Nerve growth factor-induced alteration in the response of PC12 pheochromocytoma cells to epidermal growth factor

PC12 cells, which differentiate morphologically and biochemically into sympathetic neruonlike cells in response to nerve growth fact, also respond to epidermal growth factor. The response to epidermal growth factor is similar in certain respects to the response to nerve growth fact. Both peptides produce rapid increases in cellular adhesion and 2-deoxyglucose uptake and both induce ornithine decarboxylase. But nerve growth factor causes a decreased cell proliferation and a marked hypertrophy of the cells. In contrast, epidermal growth factor enhances cell proliferation and does not cause hypertrophy. Nerve growth factor induces the formation of neuritis; epidermal growth factor does not. When both factors are presented simultaneously, the cells form neurites. Furthermore, the biological response to epidermal growth fact, as exemplified by the induction of ornithine decarboxylase, is attenuated by prior treatment of the cells with nerve growth factor. PC12 cells have epidermal growth factor receptors. The binding of epidermal growth factor to these receptors is rapid and specific, and exhibits an equilibrium constant of 1.9 x 10(-9) M. Approximately 80,000 receptors are present per cell, and this number is independent of cell density. Treatment of the cells with nerve growth factor reduces the amount of epidermal growth factor binding by at least 80 percent. The decrease in receptor binding begins after approximately 12-18 h of nerve growth factor treatment and is complete within 3 d. Scratchard plots indicate that the number of binding sites decreases, not the affinity of the binding sites for epidermal growth factor.     

Regulation of adenylate cyclase in cultured cardiocytes from neonatal rats by adenosine and other agonists

The presence of adenosine receptors coupled to adenylate cyclase in cultured cardiocytes from atria and ventricles from neonatal rats is demonstrated in these studies. N-Ethylcarboxamideadenosine (NECA), l-N⁶-phenylisopropyladenosine (PIA), and 2-chloroadenosine (2-cl-Ado) stimulated adenylate cyclase in a concentration-dependent manner in both cultured atrial and ventricular cells. The order of potency of stimulation was NECA > PIA > 2-cl-Ado. The stimulation of adenylate cyclase by NECA was enhanced by guanine nucleotides and was blocked by 3-isobutyl-1-methylxanthine in both these cells. Other agonists such as epinephrine, norepinephrine, dopamine, F^?, and forskolin were also able to stimulate adenylate cyclase, although the extent of stimulation by these agents was higher in ventricular than in atrial cells. The stimulation of adenylate cyclase by epinephrine and norepinephrine was inhibited by propranolol but not by phentolamine. On the other hand, phentolamine, propranolol, and haloperidol inhibited dopamine-stimulated adenylate cyclase activity to the same extent. Forskolin, at its maximal concentration, potentiated the stimulatory effect of epinephrine, norepinephrine, and dopamine on adenylate cyclase in both atrial and ventricular cardiocytes, but the interaction of NECA with epinephrine, norepinephrine, or dopamine was different in atrial and ventricular cells. The stimulation by an optimal concentration of NECA was additive with maximal stimulation by the catecholamines in atrial cells but not in ventricular cells. The data suggest the existence of adenosine “Ra” and catecholamine receptors in cultured atrial and ventricular cardiocytes. It can be postulated that adenosine in addition to its role as a potent vasodilator might regulate cardiac performance through its interaction with “Ra” receptors associated with adenylate cyclase. The difference in the mode of interaction of adenosine with catecholamines in atrial and ventricular cells suggests that the mechanism by which these agents activate adenylate cyclase may be different in these cells.     

Analogs of caffeine: antagonists with selectivity for A2 adenosine receptors

Several analogs of caffeine have been investigated as antagonists at A2 adenosine receptors stimulatory to adenylate cyclase in membranes from rat pheochromocytoma PC12 cells and human platelets and at A1 adenosine receptors inhibitory to adenylate cyclase from rat fat cells. Among these analogs, 1-propargyl-3,7-dimethylxanthine was about 4- to 7-fold and 7-propyl-1,3-dimethylxanthine about 3- to 4-fold more potent than caffeine at A2 receptors of PC12 cells and platelets. At A1 receptors of fat cells, both compounds were about 2-fold less potent than caffeine. These caffeine analogs have an A1/A2 selectivity ratio of about 10-20 and are the first selective A2 receptor antagonists yet reported. The results may provide the basis for the further development of highly potent and highly selective A2 adenosine receptor antagonists.     

Adenosine regulates the release of adrenocorticotropic hormone (ACTH) from cultured anterior pituitary cells

We have recently shown the presence of adenosine receptors coupled to adenylate cyclase in anterior pituitary and in the present studies we have investigated the effects of adenosine on ACTH release. The R-site specific analogs of adenosine such as N-Ethylcarboxamide adenosine (NECA), L-N⁶-phenylisopropyl adenosine (PIA), 2-chloro-adenosine (2-Cl-Ado) all stimulated ACTH release in a dose-dependent manner. NECA was the most potent analog and stimulated ACTH release by about 170% with an apparent Ka of 0.1 µM, whereas PIA and 2-Cl-Ado were less potent and stimulated the release by about 110% and 125% with an apparent Ka of 0.2 and 0.4 µ-M respectively. The stimulation of ACTH release by NECA was inhibited by 3-isobutyl-1-methylxanthine (IBMX). On the other hand, adenosine deaminase (ADA) treatment of the cells also stimulated ACTH release as well as adenylate cyclase activity by about 2-fold, suggesting that endogenous adenosine plays an inhibitory role in the release of ACTH. Other agents, such as corticotropin-releasing factor (CRF), vasoactive intestinal peptide (VIP) and forskolin (FSK) also stimulated ACTH release from these cells. In addition, the stimulation by an optimal concentration of NECA was almost additive with maximal stimulation caused by VIP and FSK. These data suggest that adenosine modulates ACTH release from anterior pituitary through its interaction with adenosine receptors coupled to adenylate cyclase.Abbreviations NECA N-Ethylcarboxamideadenosine - PIA L-N⁶-Phenylisopropyladenosine - 2-Cl-Ado 2-chloroadenosine - FSK Forskolin - VIP Vasoactive Intestinal Peptide - CRF Corticotropin Releasing Factor - ADA Adenosine Deaminase - IBMX 3-Isobutyl-1-methylxanthine     

Adenosine receptor-mediated changes in cyclic AMP production and DNA synthesis in cultured arterial smooth muscle cells

The effects of adenosine and two analogs, L-phenylisopropyladenosine (L-PIA) and 5'-N-ethylcarboxamidoadenosine (NECA), on cAMP production and on platelet-derived growth factor (PDGF)-stimulated initiation of DNA synthesis in growth-arrested cultures of rat arterial smooth muscle cells (SMC) were studied. The intracellular cAMP concentration was dose-dependently enhanced by micromolar concentrations of adenosine and its analogs, with the potency order NECA greater than adenosine greater than L-PIA. The effect was antagonized, in a competitive manner, by the adenosine receptor antagonist 8-phenyltheophylline (8-PT). The stimulatory effect of adenosine was enhanced by 3 microM dipyridamole an adenosine-uptake blocker. DNA synthesis was inhibited in a parallel manner, showing the same potency order. The inhibition was antagonized by 8-PT. Forskolin, a diterpene with the ability to stimulate the catalytic unit of adenylate cyclase and thereby cAMP formation, potentiated the effects of micromolar concentrations of NECA and L-PIA. Forskolin, by itself, stimulated cAMP production and inhibited DNA synthesis. The forskolin-stimulated increase in cAMP was inhibited by L-PIA at nanomolar concentrations. L-PIA in the nanomolar concentration range also stimulated DNA synthesis when initiation was stimulated with suboptimal concentrations of PDGF. These findings suggest the presence of adenosine receptors of both the A1- and A2-subtype on SM-mediating bidirectional changes of cAMP and DNA synthesis.     

Stimulation of adenylate cyclase by adenosine and other agonists in mesenteric artery smooth muscle cells in culture

An adenosine-sensitive adenylate cyclase has been characterized in cultured mesenteric artery smooth muscle cells. N-Ethylcarboxamide-adenosine (NECA), N-Methylcarboxamide-adenosine (MECA), L-N6-phenylisopropyladenosine (PIA) and 2-chloroadenosine (2-cl-Ado) all stimulated adenylate cyclase in a concentration dependent manner. NECA was the most potent analog (EC50, 1 microM), whereas PIA (EC50, 15 microM), 2-Cl-Ado (EC50, 15 microM) and MECA (EC50, 24 microM), were less potent and had efficacies relative to NECA of 0.61, 0.61 and 0.65, respectively. Adenosine showed a biphasic effect: stimulation at lower concentrations and inhibition at higher concentrations, whereas 2' deoxyadenosine only inhibited adenylate cyclase activity. The stimulatory effect of NECA on adenylate cyclase was dependent on metal ion concentration and was blocked by 3-isobutyl-l-methylxanthine (IBMX) and 8-phenyltheophylline (8-PT). Adenylate cyclase from these cultured cells was also stimulated by other agonists such as epinephrine, norepinephrine, prostaglandins, dopamine, NaF and forskolin. The stimulation of adenylate cyclase by isoproterenol, epinephrine and norepinephrine was blocked by propranolol but not by phentolamine. On the other hand, phentolamine, propranolol and flupentixol all inhibited dopamine-stimulated adenylate cyclase activity. In addition, the stimulation by an optimal concentration of PIA was additive or almost additive with maximal stimulation caused by catecholamines and prostaglandins. These data indicate the presence of adenosine (Stimulatory "Ra"), catecholamine and prostaglandin receptors in mesenteric artery smooth muscle cells and suggest that these agents may exert their physiological actions through their interaction with their respective receptors coupled to adenylate cyclase.     

Regulation of adenylate cyclase by adenosine and other agonists in rat myocardial sarcolemma

The presence of adenosine receptors coupled to adenylate cyclase in rat heart sarcolemma is demonstrated in these studies. Heart sarcolemma was isolated by the hypotonic shock-Lithium bromide treatment method. This preparation contained negligible amounts (2-4%) of contamination by other subcellular organelles such as mitochondria, sarcoplasmic reticulum, and myofibrils as verified by electron microscopic examination. In addition this preparation was also devoid of endothelial cells, since angiotensin-converting enzyme activity was not detected in this preparation. N-Ethylcarboxamide adenosine (NECA), L-N6-phenylisopropyladenosine (PIA), and adenosine N'-oxide (Ado N'-oxide) were all able to stimulate adenylate cyclase in heart sarcolemma, but not in crude homogenate, with an apparent Ka of 3-7 microM. The activation of adenylate cyclase by NECA was dependent on the concentrations of metal ions such as Mg2+ or Mn2+. The maximal stimulation was observed at lower concentrations of the metal ions (0.2-0.5 mM). At 5 mM Mg2+ or Mn2+, the stimulation by NECA was completely abolished. The stimulatory effect of NECA on adenylate cyclase was also dependent on guanine nucleotides and was blocked by 3-isobutyl-1-methylxanthine. In addition, 2'-deoxyadenosine showed an inhibitory effect on adenylate cyclase. The myocardial adenylate cyclase was also stimulated by beta-adrenergic agonists, dopamine and glucagon, and inhibited by cholinergic agonists such as carbachol and oxotremorine. The stimulation of adenylate cyclase by NECA was found to be additive with maximal stimulation obtained by epinephrine. These data suggest that rat heart sarcolemma contains adenosine (Ra), beta-adrenergic, dopaminergic, glucagon, and cholinergic receptors, and the stimulation of adenylate cyclase by epinephrine and adenosine occurs by distinctly different mechanism or adenosine and epinephrine stimulate different cyclase populations.     

Both A1 and A2a Purine Receptors Regulate Striatal Acetylcholine Release

The receptors responsible for the adenosine-mediated control of acetylcholine release from immunoaffinity-purified rat striatal cholinergic nerve terminals have been characterized. The relative affinities of three analogues for the inhibitory receptor were (R)-phenylisopropyladenosine greater than cyclohexyladenosine greater than N-ethylcarboxamidoadenosine (NECA), with binding being dependent of the presence of Mg2+ and inhibited by 5'-guanylylimidodiphosphate [Gpp(NH)p] and adenosine receptor antagonists. Adenosine A1 receptor agonists inhibited forskolin-stimulated cholinergic adenylate cyclase activity, with an IC50 of 0.5 nM for (R)-phenylisopropyladenosine and 500 nM for (S)-phenylisopropyladenosine. A1 agonists inhibited acetylcholine release at concentrations approximately 10% of those required to inhibit the cholinergic adenylate cyclase. High concentrations (1 microM) of adenosine A1 agonists were less effective in inhibiting both adenylate cyclase and acetylcholine release, due to the presence of a lower affinity stimulatory A2 receptor. Blockade of the A1 receptor with 8-cyclopentyl-1,3-dipropylxanthine revealed a half-maximal stimulation by NECA of the adenylate cyclase at 10 nM, and of acetylcholine release at approximately 100 nM. NECA-stimulated adenylate cyclase activity copurified with choline acetyltransferase in the preparation of the cholinergic nerve terminals, suggesting that the striatal A2 receptor is localized to cholinergic neurones. The possible role of feedback inhibitory and stimulatory receptors on cholinergic nerve terminals is discussed.     

Adenosine receptor-adenylate cyclase system in the trout testis: involvement in the regulation of germ cell proliferation

To ascertain the presence of adenosine receptors in the trout testis, cells isolated from testes at different spermatogenetic stages were cultured in the presence or absence of adenosine, adenosine receptor agonists, or antagonists and of cAMP analogs, for up to 20 min, or 20 hr, or 4.5 days. Cyclic AMP production was then assayed or 3H-thymidine incorporation was measured. Cellular content of cAMP was enhanced by adenosine, by the adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA), and by 2-p(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS-21680), an adenosine A2A receptor-selective agonist. The increase in cAMP induced by the adenylate cyclase activator L-858051 was inhibited by the adenosine A1)receptor-selective agonists R-N6-(2-phenylisopropyl)adenosine (R-PIA) and N6-cyclopentyladenosine (CPA). These effects were antagonized by the two adenosine A2)receptor antagonists 3,7-dimethyl-1-propargylxanthine (DMPX) and 8-(3-chlorostyryl)caffeine (CSC), and by the adenosine A1)receptor-selective antagonist 8-cyclopentyl-1,3dipropylxanthine (CPX), respectively. Increase in the cAMP content induced by adenosine was inhibited by the cell permeable adenylate cyclase inhibitor 2',5'-dideoxyadenosine. These data suggest that A(1) and A(2) adenosine receptors which respectively inhibit and stimulate adenylate cyclase activity are present on trout testicular cells (unidentified), while the presence of A3 adenosine receptor subtype was not apparent. 3H-thymidine incorporation decreased in the presence of the adenylate cyclase activator L-858051 and of the cAMP analogs 8-CPT cAMP and Sp-5,6-DCI-cBiMPS, regardless of the presence or absence of the phosphodiesterase inhibitor RO 20-1724. This suggests that an increase in testicular cAMP may act as a negative growth regulator for the mitotic germ cells. In agreement with these data, the activation of A2 stimulatory receptors inhibited short-term (20 hr) DNA synthesis. However, the activation of A1 inhibitory receptors had the same effect. This suggests that events, cAMP-dependent or independent, induced by the activation of testicular adenosine receptors, may participate in the regulation of trout male germ cell proliferation.     

Nerve growth factor-induced reduction in epidermal growth factor responsiveness and epidermal growth factor receptors in PC12 cells: an aspect of cell differentiation.

The rat pheochromocytoma clone PC12 responds to nerve growth factor through the expression of a number of differentiated neuronal properties. One of the most rapid changes is a large, transient increase in the activity of ornithine decarboxylase. These cells also show an increase in ornithine decarboxylase activity in response to the mitogen, epidermal growth factor, but do not respond morphologically as they do to nerve growth factor. Specific, high-affinity epidermal growth factor receptors are present on the cells. When the cells are differentiated with nerve growth factor, the response to epidermal growth factor is markedly diminished and there is a marked reduction in the binding of epidermal growth factor to the cells.     

Pharmacological profile of adenosine A2 receptor in PC12 cells

The PC12 cell line, a clone isolated from a pheochromocytoma tumor of rat adrenal medulla, was shown to exclusively contain stimulatory adenosine (A2) receptors linked to adenylate cyclase (AC). AC was stimulated 6-7 fold by several agonists with a rank order of potency of 5'-N-Ethyl carboxamidoadenosine (NECA) greater than 2-Chloroadenosine (2-CADO) greater than (R)-N-Phenylisopropyladenosine (R-(-)-PIA) greater than N6-Cyclopentyladenosine (CPA) greater than N6-Cyclohexyladenosine (CHA) greater than S-(+)-PIA. AC activity was antagonized by a variety of adenosine receptor antagonists with a potency order of 1,3,-Dipropyl-8-(2-amino-4-chlorophenyl)xanthine (PACPX) greater than 1,3,-Diethyl-8-phenylxanthine (DPX) greater than 8-Phenyltheophylline greater than 3-Isobutyl-1-methylxanthine (IBMX) greater than 8-(p-sulfophenyl)theophylline (PST) greater than 7-(beta-chloroethyl)theophylline greater than theophylline = enprofylline = caffeine. Under conditions known to favour receptor-mediated Ni-coupled inhibition of AC, R-(-)-PIA failed to inhibit both basal and forskolin stimulated AC activity in PC12 cells, confirming the absence of an A1 mediated response. On the other hand, adenosine agonists inhibited AC activity in rat cortical membranes with a rank order of potency of CPA greater than R-(-)-PIA greater than CHA greater than NECA greater than S-(+)-PIA greater than 2-CADO. These findings suggest that PC12 cells are functionally deficient in an A1 receptor linked AC response but are efficiently coupled to A2 stimulatory receptors. The cells should prove useful for further study of A2 adenosine receptors and to establish selectivity profiles of compounds acting at both A1 and A2 receptors.     

Histidine regulation of cyclic AMP metabolism in cultured renal epithelial LLC-PK1 cells.

L-Histidine and imidazole (the histidine side chain) significantly increase cAMP accumulation in intact LLC-PK1 cells. This effect is completely inhibited by isobutylmethylxanthine (IBMX). Histidine and imidazole stimulate cAMP phosphodiesterase activity in soluble and membrane fractions of LLC-PK1 cells suggesting that the IBMX-sensitive effect of these agents to stimulate cAMP formation is not due to inhibition of cAMP phosphodiesterase. Histidine and imidazole but not alanine (the histidine core structure) increase basal, GTP-, forskolin-, and AVP-stimulated adenylate cyclase activity in LLC-PK1 membranes. Two other amino acids with charged side chains (aspartic and glutamic acids) increase AVP-stimulated but neither basal- nor forskolin-stimulated adenylate cyclase activity. This suggests that multiple amino acids with charged side chains can regulate selected aspects of adenylate cyclase activity. To better define the mechanism of histidine regulation of adenylate cyclase, membranes were detergent-solubilized which prevents histidine and imidazole potentiation of forskolin-stimulated adenylate cyclase activity and suggests that an intact plasma membrane environment is required for potentiation. Neither pertussis toxin nor indomethacin pretreatment alter imidazole potentiation of adenylate cyclase. IBMX pretreatment of LLC-PK1 membranes also prevents imidazole to potentiate adenylate cyclase activity. Since IBMX inhibits adenylate cyclase coupled adenosine receptors, LLC-PK1 cells were incubated in vitro with 5'-N-ethylcarboxyamideadenosine (NECA) which produced a homologous pattern of desensitization of NECA to stimulate adenylate cyclase activity. Despite homologous desensitization, histidine and imidazole potentiation of adenylate cyclase was unaltered. These data suggest that histidine, acting via an imidazole ring, potentiates adenylate cyclase activity and thereby increases cAMP formation in cultured LLC-PK1 epithelial cells. This potentiation requires an intact plasma membrane environment, occurs independent of a pertussis toxin-sensitive substrate and of products of cyclooxygenase, and is inhibited by IBMX. This IBMX-sensitive pathway does not involve either inhibition of cAMP phosphodiesterase activity or a stimulatory adenosine receptor coupled to adenylate cyclase.     

Regulation by adenosine of the vasopressin-sensitive adenylate cyclase in pig-kidney cells (LLC-PK1L) grown in defined media

LLC-PK1L cells, a kidney-derived cell line, had sustained growth in a defined medium. When compared to the parent cell line growing with 10% fetal bovine serum, LLC-PK1L cells had about 100-times fewer vasopressin receptors. Upon modifications of the cell culture medium, the vasopressin response of the adenylate cyclase could be increased by more than 10-fold with a parallel increase in vasopressin receptor number. Using cells with high or low receptor densities, the stimulatory and inhibitory effects of N6-L-2-phenylisopropyl-adenosine on the modulation of the adenylate cyclase responsiveness to vasopressin were investigated. When high concentrations of GTP were added, low concentrations of phenylisopropyladenosine inhibited the enzyme, while higher concentrations were found to be stimulatory. The adenylate cyclase activity stimulated by vasopressin could only be inhibited by phenylisopropyladenosine under these conditions in membranes with high receptor density; only the increase in enzyme activity due to high GTP concentration was inhibitable. The analysis of the dependency of the adenylate cyclase activity as a function of the vasopressin concentration showed that, besides reducing the maximum velocity of the system for vasopressin, the addition of phenylisopropyladenosine generated an heterogeneity in the adenylate cyclase response to vasopressin (as judged by a curvilinear Eadie plot). A high-affinity component in the adenylate cyclase response appeared when phenylisopropyladenosine was added. The growth of the cells in a medium containing adenosine deaminase gave results identical to those obtained for control cells. However, growing the cells with both phenylisopropyladenosine and adenosine deaminase abolished the inhibitory effects of the former on the adenylate cyclase and greatly reduced its stimulatory action. Under these conditions, the vasopressin response of the adenylate cyclase was not further regulated by phenylisopropyladenosine. These results indicate a role of adenosine on vasopressin response, especially at low physiological concentrations of the hormone where a high-affinity component of the hormonal response could be demonstrated.     

Definition of subclasses of adenosine receptors associated with adenylate cyclase: interaction of adenosine analogs with inhibitory A1 receptors and stimulatory A2 receptors

The structure-activity relationships of 63 adenosine analogs as agonists for the A1 adenosine receptors that mediate inhibition of adenylate cyclase activity in rat fat cells and for the A2 adenosine receptors that mediate stimulation of adenylate cyclase in rat pheochromocytoma PC12 cells and human platelets were determined. The lack of correspondence between the structure-activity relationships of these analogs at the A1 and A2 receptors appear definitive in terms of establishing the existence of A1 and A2 subclasses of adenosine receptors. However, significant differences in the agonist profiles at A2 receptors of platelet and PC12 indicate a certain degree of structural heterogeneity within the members of the A2 adenosine receptor subclass. Whether such differences are due to different species or different cell types is not known. A set of adenosine analogs, such as N6-cyclohexyl-, N6-R-, and S-1-phenyl-2- propyladenosines, 5'-N-ethylcarboxamidoadenosine and its N6-cyclohexyl derivative, 2-chloroadenosine, and 2-phenylaminoadenosine, appear to represent a series of analogs useful for pharmacological characterization of A1 and A2 classes of adenosine receptors.     

Thyroid hormone differentially regulates development of beta-adrenergic receptors, adenylate cyclase and ornithine decarboxylase in rat heart and kidney.

In mature animals, thyroid hormone produces parallel up-regulation of beta-adrenergic receptor binding sites and their linkage to adenylate cyclase; during development, these same processes may be critical in establishing the set-point for subsequent adrenergic reactivity. In the current study, we administered triiodothyronine to neonatal rats for the first five days postpartum and evaluated [125I]pindolol binding capabilities and adenylate cyclase activity in membrane preparations from heart and kidney. In the heart, hyperthyroidism elicited an initial increase in receptor density, with subsequent deficits and an eventual return to normal values by young adulthood. In contrast, the ability of isoproterenol, a beta-adrenergic agonist, to stimulate adenylate cyclase was enhanced regardless of whether receptor numbers were increased or decreased; the same effects were also present for basal adenylate cyclase activity and non-receptor-mediated stimulation by forskolin. Enhanced cyclase activity involved both increases in the magnitude of response as well as accelerated onset of the postweaning peak of enzyme activity, results which suggest a direct impact of thyroid status on the ontogenetic expression of adenylate cyclase itself. The kidney, which possesses less efficient beta-receptor coupling to adenylate cyclase in the neonate, was less drastically affected by triiodothyronine for either beta-receptor binding sites or enzyme activity. As we had previously shown that neonatal hyperthyroidism uncouples beta-receptors from growth-related enzymes, such as ornithine decarboxylase, we also evaluated whether the promotion of adenylate cyclase responses was mechanistically linked to effect on ornithine decarboxylase; administration of cyclic AMP analogs to 5 days-old rats led to inhibition of the enzyme in the heart, whereas the same treatment in 9 days-old animals was ineffective. These data suggest that thyroid hormone differentially regulates the development of beta-receptors as well as adenylate cyclase and ornithine decarboxylase, with preferential effects on tissues, such as the heart, that already possess efficient linkage of the receptors to cell transduction mechanisms at birth.     

An Investigation of the Low Intrinsic Activity of Adenosine and Its Analogs at Low Affinity (A2) Adenosine Receptors in Rat Cerebral Cortex

The potencies and intrinsic activities of adenosine analogs for stimulating cyclic AMP accumulation in slices of rat cerebral cortex were examined. 5'-N-Ethylcarboxamidoadenosine (NECA) caused the greatest increase in cyclic AMP accumulation (19.2-fold). 2-Chloroadenosine (2-CAD) induced a similar increase, but adenosine and six other analogs caused much smaller increases. All agonists tested had similar potencies in activating this response. Inhibition of adenosine uptake with 10 microM dipyridamole did not affect the maximal response to any agonist, although the potency of adenosine was increased approximately threefold. Each analog was also able to block partially the stimulation of cyclic AMP accumulation caused by NECA. Levels of cyclic AMP accumulation in the presence of NECA plus another analog were similar to those observed when the analog alone was present, as expected for partial agonists. Furthermore, the EC50 value for R-(-)-N6(2-phenylisopropyl)adenosine in increasing cyclic AMP accumulation was similar to the KI value for inhibiting the response to NECA. The EC50 value for adenosine was substantially higher than the KI value for inhibiting the response to NECA; however, in the presence of dipyridamole, the two values were more closely correlated. The response to NECA was blocked by 8-phenyltheophylline, 1,3-diethyl-8-phenylxanthine, and 8-p-sulfophenyltheophylline, with KI values from 1 to 10 microM. The results suggest that adenosine analogs stimulate cyclic AMP accumulation in cerebral cortex through low-affinity receptors, but that some analogs only partially activate these receptors. Adenosine itself may also be a partial agonist, or its actions may be obscured by simultaneous activation of another receptor.     

Functionalized congeners of 1,3-dipropyl-8-phenylxanthine: potent antagonists for adenosine receptors that modulate membrane adenylate cyclase in pheochromocytoma cells, platelets and fat cells

Six amine, amino acid and peptide derivatives derived from 1,3-dipropyl-8-(p-carboxymethylphenyl)xanthine, a functionalized congener of 1,3-dipropyl-8-phenylxanthine, have been investigated as antagonists at A2 adenosine receptors stimulatory to adenylate cyclase in membranes from rat pheochromocytoma PC 12 cells and human platelets and at A1 adenosine receptors inhibitory to adenylate cyclase from rat fat cells. The functionalized congeners and conjugates have affinity constants ranging from 80 to 310 nM at A2 receptors of PC 12 cells and from 25 to 135 nM at those of platelets. The affinity of the xanthine derivatives at A1 receptors of fat cells are in the 15 to 30 nM range. Thus, the amino acid and peptide conjugates have high potencies at both receptor subclasses and show some selectivity toward A1 adenosine receptors. Derivatives of the congeners should be useful as receptor probes and as radioiodinated ligands.     

Activation of phospholipase C via adenosine receptors provides synergistic signals for secretion in antigen-stimulated RBL-2H3 cells. Evidence for a novel adenosine receptor

5'-(N-Ethyl)carboxamidoadenosine (NECA), an analog of adenosine, transiently stimulated a rat tumor mast cell (RBL-2H3 cells) to cause a release of inositol phosphates and an increase in levels of Ca2+ in the cytosol. It failed, however, to stimulate a sustained uptake of 45Ca2+ or secretion. The effects of other agents that act on P1- or P2-purinergic receptors suggested that NECA and other adenosine agonists acted via a novel subtype of adenosine membrane receptor. Although the order of potency of agonists was characteristic of A2-adenosine receptors, there was no indication of the involvement of adenylate cyclase, and antagonists such as isobutylmethylxanthine, 8-phenyltheophylline, and 8-p-sulfophenyltheophylline inhibited the responses to either NECA or antigen. The fact that stimulation of inositol phospholipid hydrolysis by NECA in washed, permeabilized RBL-2H3 cells was blocked by pertussis toxin as well as by cholera toxin suggested instead that the NECA-sensitive receptor activated phospholipase C via a G-protein. In contrast to NECA, antigen stimulation resulted in a pertussis toxin-resistant, sustained hydrolysis of inositol phospholipids, increases in free intracellular Ca2+, accelerated influx of 45Ca2+, and secretion from RBL-2H3 cells. In combination with NECA, all responses to antigen were markedly enhanced, and the enhancement was selectively blocked by pertussis toxin. The ability of antigen, but not NECA, to provoke secretion may be dependent primarily on the sustained activation of a cholera toxin-sensitive Ca2+ influx pathway that serves to amplify stimulatory signals for secretion. These studies also suggested that phospholipase C could be activated through different G-proteins via different receptors within the same cell.     

The effects of nerve growth factor on polyamine metabolism in PC12 cells

Nerve growth factor treatment produces a large increase in the activity of ornithine decarboxylase and a moderate decrease in the activity of S-adenosylmethionine decarboxylase in PC12 cells. These changes are reflected weakly, if at all, in the levels of putrescine, spermidine, and spermine in the cells. The rates of polyamine synthesis are increased somewhat more than the overall levels, but still are not comparable in extent to the increase in the ornithine decarboxylase activity. Inhibitors of ornithine decarboxylase and S-adenosylmethionine decarboxylase have their expected effects on the induction of ornithine decarboxylase and on the activities of both enzymes. Neither inhibitor alone, nor a combination of inhibitors, altered the rate or extent of nerve growth factor-induced neurite outgrowth in the cells.