibitory pathway is cAMP. To estimate the amplification of this step, we determined the cAMP concentration necessary to maximally inhibit superoxide anion production of formyl peptide-stimulated electropermeabilized cells, and we compare these concentrations to previously determined values of cAMP production in neutrophils. We conclude that each receptor may generate up to 10,000 molecules of cAMP.     

The effect of polar head group substitution on phospholipid methylation and the beta-adrenergic response in C6 glial cells.

The membranes of intact C6 cells were enriched with phosphatidyldimethylethanolamine or phosphatidylmonomethylethanolamine. These cells showed enhanced rates of phospholipid methylation but this was not accompanied by an increased beta-adrenergic response. We conclude that phospholipid methylation is not coupled to the activation of adenylate cyclase in the beta-adrenergic response of C6 glial cells.     

Characteristics of the alpha 2/beta-adrenoceptor-coupled adenylate cyclase system and their relationship with adrenergic responsiveness in hamster fat cells from different anatomical sites.

Various studies have shown that the lipolytic response of white adipocytes to catecholamines was dependent on the anatomical origin of these cells. To provide a biological explanation for this phenomenon, we compared hamster white adipocytes, from femoral subcutaneous and epididymal fat, for their lipolytic activities, cAMP responses and adrenoceptor-coupled adenylate cyclase system. Basal and maximal lipolytic responses to the beta-adrenergic (isoproterenol) and the mixed alpha 2/beta-adrenergic (epinephrine) agonists were lower in femoral subcutaneous cells than in epididymal cells, but the alpha 2-adrenergic antilipolytic response to 5-bromo-6-(2-imidazolin-2-ylamino)quinoxaline bi-tartate (UK14304) was slightly greater in femoral subcutaneous fat cells than in epididymal fat cells. Identical results were observed for cAMP responses, except for the alpha 2-adrenergic inhibitory response which was identical in both fat deposits. Adrenoceptors studies revealed higher density of inhibitory alpha 2-adrenoceptors 2-(2-methoxy-1,4-benzodioxan-2-yl)-2-imidazoline ([3H]RX821002-binding sites) in femoral subcutaneous fat cells than in epididymal fat cells, but identical density of stimulatory beta-adrenoceptors (125I-cyanopindolol-binding sites) and similar subdivision into beta-adrenoceptor subtypes in both adipose deposits. Finally, the level of the alpha-subunits of the stimulatory and inhibitors guanine-nucleotide-binding regulatory proteins, as well as the adenylate cyclase catalytic activity were 40-50% lower in femoral subcutaneous fat cell membranes than in epididymal fat cell membranes. These results suggest that the differences in cAMP and lipolytic responses to catecholamines between epididymal and femoral subcutaneous adipocytes result at least in part from site-related differences in the adenylate cyclase system rather than in the adrenoceptor status.     

Acquisition of a beta-adrenergic response by adult rat hepatocytes during primary culture

In freshly isolated parenchymal hepatocytes of adult rats, the beta-adrenergic agonist isoproterenol (Ip) did not stimulate cAMP formation, protein kinase activity, or glycogenolysis, although glucagon markedly stimulated all these activities. However, the beta-adrenergic response appeared when rat hepatocytes were cultured as monolayers. This response had already appeared after 2-h culture and increased during further culture. The appearance of the beta-adrenergic response during culture was blocked by cycloheximide, actinomycin D, or alpha-amanitin. Thus adult rat hepatocytes acquired marked ability to respond to Ip during culture through the syntheses of mRNA and protein. Freshly isolated hepatocytes from postnatal rats showed a high beta-adrenergic response that did not increase further during culture. This response gradually decreased during development and had almost disappeared about 60 days after birth. In plasma membranes prepared from freshly isolated cells of adult rats the basal and NaF-stimulated activities of adenylate cyclase (EC 4.6.1.1) were similar to those of cultured cells and the enzyme activity was also stimulated by guanyl-5'-yl imidodiphosphate. However, in plasma membranes of freshly isolated cells Ip scarcely stimulated adenylate cyclase, but glucagon did. The intact cells, whether they were freshly isolated or cultured, accumulated cAMP when exposed to cholera toxin. Moreover, the two subunits of GTP-binding regulatory protein (also named G/F or Ns site) were detected by [32P]ADP ribosylation with cholera toxin and [32P]NAD+ in freshly isolated cells as well as in cultured cells. These results indicate that freshly isolated and cultured hepatocytes of adult rats contain sufficient levels of all the components of the postreceptor-adenylate cyclase system for activity. However, the number of beta-adrenergic receptors measured by binding of [125I]iodocyanopindolol, a potent beta-adrenergic antagonist, was very low in purified plasma membranes of freshly isolated cells (20 fmol/mg of protein), and the number increased about 6-fold without change in the dissociation constant (Kd = 132 pM) when the cells were cultured for 7 h. This increase in beta-adrenergic receptor sites was completely abolished by cycloheximide and alpha-amanitin. Thus it is concluded that the unresponsiveness of adult rat hepatocytes to Ip was due to a very low amount of beta-adrenergic receptor and that the appearance of a beta-adrenergic response during primary culture was due to new synthesis of beta-adrenergic receptor through synthesis of mRNA.     

Enhancement of adenosine A2 and prostaglandin E1 receptor-mediated cAMP generation by prior exposure of Swiss 3T3 fibroblasts to Ca2+-mobilizing receptor agonists or phorbol ester. Activation of protein kinase C triggers increases in the receptor density in cell membranes

Production of cAMP in response to adenosine A2 or prostaglandin E1 receptor stimulation was, but the production induced by a beta-adrenergic agonist or forskolin was not, enhanced by prior exposure of Swiss 3T3 fibroblasts to agonists of Ca2+-mobilizing receptors or phorbol ester for 3 h. The enhancement reflected potentiation of the receptor-coupled activation of adenylate cyclase and the 2-fold increase in the adenosine A2 receptor number in membranes under these conditions. No enhancement was observed, however, when the medium used for the prior exposure was further supplemented with 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) or staurosporin, inhibitors of protein kinase C, neither of which affected the cAMP responses of the nonexposed cells. It is very likely, therefore, that activation of protein kinase C triggers the increase in certain receptor density in membranes, thereby enhancing the receptor-coupled cAMP-generating responses. The physiological significance of such cross-talk between cellular signaling systems is discussed in comparison with similar previous observations.     

Interactions of formylmethionyl-leucyl-phenylalanine, adenosine, and phosphodiesterase inhibitors in human monocytes. Effects on superoxide release, inositol phosphates and cAMP

Cessation of the fMLF-induced burst of human monocyte superoxide release was associated with a rise in cAMP. This was not due to inhibition of phosphodiesterase (PDE), the major form of which was the PDE IV isozyme. The action of burst inhibitors did not correlate with cAMP levels: Rolipram, a PDe IV inhibitor, increased cAMP 6-fold, with minimal effects on the burst; whereas theophylline increased cAMP less than 2-fold but decreased the burst to less than half. Although theophylline and the adenylate cyclase activator, adenosine, inhibited fMLF-induced superoxide release, they did not inhibit production of inositol phosphates. Thus, these studies on inhibition of superoxide release implicated neither cAMP nor inositol phosphates.     

Desensitization of beta receptor mediated cyclic AMP response of cultured fibroblasts by partial agonists.

Two novel beta-adrenergic myocardial stimulants of general structure: Formula: (see text), where R is a phenyl or benzyl group were investigated for their ability to stimulate and desensitize the cyclic AMP response of C6 glioma cells. Compound ICI 89, 963 (R:phenyl) which elicited less than 1% of the maximum increase in cAMP produced by isoproterenol, was strikingly effective as a desensitizer of the isoproterenol response. This desensitization was markedly reduced by propranolol. Compound ICI 119,033 (R: benzyl) which was a more effective stimulant of cAMP synthesis than ICI 89,963, was also a more effective desensitizer of the isoproterenol response of C6 cells. The kinetics of the desensitization by ICI 89,963 were comparable with those for isoproterenol reaching a maximum in 2 to 3 hours. The data indicate that beta-adrenergic agonists are more potent as desensitizers of the cyclic AMP response than as stimulants of that response.     

cAMP has distinct acute and chronic effects on aquaporin-5 in lung epithelial cells

Aquaporin-5 (AQP5) is present on the apical membrane of epithelial cells in various secretory glands as well as on the apical membrane of the airway epithelium, airway submucosal glands, and type 1 pneumocytes, where it can participate in respiratory tract water homeostasis. We examined the effects of cAMP on AQP5 distribution and abundance. When AQP5-expressing mouse lung epithelial cells were treated with cAMP or the beta-adrenergic agonist terbutaline, a biphasic AQP5 response was observed. Short term (minutes) exposure to cAMP produced internalization of AQP5 off of the membrane and a decrease in protein abundance. Both of these responses were blocked by inhibition of protein kinase A and the decrease in abundance was blocked by chloroquine, indicating lysosome-mediated degradation. Sustained cAMP exposure (hours) produced an increase in membrane localization and increased abundance; these effects were also blocked by protein kinase A inhibition. The beta-adrenergic agonist terbutaline produced changes in AQP5 abundance in mouse trachea and lung, consistent with our findings in cultured epithelial cells. Purified AQP5 protein was phosphorylated by protein kinase A but not protein kinase C or casein kinase II, and aquaporin-5 was phosphorylated in cultured cells after long term (but not short term) exposure to cAMP. These studies indicate that cAMP and beta-adrenergic agonists produce distinct short and long term effects on AQP5 distribution and abundance that may contribute to regulation of lung water homeostasis.     

Activators of Protein Kinase C Act at a Postreceptor Site to Amplify Cyclic AMP Production in Rat Pinealocytes

Activation of alpha 1-adrenoceptors appears to amplify beta-adrenergic stimulation of cyclic AMP (cAMP) accumulation in rat pinealocytes severalfold by a mechanism involving activation of a Ca2+-, phospholipid-dependent protein kinase (protein kinase C). The mechanism of action of protein kinase C was investigated in this report using intact cells. Activation of protein kinase C with 4 beta-phorbol 12-myristate 13-acetate (PMA; 10(-7) M) or the alpha 1-adrenergic agonist phenylephrine (PE; 10(-6) M) did not inhibit cAMP efflux in beta-adrenergically stimulated cells. The amplification of the beta-adrenergic cAMP response by these agents also occurred in the presence of isobutylmethylxanthine (10(-3) M) and Ro 20-1724 (10(-4) M), an observation suggesting that inhibition of cAMP phosphodiesterase activity is not the mechanism of action. Furthermore, although PMA (10(-7) M) caused a sixfold increase in the magnitude of the cAMP response to isoproterenol, it did not alter the EC50 of the response (1.7 X 10(-8) M), a result indicating that protein kinase C activation does not alter beta-adrenoceptor sensitivity. The cAMP response following cholera toxin pretreatment (60-120 min) was rapidly and markedly enhanced by alpha 1-adrenergic agonists (cirazoline greater than PE greater than methoxamine), by phorbol esters (PMA greater than 4 beta-phorbol 12,13,-dibutyrate much greater than 4 alpha-phorbol 12,13-didecanoate), and by synthetic diacylglycerols (1,2-dioctanoylglycerol greater than 1-oleoyl 2-acetylglycerol much greater than diolein). The cAMP response to forskolin (10(-5)-10(-3) M) was also increased by PE (3 X 10(-6) M) and PMA (10(-7) M).(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein tyrosine kinase-dependent regulation of adenylate cyclase and phosphatidylinositol 3-kinase activates the expression of glial fibrillary acidic protein upon induction of differentiation in rat c6 glioma

Glial fibrillary acidic protein (GFAP) is expressed upon cAMP-mediated induction of differentiation of glial progenitor cells into type II astrocytes. The protein is regulated by hormones, growth factors and cytokines but the signal transduction pathways involved in the regulation of GFAP expression are largely unknown. Specific protein kinase inhibitors were used to study their effect on the expression of GFAP in rat C6 glioma cells. Herbimycin A, a selective protein tyrosine kinase inhibitor, reduced GFAP mRNA and protein expression upon cAMP analog or beta-adrenergic receptor-mediated induction of differentiation. The latter inhibitor attenuated the elevation of cAMP by adenylate cyclase and abolished the activity of phosphatidylinositol 3-kinase (PI 3-K). These data indicate that GFAP expression is regulated by protein tyrosine phosphorylations, modulating the cAMP concentration and PI 3-K activity in C6 glioma cells.     

Modulation by islet-activating protein of adenylate cyclase activity in C6 glioma cells

The cAMP content of intact cells as well as adenylate cyclase of the membrane-rich particulate fractions was studied with C6 glioma cells that had been exposed to the culture medium supplemented with islet-activating protein (IAP), one of the pertussis toxins. Both the increase in the cellular cAMP content in response to a beta-adrenergic agonist and the stimulation of membrane adenylate cyclase by the beta-agonist and/or GTP were markedly enhanced by the IAP treatment of C6 cells, but no change was induced in affinities of the agonist (or an antagonist) or GTP for their respective sites of action (or binding). The concentration of IAP required for the half-maximal enhancement was as low as 1 pg/ml, when the time of cell exposure to the toxin was prolonged to 18 h. No enhancement was observed for the basal cAMP content or basal enzyme activity, nor was activation of adenylate cyclase by Gpp(NH)p (or NaF) affected by IAP treatment. The Vmax value of a specific and low Km GTPase was significantly smaller in the membranes of IAP-treated cells than in those of control cells. Cholera toxin treatment of cells activated adenylate cyclase without exerting any influence on these IAP actions. Thus, IAP would appear to enhance beta-receptor-coupled stimulation of adenylate cyclase, in a manner distinct from cholera toxin, by rendering more GTP available to the GTP sites on the regulatory subunit of the receptor-enzyme system.     

Agonist interactions at hepatic alpha 1- and beta-adrenergic receptors: affinity-state regulation by guanine nucleotides and temperature

We investigated the binding characteristics of agonists to alpha 1- and beta-adrenergic receptors of intact liver cells, broken rat liver cell membranes, and detergent-solubilized preparations under varying experimental conditions, focusing on the different "states" of the receptor for agonists and the regulation of these states by temperature and guanine nucleotides. While only low-affinity binding of agonists to both receptor subtypes was evident in studies performed at 37 degrees C with solubilized preparations, biphasic competition curves for agonists were observed in both intact cells and membrane preparations; the majority of sites were of low affinity. In membrane preparations, the nonhydrolyzable GTP analogue Gpp(NH)p caused a rightward shift of agonist competition curves and a loss of high-affinity binding. These results are consistent with the involvement of guanine nucleotide binding proteins in both alpha 1- and beta-adrenergic transduction pathways. When competition studies were performed at 4 degrees C, receptor sites existed predominantly in the high-affinity configuration, in intact cells and membranes, as well as in soluble preparations. In contrast to the studies conducted at 37 degrees C, no Gpp(NH)p-induced conversion to the lower affinity state could be demonstrated in studies performed with membrane preparations at 4 degrees C. Thus, the high-affinity state of alpha 1- and beta-adrenergic receptors is stabilized at 4 degrees C in intact cells, membranes, and soluble preparations. After incubations had been performed at 37 degrees C, high-affinity binding of agonists could not be restored by subsequent incubation at 4 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hormonal activation of adenylate cyclase in macrophage membranes is regulated by guanine nucleotides

Many macrophage functions such as chemotaxis, phagocytosis, enzyme secretion, and cytotoxicity are influenced by intracellular cyclic nucleotide levels, but the regulatory mechanisms involved are poorly defined. We have developed methods that allowed us to study the activation of AC in isolated guinea pig (g.p.) macrophage membranes. AC in these membrane preparations could be stimulated approximately twofold by guanine nucleotides. We could not obtain any hormonal activation of membrane-bound AC in the absence of guanine nucleotides. In the presence of GTP, however, the hormones isoproterenol and PGE1 elicited an additional threefold rise in AC activity, which subsided after approximately 15 min. As little as 10(-8) M concentrations of these two hormones induced significant elevations of AC activity. Replacement of GTP by its nonhydrolyzable analogue Gpp(NH)p resulted in a persistent hormone-independent activation of AC, and addition of hormones enhanced this level of activation. Thus, GTP-ase activity is present in macrophage membrane preparations and serves to regulate AC activation. Hormonal stimulation of AC was receptor mediated, because the effect of the beta-adrenergic agonist isoproterenol, but not PGE1, was inhibited by the beta-adrenergic blocker propranolol. In addition, the potency series of PG corresponded to that observed for stimulation of cAMP production in intact g.p. macrophages, i.e., PGE1 = PGE2 greater than PGA1 greater than PGF2 alpha. AC activation by PG in the membrane preparation was inhibited by an alpha-adrenergic agonist, thus demonstrating one means for down regulating cAMP production in g.p. macrophages. Our studies also showed that certain hormones (e.g., beta-adrenergic agonists, PG) can exert their effect on cAMP production by stimulation of membrane-bound AC, whereas other agents such as lectins or arachidonic acid require additional intracellular components to elevate cAMP levels in macrophages. The mechanism of activation of AC by hormones in g.p. macrophage membranes appears to fit the model of a ternary complex, the components of which include the hormone receptor, AC, and guanine nucleotide regulatory protein, which transmits the signal from the receptor to AC.     

beta-Adrenergic receptors and cyclic AMP responses to epinephrine in cultured human fibroblasts at various population densities

The beta-adrenergic receptors of the intact human lung diploid fibroblast line Wl-38 and an SV-40 transformed clone of Wl-38, Wl-38-VA-13-2RA (VA13), were estimated in experiments utilizing the beta-adrenergic ligand, 125l-hydroxybenzylpindolol (125IHYP). When specific 125IHYP binding was measured in cells grown to relatively low population densities (0.15x10(6)cells/35mm dish), both Wl-38 and VA13 cells had approximately 40,000 beta-adrenergic receptors per cell. Wl-38 cells, when cultured to a high population density (0.5x10(6) cells/35/mm dish) had clearly diminished numbers of beta-adrenergic receptors and greatly decreased cAMP responses to epinephrine stimulation. On the other hand, in VA13 cells, neither the receptor number nor the beta-adrenergic response was affected by cell population density. In Wl-38 cells, the diminished cAMP response to epinephrine paralleled the decrease in number of beta-adrenergic receptors. Prostaglandin E1 (PGE1) stimulation of cAMP levels was unaffected by cell population density in either Wl-38 or VA13 cells. Thus, increased cell population density, perhaps related to density dependent inhibition of growth, caused a specific diminution in 125IHYP binding concomitant with decreased cAMP responses to epinephrine.     

Propionic and methylmalonic acids increase cAMP levels in slices of cerebral cortex of young rats via adrenergic and glutamatergic mechanisms

We have previously described that propionic (PA) and methylmalonic (MMA) acids increased the in vitro phosphorylation of cytoskeletal proteins through cAMP-dependent protein kinase and glutamate. In the present study we investigated the in vitro effects of 1 mM glutamate, 2.5 mM MMA and 2.5 mM PA on cAMP levels in the slices of cerebral cortex of young rats. Results showed that PA, MMA and glutamate increased cAMP levels after 30 min of incubation, while the beta-adrenergic agonist epinephrine elicited a similar effect only at a shorter incubation time. Then effects were prevented by the beta-adrenergic antagonist propranolol, rather than by glutamate antagonists (AP5, CNQX and MCPG), suggesting that they were mediated by beta-adrenergic receptors. In addition, glutamate antagonists per se induced increased cAMP levels; however propranolol prevented only the effect elicited by the metabotropic glutamate antagonist MCPG. Taken together, it is feasible that PA and MMA increase cAMP synthesis via a beta-adrenergic/G protein coupled pathway, in a glutamate-dependent manner. Although additional studies will be necessary to evaluate the importance of these observations for the neuropathology of propionic and methylmalonic acidemias, it is possible that high brain cAMP levels may contribute to a certain extent to the neurological dysfunction of the affected individuals.     

The rainbow trout skeletal muscle beta-adrenergic system: characterization and signaling

The presence and functionality of beta-adrenoceptors (beta-ARs) were examined in red (RM) and white muscle (WM) membranes isolated from the rainbow trout Oncorhynchus mykiss. Specific binding assays revealed the presence of a single class of binding sites with similar affinities in both muscle types (K(d) in nM: 0.14 +/- 0.03 and 0.18 +/- 0.03 for RM and WM, respectively) but with a significantly higher number of binding sites in RM compared with WM (B(max) in fmol/mg protein: 3.22 +/- 0.11 and 2.60 +/- 0.13, respectively). Selective and nonselective beta-adrenergic agonists (beta-AAs) and antagonists indicated an atypical beta-AR pharmacology. This result may represent a nonmammalian beta-AR classification or, more likely, the presence of more than one beta-AR subtype in trout muscles with similar affinities that could not be kinetically resolved. Adenylyl cyclase (ACase) assays showed a dose-dependent increase in cAMP production as concentrations of beta(2)-AAs increased in both muscle membranes with significantly higher basal cAMP production in RM compared with WM (cAMP production in pmol cAMP. mg protein(-1). 10 min(-1): 24.67 +/- 3.06 and 9.64 +/- 3.45, respectively). The agonist-induced increase in cAMP production was blocked by the beta-adrenergic antagonist propranolol, while the ACase activator forskolin increased cAMP production by 7- to 14-fold above basal and approximately 3-fold above all beta-AAs tested. This study demonstrated the presence of atypical beta(2)-ARs on RM and WM membranes of trout, suggesting that beta(2)-AAs may be a tool to enhance protein accretion through this signaling pathway.     

Inhibition by verapamil of neutrophil responses to formylmethionylleucylphenylalanine and phorbol myristate acetate. Mechanisms involving Ca2+ changes, cyclic AMP and protein kinase C

Verapamil inhibits in human neutrophils the respiratory burst, the secretion and the change of transmembrane potential induced by formylmethionylleucylphenylalanine, a Ca2+-dependent stimulus, and by phorbol myristate acetate, a Ca2+-independent stimulus. Besides the blocking of Ca2+ channels, many mechanisms are responsible for the inhibition of neutrophil responses. In fact, verapamil (i) increases the intracellular cAMP concentration, potentiates the cAMP response induced by the chemotactic peptide and induces the appearance of a cAMP response also when the stimulant is phorbol myristate acetate; (ii) causes a decrease of Ca2+ association to cell membranes, so depleting the pools of exchangeable Ca2+ and depressing the 'Ca2+ response' in terms of rise in [Ca2+]i monitored with Quin 2 and of rapid mobilization from cell membranes monitored by chlorotetracycline fluorescence change; (iii) inhibits the Ca2+-activated phospholipid-dependent protein kinase C. The data, discussed in relation to the biochemical mechanisms of the stimulus-response coupling, are compatible with the hypothesis of an involvement of the activation of protein kinase C as key step in the sequence of transduction events for the induction of many neutrophil functions.     

Desensitization of the turkey erythrocyte beta-adrenergic receptor in a cell-free system. Evidence that multiple protein kinases can phosphorylate and desensitize the receptor

We have used a recently developed cell-free system (cell lysate) derived from turkey erythrocytes to explore the potential role of cAMP-activated and other protein kinase systems in desensitizing the adenylate cyclase-coupled beta-adrenergic receptor. Desensitization by the agonist isoproterenol required more than simple occupancy of the receptor by the agonist since under conditions where adenylate cyclase was not activated, no desensitization occurred. As in whole cells, addition of cyclic nucleotides to the cell lysate produced only approximately 50% of the maximal isoproterenol-induced desensitization obtainable. Addition of the purified cAMP-dependent protein kinase holoenzyme plus isoproterenol to isolated turkey erythrocyte plasma membranes mimicked the submaximal desensitization induced in lysates by cAMP. This effect was entirely blocked by the specific inhibitor of the cAMP-dependent protein kinase. By contrast, maximal desensitization induced in lysates by isoproterenol was only approximately 50% attenuated by the protein kinase inhibitor. In the lysate preparations, isoproterenol was also shown to induce, in a stereospecific fashion, phosphorylation of the beta-adrenergic receptor. Phosphorylation promoted by isoproterenol was attenuated by cAMP-dependent protein kinase inhibitor to the same extent as desensitization (i.e. approximately 50%). Phorbol diesters also promoted receptor desensitization and phosphorylation in cell lysates. The desensitization was mimicked by incubation of isolated turkey erythrocyte membranes with partially purified preparations of protein kinase C plus phorbol diesters. In the cell lysate, calmodulin also promoted receptor phosphorylation and desensitization which was blocked by EGTA. Desensitization of adenylate cyclase by isoproterenol, phorbol diesters, and calmodulin was not observed to be additive. These findings suggest that: (a) multiple protein kinase systems, including cAMP-dependent, protein kinase C-dependent, and Ca2+/calmodulin-dependent kinases, are capable of regulating beta-adrenergic receptor function via phosphorylation reactions and that (b) cAMP may not be the sole mediator of isoproterenol-induced phosphorylation and desensitization in these cells.     

Coordinate regulation of membrane cAMP by Ca2+-inhibited adenylyl cyclase and phosphodiesterase activities

Activation of store-operated Ca(2+) entry inhibits type 6 adenylyl cyclase (EC; AC(6); Yoshimura M and Cooper DM. Proc Natl Acad Sci USA 89: 6712-6720, 1992) activity in pulmonary artery endothelial cells. However, in lung microvascular endothelial cells (PMVEC), which express AC(6) and turn over cAMP at a rapid rate, inhibition of global (whole cell) cAMP is not resolved after direct activation of store-operated Ca(2+) entry using thapsigargin. Present studies sought to determine whether the high constitutive phosphodiesterase activity in PMVECs rapidly hydrolyzes cAMP so that Ca(2+) inhibition of AC(6) is difficult to resolve. Direct stimulation of adenylyl cyclase using forskolin and inhibition of type 4 phosphodiesterases using rolipram increased cAMP and revealed Ca(2+) inhibition of AC(6). Enzyme activity was assessed using PMVEC membranes, where Ca(2+) and cAMP concentrations were independently controlled. Endogenous AC(6) activity exhibited high- and low-affinity Ca(2+) inhibition, similar to that observed in C6-2B cells, which predominantly express AC(6). Ca(2+) inhibition of AC(6) in PMVEC membranes was observed after enzyme activation and inhibition of phosphodiesterase activity and was independent of the free cAMP concentration. Thus, under basal conditions, the constitutive type 4 phosphodiesterase activity rapidly hydrolyzes cAMP so that Ca(2+) inhibition of AC(6) is difficult to resolve, indicating that high phosphodiesterase activity works coordinately with AC(6) to regulate membrane-delimited cAMP concentrations, which is important for control of cell-cell apposition.