Overexpression of myocardial Gsalpha prevents full expression of catecholamine desensitization despite increased beta-adrenergic receptor kinase.

Inotropic and chronotropic responses to catecholamines in young adult transgenic mice overexpressing myocardial Gsalpha are enhanced. One might predict that over the life of the animal, this chronically enhanced beta-adrenergic receptor stimulation would result in homologous catecholamine desensitization. To test this hypothesis, old transgenic Gsalpha mice and age-matched controls were studied physiologically in terms of responsiveness of left ventricular function (ejection fraction) to isoproterenol in vivo and in vitro in terms of beta-adrenergic receptor signaling. Old transgenic mice still responded to isoproterenol with augmented (P < 0.05) left ventricular ejection fraction (+44+/-3%) compared with age-matched controls (+24+/-1%). Although total beta-adrenergic receptor density was reduced in the old transgenic mice, and G protein receptor kinase 2 (beta-adrenergic receptor kinase) levels were increased, the fraction of receptors binding agonist with high affinity as well as isoproterenol- and G protein-stimulated adenylyl cyclase activities were enhanced. Thus, classical catecholamine desensitization is not effective in attenuation of persistently enhanced responses to sympathetic stimulation in mice overexpressing myocardial Gsalpha. To support this conclusion further, experiments were performed with chronic isoproterenol, which elicited effective desensitization in wild-type controls, but failed to elicit desensitization in overexpressed Gsalpha mice. The results of this study suggest that the lack of protective desensitization mechanisms may be responsible in part for the dilated cardiomyopathy which develops with chronic sympathetic stress over the life of these animals.     

Reduced beta-adrenergic receptor activation decreases G-protein expression and beta-adrenergic receptor kinase activity in porcine heart.

To determine whether beta-adrenergic receptor agonist activation influences guanosine 5'-triphosphate-binding protein (G-protein) expression and beta-adrenergic receptor kinase activity in the heart, we examined the effects of chronic beta 1-adrenergic receptor antagonist treatment (bisoprolol, 0.2 mg/kg per d i.v., 35 d) on components of the myocardial beta-adrenergic receptor-G-protein-adenylyl cyclase pathway in porcine myocardium. Three novel alterations in cardiac adrenergic signaling associated with chronic reduction in beta-adrenergic receptor agonist activation were found. First, there was coordinate downregulation of Gi alpha 2 and Gs alpha mRNA and protein expression in the left ventricle; reduced G-protein content was also found in the right atrium. Second, in the left ventricle, there was a twofold increase in beta-adrenergic receptor-dependent stimulation of adenylyl cyclase and a persistent high affinity state of the beta-adrenergic receptor. Finally, there was a reduction in left ventricular beta-adrenergic receptor kinase activity, suggesting a previously unrecognized association between the degree of adrenergic activation and myocardial beta-adrenergic receptor kinase expression. The heart appears to adapt in response to chronic beta-adrenergic receptor antagonist administration in a manner that would be expected to offset reduced agonist stimulation. The mechanisms for achieving this extend beyond beta-adrenergic receptor upregulation and include alterations in G-protein expression, beta-adrenergic receptor-Gs interaction, and myocardial beta-adrenergic receptor kinase activity.     

Uncoupling of the beta-adrenergic receptor as a mechanism of in vitro neutrophil desensitization

Human leukocytes have been useful in studying desensitization phenomena to beta-adrenergic agonists in a number of clinical conditions. For example, we have previously shown that oral terbutaline causes a time-dependent decrease in neutrophil (PMN) beta receptor number, using the beta antagonist ligand [3H]dihydroalprenolol (DHA), in conjunction with a significant loss of isoproterenol-induced adenylate cyclase activity. In the present in vitro study we have explored the mechanism for beta-adrenergic desensitization and have compared conditions for homologous and heterologous desensitization, using the intact PMN model. PMN preincubated with isoproterenol (10(-4)M), washed thoroughly, then restimulated, desensitize rapidly so that within 10 min 80% of control isoproterenol-induced cyclic AMP stimulation is lost. Cells washed free of isoproterenol recover full responsiveness in 1 to 2 hr. The estimated isoproterenol desensitization EC50 in cells washed and then restimulated is 1 X 10(-5)M, and the EC50 in unwashed cells that are restimulated is 9 X 10(-8)M. Rank-order potency studies of catecholamine desensitization show isoproterenol greater than epinephrine greater than norepinephrine, a beta-2 pattern. Isoproterenol-induced desensitization results in a small reduction in [3H]DHA binding sites, which becomes statistically significant (p less than 0.05) from control values at 1 hr (67% of control) and 3 hr (64%). Since the change in number of beta receptors did not explain the profound, rapid loss of beta agonist-induced cyclic AMP responsiveness, we explored the possibility of an uncoupling phenomenon. In the absence of GTP, isoproterenol binding is characterized by an EC50 of 6.6 +/- 2.6 X 10(-7)M, which is significantly different (p less than 0.05) from the EC50 of 38.1 +/- 9.1 X 10(-7)M found when cells are previously desensitized with isoproterenol for 10 min. GTP does not affect the EC50 of desensitized cells. These findings are consistent with the uncoupled receptor state fitting the model described by Su et al. Finally, prolonged (3 hr) isoproterenol preincubation results in a small but significant (p less than 0.05) loss of cyclic AMP responsiveness to histamine (67.7% +/- 11.7 of control) and PGE1 (59.3% +/- 7.4), suggesting heterologous desensitization. These studies suggest that the human PMN is a suitable model to study both homologous and heterologous desensitization in vitro.     

Regulation of G protein-coupled receptor kinase subtypes in activated T lymphocytes. Selective increase of beta-adrenergic receptor kinase 1 and 2.

Beta-adrenergic receptor kinase (beta ARK) is a serine-threonine kinase involved in the process of homologous desensitization of G-coupled receptors. beta ARK is a member of a multigene family, consisting of six known subtypes, also named G protein-coupled receptor kinases (GRK 1-6). In this study we investigated the expression of GRKs during the process of T cell activation, which is of fundamental importance in regulating immune responses. T cell activation was induced by exposing mononuclear leukocytes (MNL) to PHA and confirmed by tritiated thymidine incorporation measurement. A substantial increase of GRK activity (as measured by in vitro phosphorylation of rhodopsin) was found after 48 h (331 +/- 80% of controls) and 72 h (347 +/- 86% of controls) of exposure to PHA. A threefold increase of beta ARK1 immunoreactivity was found in MNL exposed to PHA for 72 h. Persistent activation of protein kinase C (PKC) by 10 nM 12-O-tetradecanoylphorbol-13-acetate (TPA) was able to increase beta ARK activity to the same extent as PHA, suggesting a PKC-mediated mechanism. The kinetic of beta-adrenergic-stimulated cAMP production was substantially modified in TPA and PHA-activated cells, indicating that the increased GRK activity resulted in an increased beta-adrenergic homologous desensitization. A three- to fourfold increase in GRK activity was also observed in a population of T cell blasts (> 97% CD3+) exposed to PHA for 48-72 h. A significant increase in beta ARK1 and beta ARK2 mRNA expression was observed 48 h after mitogen stimulation, while mRNA expression of GRK5 and GRK6 was not changed. In conclusion our data show that the expression of GRK subtypes is actively and selectively modulated according to the functional state of T lymphocytes.     

Role of beta-adrenergic receptor signaling and desensitization in heart failure: new concepts and prospects for treatment

The use of beta-blockers to antagonize beta-adrenergic receptor signaling in the heart has become a standard method of treatment for heart failure, resulting in positive clinical outcomes alone and in conjunction with other modulators of cardiomyocyte contractility. However, an entire explanation for improved cardiac function in patients using beta-blockers is unknown, and in fact may be quite complicated, considering the numerous intracellular signaling pathways associated with beta-adrenergic receptors. Stimulation of beta-adrenergic receptors during both normal conditions and during heart failure activate several distinct signaling cascades, which influence cardiomyocyte contraction, hypertrophy and apoptosis. This review explores the signaling cascades induced by beta-adrenergic receptor activation in normal and desensitized states to provide new insight into the effective treatment of cardiac dysfunction.     

Development of glucagon sensitivity in neonatal rat liver.

The ontogenesis of the hepatic glucagon-sensitive adenylate cyclase system has been studied in the rat. With a partially purified liver membrane preparation, fetal adenylate cyclase was less responsive to glucagon than the enzyme from neonatal or adult livers. Similar results were obtained in gently prepared liver homogenates, suggesting that destruction of essential components of the fetal liver membrane did not account for the relative unresponsiveness of the adenylate cyclase enzyme to glucagon. Investigation of other factors that might account for diminished fetal hepatic responsiveness to glucagon indicate (a) minimal glucagon degradation by fetal membranes relative to 8-day or adult tissue; and (b) available adenylate cyclase enzyme, as suggested by a 13-fold increase over basal cyclic AMP formation with NaF in fetal liver membranes. These results indicate that neither enhanced glucagon degradation nor adenylate cyclase enzyme deficiency accounts for the relative insensitivity of the fetal hepatic adenylate cyclase system to glucagon. In early neonatal life, hepatic adenylate cyclase responsiveness to glucagon rapidly developed and was maximal 6 days after birth. These changes were closely paralleled by a fivefold increase in glucagon binding and the kinetically determined Vmax for cyclic AMP formation. These observations suggest that (a) fetal hepatic unresponsiveness to glucagon may be explained by a limited number of glucagon receptor sites; (b) during the neonatal period, the development of glucagon binding is expressed primarily as an increase in adenylate cyclase Vmax; (c) the ontogenesis of hepatic responsiveness to glucagon may be important in the resolution of neonatal hypoglycemia.     

Agonist-induced serotonin 2A receptor desensitization in the rat frontal cortex and hypothalamus

This study examined the time course and possible mechanisms of agonist-induced desensitization of 5-hydroxytryptamine serotonin 2A receptors in the rat frontal cortex and hypothalamic paraventricular nucleus after 1, 4, and 7 days of treatment with (-)-1-(2,5-dimethoxy-4-iodophenyl)2-aminopropane HCl [(-)-DOI] (1 mg/kg i.p.), a selective 5-HT(2A/2C) receptor agonist. In the frontal cortex, 5-HT-mediated phospholipase C (PLC) enzyme activity decreased by 24 to 30% after 4 to 7 days of (-)-DOI treatment without any significant changes in the guanosine 5'-3-O-(thio)triphosphate-mediated PLC enzyme activity. Additionally, treatment with (-)-DOI did not significantly change the levels of G(alpha11), regulator of G protein signaling (RGS)4, or RGS7 proteins in the frontal cortex, whereas G(alphaq) protein levels in the frontal cortex decreased (47%) only after 7 daily (-)-DOI injections. The functional status of 5-HT(2A) receptors in the hypothalamic paraventricular nucleus was examined using 5-HT(2A) receptor-mediated increases in plasma hormone levels. Plasma adrenocorticotrophic hormone (ACTH) and oxytocin measurements showed that 5-HT(2A) receptor desensitization began after only 1 day of (-)-DOI treatment, and the desensitization continued to increase after 4 and 7 days of treatment (ACTH response decreased 64.2-67.7%; oxytocin response decreased 82.3-90.1%). There were no significant alterations in levels of G(alphaq) or G(alpha11) lamic paraventricular proteins in the hypothanucleus. In conclusion, these results suggest that chronically administered (-)-DOI induces desensitization of 5-HT(2A) receptors in vivo, via a reduction in the ability of 5-HT(2A) receptors to activate G proteins without consistently altering levels of G(alpha) proteins or RGS proteins.     

Reduction of beta-adrenergic receptors by tertatolol: an additional mechanism for beta-adrenergic blockade

Tertatolol is a potent new beta-blocker with no intrinsic sympathomimetic activity or beta 1/beta 2-receptor subtype selectivity. When given at therapeutic doses (5 mg/day) to human subjects it induced a reduction in the beta-adrenergic receptor number measured by 3H-CGP 12177 specific binding, without any change in the affinity on intact lymphocytes. This reduction was seen 7 hours (54%), 24 hours (35%), and 48 hours (30%) after a single drug dose. A similar receptor reduction was observed 7 hours (42%), 24 hours (37%), and 48 hours (15%) after 14 doses of the drug. In parallel, the pharmacologic efficacy of the drug was evident from the reduction in supine and upright heart rates and after submaximal exercise; heart rate was reduced to the same extent after single or repeated drug doses. The reduction of receptor number correlated well with the reduction in heart rate in the supine (P less than 0.001) and upright (P less than 0.01) positions and after exercise (P less than 0.02). In in vitro competitive binding experiments tertatolol was found to be a competitive inhibitor of beta-adrenergic receptors. However, on intact human lymphocytes preincubated with this drug, tertatolol reduced the density of beta-adrenergic receptors. We conclude that tertatolol, besides competitively inhibiting beta-adrenergic receptors, induced a marked and lasting decrease in the beta-adrenergic receptor number. This effect may be important for its beta-blocking effects.     

Orphanin FQ/nociceptin-mediated desensitization of opioid receptor-like 1 receptor and mu opioid receptors involves protein kinase C: a molecular mechanism for heterologous cross-talk

Morphine tolerance in vivo is reduced following blockade of the orphanin FQ/nociceptin (OFQ/N)/opioid receptor-like 1 (ORL1) receptor system, suggesting that OFQ/N contributes to the development of morphine tolerance. We previously reported that a 60-min activation of ORL1 receptors natively expressed in BE(2)-C cells desensitized both mu and ORL1 receptor-mediated inhibition of cAMP. Investigating the mechanism(s) of OFQ/N-mediated mu and ORL1 receptor cross-talk, we found that pretreatment with the protein kinase C inhibitor, chelerythrine chloride (1 microM), blocked OFQ/N-mediated homologous desensitization of ORL1 and heterologous desensitization of mu opioid receptors. Furthermore, depletion of PKC by 12-O-tetradecanoylphorbol-13-acetate exposure (48 h, 1 microM) also prevented OFQ/N-mediated mu and ORL1 desensitization. OFQ/N pretreatment resulted in translocation of PKC-alpha, G protein-coupled receptor kinase 2 (GRK2) and GRK3 from the cytosol to the membrane, and this translocation was also blocked by chelerythrine. Reduction of GRK2 and GRK3 levels by antisense, but not sense DNA treatment blocks ORL1 and mu receptor desensitization. This suggests that PKC-alpha is required for GRK2 and GRK3 translocation to the membrane, where GRK can inactivate ORL1 and mu opioid receptors upon rechallenge with the appropriate agonist. Our results demonstrate for the first time the involvement of conventional PKC isozymes in OFQ/N-induced mu-ORL1 cross-talk, and represent a possible mechanism for OFQ/N-induced anti-opioid actions.     

In vivo desensitization to beta receptor mediated bronchodilator drugs in the rat: decreased beta receptor affinity.

When tracheas were isolated from rats pretreated with isoproterenol (ISO) or terbutaline, they were found to be considerably less sensitive to the relaxant action of ISO than tracheas which were isolated from saline-pretreated rats. The dissociation constant (Kb) for the propranolol-beta receptor complex was determined to be up to 400-fold larger in the tracheas isolated from beta agonist-pretreated rats (1.1 +/- 0.1 X 10(-6) M) than in tracheas isolated from saline-pretreated rats (3.0 +/- 0.3 X 10(-9) M). The longer the duration of pretreatment and the higher the dose of ISO or terbutaline used, the more attenuated was the response of tracheal smooth muscle to ISO, and the greater was the Kb for propranolol-beta receptor complex. These findings provide strong evidence which shows that desensitization, which occurs as a result of in vivo pretreatment with beta agonist drugs, results from pronounced reduction in this affinity of the beta receptors for beta agonist drugs. We observed that the in vivo treatment of rats with aminophylline (Amino), a phosphodiesterase inhibitor, did not affect the responsiveness of their isolated tracheas to either ISO or Amino. In addition, the responsiveness to Amino was determined in tracheal preparations taken from rats desensitized to ISO in vivo. The response to ISO was attenuated and the Kb for the propranolol-beta receptor complex was elevated (1.1 +/- 0.1 X 10(-6) M); however, Amino was half as effective in these tissues as in the saline control tissues. It is postulated, therefore, that the intracellular enzymes controlling the levels of cyclic adenosine monophosphate may be affected by the ISO-induced desensitization process, but are not affected by pretreatment with Amino.     

In vivo desensitization of glycogenolysis to Ca2+-mobilizing hormones in rat liver cells.

Rat hepatocytes contain several types of Ca2+-linked receptors, all of which stimulate glycogen breakdown by increasing cytosolic free Ca2+ concentration [( Ca2+]c). In vivo desensitization of this Ca2+ messenger system was studied in hepatocytes isolated from either pheochromocytoma (PHEO)-harboring and chronically norepinephrine (NE)-infused rats. Homologous desensitization for alpha 1-adrenergic receptor-mediated phosphorylase activation developed in the early stage of PHEO rats (3-4 wk after implantation), whereas, in the later stage of tumor development or in the NE-infused rats, phosphorylase responses to all Ca2+-mobilizing stimulations were subsensitive (heterologous desensitization). In the homologous desensitization, the [Ca2+]c response to alpha 1-adrenergic stimulation was selectively reduced. We found, using the phenoxybenzamine inactivation method, that there was a linear relationship between alpha 1 receptor density and the [Ca2+]c response; consequently, the blunted [Ca2+]c response to alpha 1-adrenergic stimulation could not be explained by the 34% downregulation of alpha 1 receptors seen in these rats. These results indicated that uncoupling at a step proximal to alpha 1 receptor-stimulated [Ca2+]c increase is also of primary importance in homologous desensitization of phosphorylase activation. On the other hand, heterologous desensitization also involved alteration(s) at steps distal to the rise in [Ca2+]c. Our data demonstrate that prolonged exposure to catecholamines results in desensitization of the [Ca2+]c mobilization pathway and may involve multiple mechanisms.     

Mediation of corticotropin releasing factor type 1 receptor phosphorylation and desensitization by protein kinase C: a possible role in stress adaptation

Protein kinase C (PKC)-mediated desensitization of the corticotropin releasing factor type 1 (CRF1) receptor was investigated in human retinoblastoma Y79 and transfected COS-7 cells. Because stimulation of Y79 cells with CRF resulted in large ( approximately 30-fold) increases in intracellular cAMP accumulation without changing inositol phosphate levels, the CRF1 receptor expressed in retinoblastoma cells couples to Gs, but not to Gq, and predominantly signals via the protein kinase A cascade. Direct activation of PKC by treatment with the phorbol ester phorbol 12-myristate 13-acetate (PMA) or 1,2-dioctanoyl-sn-glycerol (DOG) desensitized CRF1 receptors in Y79 cells, reducing the maximum for CRF- (but not forskolin)-stimulated cAMP accumulation by 56.3 +/- 1.2% and 40.4 +/- 2.1%, respectively (p < 0.001). Pretreating Y79 cells with the PKC inhibitor bisindolylmaleimide I (BIM) markedly inhibited PMA's desensitizing action on CRF-stimulated cAMP accumulation, but did not affect homologous CRF1 receptor desensitization. Retinoblastoma cells were found to express PKCalpha, betaI, betaII, delta, lambda, and RACK1. When alpha and beta isoforms of PKC were down-regulated 80 to 90% by a 48-h PMA exposure, PMA-induced CRF1 receptor desensitization was abolished. In transfected COS-7 cells the magnitude of CRF1 receptor phosphorylation after a 5-min exposure to PMA was 2.32 +/- 0.21-fold greater compared with the basal level. Pretreating COS-7 cells with BIM abolished PMA-induced CRF1 receptor phosphorylation. These studies demonstrate that protein kinase C (possibly alpha and beta isoforms) has an important role in the phosphorylation and heterologous desensitization of the CRF1 receptor.     

Antagonistic control of tumor necrosis factor receptors by protein kinases A and C. Enhancement of TNF receptor synthesis by protein kinase A and transmodulation of receptors by protein kinase C

We have investigated control mechanisms of TNF receptor expression (TNF-R) in various human tumor cells and normal peripheral blood monocytes. Activators of protein kinase A (PKA) signal transduction pathways were found to enhance TNF-R expression up to sevenfold, whereas in the same cells, IFN-alpha and -gamma receptors remained unaffected. Inhibitors of protein kinases downregulate both constitutive and cAMP-enhanced TNF-R expression. Binding studies revealed an increase in TNF-R numbers without a change in receptor affinity. Both, direct activators of PKA and inhibitors of phosphodiesterase, raising intracellular levels of cAMP, were found to be effective. As activation of PKA does not slow down the degradation rate of TNF-Rs, but rather enhances protein synthesis-dependent reexpression of TNF-Rs after transient PKC-mediated transmodulation and after tryptic digestion of TNF-Rs, it is concluded that PKA stimulates TNF-R synthesis. Maximum TNF-Rs enhancement is reached after 24 h of stimulation and is reversible, suggesting that receptor upregulation is not linked to irreversible steps of cellular differentiation. PKA-mediated enhancement of TNF-R expression was predominantly observed in normal peripheral blood monocytes and tumor cell lines of myeloid origin. As in these typical TNF producer cells, the production of TNF is also controlled by PKA and PKC, a regulatory circuit is proposed, by which these two independent signal pathways antagonistically regulate TNF production and, at the receptor level, TNF sensitivity.     

Homologous desensitization of human platelet thromboxane A2/prostaglandin H2 receptors.

Desensitization of platelet thromboxane (TX)A2/prostaglandin (PG)H2 receptors was induced by incubating platelet-rich plasma with the stable PGH2 analog 11 alpha,9 alpha-(epoxymethano)prosta-5Z,13E-dienoic acid (U46619) (1 microM). Iloprost, a stable prostacyclin analog, was included in the incubation to prevent platelet activation. The TXA2 mimetic, [1S-1 alpha,2 beta(5Z), 3 alpha(1E,3S*), 4 alpha)]-7-[3-(3-hydroxy-4-(4'-iodophenoxy)-1-butenyl)-7-oxabicyclo - [2.2.1]heptan-2-yl]-5-heptenoic acid (I-BOP), was used to induce platelet aggregation, shape change and increases in intracellular free calcium. The EC50 values for I-BOP-induced rise in intracellular free calcium (control = 10.2 +/- 1.5 nM; desensitized = 79.4 +/- 22.4 nM, n = 6, P less than .05), aggregation (control = 15.8 +/- 2.4 nM; desensitized = 51.7 +/- 11.9 nM; P less than .05, n = 5) and shape change (control = 172 +/- 37 pM; desensitized = 350 +/- 60 pM; P less than .05, n = 7) were increased by the preincubation with U46619. Aggregation responses to thrombin and the calcium ionophore, ionomycin, were unaltered by the preincubation with U46619. Equilibrium binding studies at pH 7.4 revealed a decrease in the number of binding sites for the receptor antagonist 9,11-dimethylmethano-11,12- methano-16(3-iodo-4-hydroxyphenyl)-13,14-dihydro-13-aza-15 alpha beta-omega- tetranor-TXA2 [125I]PTA-OH) (control = 3246 +/- 509 sites/platelet, desensitized = 2198 +/- 324 sites/platelet, n = 6, P less than .05) without a change in affinity.(ABSTRACT TRUNCATED AT 250 WORDS)     

整合素连接激酶高表达对新生大鼠心肌细胞增殖的影响

背景:整合素连接激酶在调节细胞生存、抗凋亡以及促进细胞迁移、增殖、分化等方面发挥重要作用。目的:观察整合素连接激酶在新生大鼠心肌细胞内高表达后对心肌细胞增殖能力的影响。方法:取新生3d内SD大鼠心脏,体外分离、培养心肌细胞72h后,分别转染重组腺病毒载体或重组腺病毒载体+整合素连接激酶基因。结果与结论:转染48h,与转染重组腺病毒载体比较,转染重组腺病毒载体+整合素连接激酶基因的新生大鼠心肌细胞内DNA合成增加(P<0.05),有丝分裂增多(P<0.05),心肌细胞数量增多(P<0.05)。在整合素连接激酶的作用下,新生大鼠心肌细胞的增殖能力明显提高。     

整合素连接激酶高表达对新生大鼠心肌细胞增殖的影响

背景：整合素连接激酶在调节细胞生存、抗凋亡以及促进细胞迁移、增殖、分化等方面发挥重要作用。目的：观察整合素连接激酶在新生大鼠心肌细胞内高表达后对心肌细胞增殖能力的影响。方法：取新生3d内SD大鼠心脏,体外分离、培养心肌细胞72h后,分别转染重组腺病毒载体或重组腺病毒载体＋整合素连接激酶基因。结果与结论：转染48h,与转染重组腺病毒载体比较,转染重组腺病毒载体＋整合素连接激酶基因的新生大鼠心肌细胞内DNA合成增加（P〈0.05）,有丝分裂增多（P〈0.05）,心肌细胞数量增多（P〈0.05）。在整合素连接激酶的作用下,新生大鼠心肌细胞的增殖能力明显提高。     

Mechanical stress activates protein kinase cascade of phosphorylation in neonatal rat cardiac myocytes.

We have previously shown that stretching cardiac myocytes evokes activation of protein kinase C (PKC), mitogen-activated protein kinases (MAPKs), and 90-kD ribosomal S6 kinase (p90rsk). To clarify the signal transduction pathways from external mechanical stress to nuclear gene expression in stretch-induced cardiac hypertrophy, we have elucidated protein kinase cascade of phosphorylation by examining the time course of activation of MAP kinase kinase kinases (MAPKKKs), MAP kinase kinase (MAPKK), MAPKs, and p90rsk in neonatal rat cardiac myocytes. Mechanical stretch transiently increased the activity of MAPKKKs. An increase in MAPKKKs activity was first detected at 1 min and maximal activation was observed at 2 min after stretch. The activity of MAPKK was increased by stretch from 1-2 min, with a peak at 5 min after stretch. In addition, MAPKs and p90rsk were maximally activated at 8 min and at 10 approximately 30 min after stretch, respectively. Raf-1 kinase (Raf-1) and (MAPK/extracellular signal-regulated kinase) kinase kinase (MEKK), both of which have MAPKKK activity, were also activated by stretching cardiac myocytes for 2 min. The angiotensin II receptor antagonist partially suppressed activation of Raf-1 and MAPKs by stretch. The stretch-induced hypertrophic responses such as activation of Raf-1 and MAPKs and an increase in amino acid uptake was partially dependent on PKC, while a PKC inhibitor completely abolished MAPK activation by angiotensin II. These results suggest that mechanical stress activates the protein kinase cascade of phosphorylation in cardiac myocytes in the order of Raf-1 and MEKK, MAPKK, MAPKs and p90rsk, and that angiotensin II, which may be secreted from stretched myocytes, may be partly involved in stretch-induced hypertrophic responses by activating PKC.     

Molecular mechanisms of homologous desensitization and internalization of muscarinic receptors in primary cultures of neonatal corticostriatal neurons

Homologous desensitization of muscarinic acetylcholine receptors (mAChR) was studied using primary cultures of corticostriatal neurons from neonatal rats. Prolonged incubation with carbachol attenuated phospholipase C responsiveness to muscarinic agonists and decreased the number of cell surface mAChR, as measured by binding of N-[3H] methylscopolamine to neuronal monolayers. When neurons were exposed to carbachol for 15 min, 40% of the mAChR lost from the membrane domain was recovered in the cytosol; a decrease of the total neuronal receptors was detected following an incubation with the agonist lasting longer than 15 min. Both 8-Br-cyclic AMP and forskolin neither affected N-[3H]methylscopolamine binding to cell monolayers or did they prevent the agonist-mediated mAChR desensitization. 8-Br-cyclic GMP also failed to decrease mAChR number. Pertussis toxin failed to prevent the homologous desensitization of mAChR under conditions that blocked the agonist-mediated inhibition of forskolin-stimulated cyclic AMP formation. The phorbol ester 12-O-tetradecanoyl-phorbol-12, 13-acetate induced a concentration-dependent decrease of N-[3H]methylscopolamine binding to neuronal monolayers. However, the protein kinase C inhibitors sphingosine and the ganglioside monosialosyl-gangliotetraglicosylceramide inhibited the 12-O-tetradecanoyl-phorbol-12,13-acetate-induced but not the agonist-induced desensitization of mAChRs. Furthermore, incubation with muscarinic agonists failed to translocate protein kinase C from cytosol to plasma membranes, as measured by binding of the phorbol ester [3H]-4-beta-phorbol-12,13-dibutyrate to neuronal monolayers. In corticostriatal neurons the agonist-induced desensitization and internalization of mAChR involves neither protein kinase C and protein kinase A activation nor changes in cyclic GMP and cyclic AMP content.     

Activation of protein kinase A is necessary but not sufficient for ethanol-induced desensitization of cyclic AMP production.

Acute addition of EtOH to PC 12 pheochromocytoma cells increases cyclic AMP production, whereas chronic exposure to EtOH results in a decrease in the stimulation of cyclic AMP production in response to 2-chloroadenosine and forskolin. This EtOH-induced desensitization was not observed after chronic EtOH treatment of A126-1B2-1 cells which are a protein kinase A-deficient mutant cell line derived from PC 12 cells. Furthermore, in the parental PC 12 cell line the cell-permeable protein kinase A inhibitor, Rp-isomer of adenosine 3',5'-monophosphorothioate, blocked the development of EtOH-induced desensitization. Thus, activation of protein kinase A is apparently necessary for EtOH-induced desensitization of cyclic AMP production. Chronic treatment of PC 12 cells with forskolin qualitatively mimicked the desensitization observed with chronic EtOH exposure. However, the degree of desensitization induced by forskolin was significantly less than that caused by EtOH even though the acute addition of forskolin caused a greater increase in cyclic AMP production. Furthermore, the acute addition of EtOH inhibited forskolin-stimulated cyclic AMP production, yet inclusion of EtOH during the chronic forskolin treatment of PC 12 cells resulted in a greater degree of desensitization. These findings indicate an obligatory role of protein kinase A in EtOH-induced desensitization of cyclic AMP production in PC 12 cells. However, because protein kinase A activation alone is not sufficient to account for the degree of desensitization, EtOH probably also acts through a mechanism in addition to activation of protein kinase A.     

Dissociation of receptor sensitivity changes in rat perifornical hypothalamus: a role for dopaminergic receptors in amphetamine anorexic tolerance

Development of a contingent tolerance to amphetamine (AMPH) anorexia has been reported with chronic s.c. injections in rats (Ghosh and Parvathy, 1973, 1976). Using this model, the present study examined the role of potential receptor sensitivity changes in the beta adrenergic and/or dopamine (DA) receptors in the perifornical hypothalamus (PFH) during chronic central and peripheral drug injections. Chronic injections of AMPH into the PFH, or l-dopa injected s.c., resulted in persistent anorexia in the daily first 2 hr of eating on all test days compared with the next 2 hr that showed a progressive increase in eating with subsequent injections, the net effect being an apparent tolerance to its 4 hr anorexic effect. The tolerance patterns obtained with these two treatments were essentially the same as that of s.c. AMPH, suggesting a role for PFH catecholamine synapses, particularly at the postsynaptic receptor level. Selective beta adrenoceptor-mediated anorexia, as obtained with chronic PFH-injected nor-adrenaline and isoprenaline, was dose-dependent and persistent on all days, suggesting that no change in the sensitivity of PFH beta adrenoceptors had occurred. This contrasted with the PFH-injected DA, which produced a rapid tolerance development due to a progressive loss of effect in the first 2 hr. Present findings suggest that although beta adrenergic and DA mechanisms act in concert in eliciting the acute anorexic effects of AMPH, DA, rather than beta adrenergic system, has a predominant influence in the development of tolerance to the anorexic effect of AMPH.