Receptor-mediated Signal Transduction Mechanisms Research Articles

Role of Vasoactive Hormone-Induced Signal Transduction in Cardiac Hypertrophy and Heart Failure.

Heart failure is the common concluding pathway for a majority of cardiovascular diseases and is associated with cardiac dysfunction. Since heart failure is invariably preceded by adaptive or maladaptive cardiac hypertrophy, several biochemical mechanisms have been proposed to explain the development of cardiac hypertrophy and progression to heart failure. One of these includes the activation of different neuroendocrine systems for elevating the circulating levels of different vasoactive hormones such as catecholamines, angiotensin II, vasopressin, serotonin and endothelins. All these hormones are released in the circulation and stimulate different signal transduction systems by acting on their respective receptors on the cell membrane to promote protein synthesis in cardiomyocytes and induce cardiac hypertrophy. The elevated levels of these vasoactive hormones induce hemodynamic overload, increase ventricular wall tension, increase protein synthesis and the occurrence of cardiac remodeling. In addition, there occurs an increase in proinflammatory cytokines and collagen synthesis for the induction of myocardial fibrosis and the transition of adaptive to maladaptive hypertrophy. The prolonged exposure of the hypertrophied heart to these vasoactive hormones has been reported to result in the oxidation of catecholamines and serotonin via monoamine oxidase as well as the activation of NADPH oxidase via angiotensin II and endothelins to promote oxidative stress. The development of oxidative stress produces subcellular defects, Ca2+-handling abnormalities, mitochondrial Ca2+-overload and cardiac dysfunction by activating different proteases and depressing cardiac gene expression, in addition to destabilizing the extracellular matrix upon activating some metalloproteinases. These observations support the view that elevated levels of various vasoactive hormones, by producing hemodynamic overload and activating their respective receptor-mediated signal transduction mechanisms, induce cardiac hypertrophy. Furthermore, the occurrence of oxidative stress due to the prolonged exposure of the hypertrophied heart to these hormones plays a critical role in the progression of heart failure.

Could Endogenous Glucocorticoids Influence SARS-CoV-2 Infectivity?

Endogenous glucocorticoids and their synthetic analogues, such as dexamethasone, stimulate receptor-mediated signal transduction mechanisms on target cells. Some of these mechanisms result in beneficial outcomes whereas others are deleterious in the settings of pathogen infections and immunological disorders. Here, we review recent studies by several groups, including our group, showing that glucocorticoids can directly interact with protein components on SARS-CoV-2, the causative agent of COVID-19. We postulate an antiviral defence mechanism by which endogenous glucocorticoids (e.g., cortisol produced in response to SARS-CoV-2 infection) can bind to multiple sites on SARS-CoV-2 surface protein, Spike, inducing conformational alterations in Spike subunit 1 (S1) that inhibit SARS-CoV-2 interaction with the host SARS-CoV-2 receptor, ACE2. We suggest that glucocorticoids-mediated inhibition of S1 interaction with ACE2 may, consequently, affect SARS-CoV-2 infectivity. Further, glucocorticoids interactions with Spike could protect against a broad spectrum of coronaviruses and their variants that utilize Spike for infection of the host. These notions may be useful for the design of new antivirals for coronavirus diseases.

Abstract 5167: Melatonin inhibition of linoleic acid transport and 13-HODE production in HeLa human cervical adenocarcinoma occurs via receptor-mediated signal transduction

Abstract Over 12,000 women in the U.S. alone this year will be diagnosed with cervical cancer, ranking third behind endometrial and ovarian cancers. Patients with an advanced stage cervical cancer have only a 15-20% survival rate at 5 years. Observational studies of the night-shift worker population have shown an increased incidence of risk in persons exposed to ocular light at night in a number of cancers, including cervical cancer. This finding was associated with an inhibition of nighttime pineal gland production of melatonin, the circadian neurohormone shown to suppress tumor metabolic and proliferative activity. This occurs via melatonin inhibition of tumor linoleic acid (LA) uptake and conversion to 13-hydroxyoctadecadienoic acid (13-HODE), a molecule that upregulates epidermal growth factor and insulin-like growth factor-I-induced mitogenesis. Here, we investigate the effects in vivo of melatonin (500 pM) on perfused, tissue-isolated HeLa human adenocarcinoma xenografts in nude rats. HeLa adenocarcinoma latency-to-onset and tumor growth rates were, respectively, 8 days and 0.09 ± 0.01 g/day; mean tumor weights were 5.9 ± 0.3 g (n = 32). Cervical adenocarcinomas perfused in situ with donor rodent blood for 150 minutes (Controls) revealed a tumor glucose uptake and lactate release of 11.4 ± 0.9 ng/min/g and −20.4 ± 2.2 nmol/min/g, respectively; control tumor LA uptake and 13-HODE release was 0.71 ± 0.08 μg/min/g and 0.35 ± 0.07 ng/min/g, respectively. Incorporation of 3H-thymidine into tumor DNA and DNA content, respectively, were 21.2 ± 0.6 dpms/μg DNA and 2.5 ± 0.1 mg/g tumor. Adenocarcinomas perfused with arterial donor blood augmented with physiological nocturnal levels of 500 pM melatonin showed over a 20% abrogation of tumor glucose uptake and lactate production, O2 consumption and CO2 production, and a complete inhibition of LA uptake, 13-HODE release, ERK 1/2, MEK, Akt, GSK3β proliferative activities, and over an 85% reduction in tumor cAMP levels and 3H-thymidine incorporation into tumor DNA. Supplementation with the non-selective MT1/MT2 melatonin antagonist S20928, forskolin, 8-Bromo-cyclic-AMP, or pertusiss toxin completely reversed the tumor growth inhibitory response. Also, addition of 13-HODE reversed 3H-thymidine into tumor DNA, but had no effect on LA uptake. The preliminary results presented here demonstrate, for the first time, that nocturnal melatonin levels suppress HeLa cervical adenocarcinoma metabolism and proliferation in vivo via a melatonin receptor mediated signal transduction mechanism. A better understanding of this signaling pathway and the regulation of LA in cancer metabolism and proliferation may lead to new therapeutic and/or chemopreventative interventions. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5167. doi:1538-7445.AM2012-5167

Abstract 165: The anticarcinogenic effects of melatonin on inhibition of fatty acid transport and 13-HODE production in HT29 human colorectal cancer occurs via receptor-mediated signal transduction

Abstract Over 620,000 people in the U.S. alone this year will be diagnosed with either breast, prostate, or colorectal cancers, which represent nearly 50% of all reported malignancies. Patients with advanced stage breast, prostate, or colorectal cancer have only a 35% survival rate at 5 years. Epidemiological studies have indicated that the risk of these cancers is increased in persons exposed to excessive sunlight or, interestingly, ocular light at night, as experienced by night-shift workers. This latter finding was shown to be associated with a suppression of the nighttime production of the circadian neurohormone melatonin. Previous work from this laboratory using perfused, tissue-isolated human breast and prostate tumors showed that tumor growth and metabolism in vivo is dependent upon uptake of linoleic acid (LA), the most prominent fatty acid in our western diet, and its conversion to 13-hydroxyoctadecadienoic acid (13-HODE). In tissue-isolated human MCF-7 (steroid responsive and non-responsive) and PC3 prostate tumors perfused in situ with arterial blood, melatonin inhibits LA-uptake and conversion to 13-HODE, a lipoxygenase product that stimulates EGF mitogenesis. The present study explores the effects in vivo of melatonin (500 pM) on perfused, tissue isolated HT29 colorectal carcinomas in nude rats. HT29 colorectal carcinoma latency-to-onset and growth rates, respectively, of 12 days and 0.12 ± 0.01 g/day; mean tumor weights were 5.6 ± 0.2 g (n = 20). Colorectal carcinomas perfused in situ with donor rodent blood for 2 hours (Controls) revealed a tumor LA uptake and 13-HODE release of 0.86 ± 0.08 μg/min/g (20.0 ± 1.6% of arterial supply) and 3.23 ± 0.81 ng/min/g, respectively. Incorporation of 3H-thymidine into tumor DNA and DNA content, respectively were 25.9 ± 1.6 dpms/μg DNA and 2.7 ± 0.1 μg/g tumor. Carcinomas perfused with arterial donor blood augmented with physiological nocturnal levels of 500 pM melatonin showed a complete inhibition of LA uptake, 13-HODE release, ERK 1/2, MEK, Akt, and GSK3β activities, and over a 90% reduction in tumor cAMP levels and [3H]thymidine incorporation into tumor DNA. Addition of the non-selective MT1/MT2 melatonin antagonist S20928, forskolin, 8-Bromo-cyclic-AMP, or pertussis toxin completely reversed the tumor growth inhibitory response. Also, addition of 13-HODE reversed the inhibition of 3H-thymidine incorporation into tumor DNA, while having no effect on LA uptake. These results are the first to demonstrate in HT29 colorectal carcinoma that nocturnal melatonin levels suppress tumor growth via a melatonin receptor-mediated signal transduction mechanism. An understanding of this signaling pathway for the control of LA uptake in cancer could lead to new approaches for therapeutic intervention and/or chemoprevention. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 165. doi:1538-7445.AM2012-165

Antineoplastic effects of melatonin on a rare malignancy of mesenchymal origin: melatonin receptor‐mediated inhibition of signal transduction, linoleic acid metabolism and growth in tissue‐isolated human leiomyosarcoma xenografts

Melatonin provides a circadian signal that regulates linoleic acid (LA)-dependent tumor growth. In rodent and human cancer xenografts of epithelial origin in vivo, melatonin suppresses the growth-stimulatory effects of linoleic acid (LA) by blocking its uptake and metabolism to the mitogenic agent, 13-hydroxyoctadecadienoic acid (13-HODE). This study tested the hypothesis that both acute and long-term inhibitory effects of melatonin are exerted on LA transport and metabolism, and growth activity in tissue-isolated human leiomyosarcoma (LMS), a rare, mesenchymally-derived smooth muscle tissue sarcoma, via melatonin receptor-mediated inhibition of signal transduction activity. Melatonin added to the drinking water of female nude rats bearing tissue-isolated LMS xenografts and fed a 5% corn oil (CO) diet caused the rapid regression of these tumors (0.17 +/- 0.02 g/day) versus control xenografts that continued to grow at 0.22 +/- 0.03 g/day over a 10-day period. LMS perfused in situ for 150 min with arterial donor blood augmented with physiological nocturnal levels of melatonin showed a dose-dependent suppression of tumor cAMP production, LA uptake, 13-HODE release, extracellular signal-regulated kinase (ERK 1/2), mitogen activated protein kinase (MEK), Akt activation, and [(3)H]thymidine incorporation into DNA and DNA content. The inhibitory effects of melatonin were reversible and preventable with either melatonin receptor antagonist S20928, pertussis toxin, forskolin, or 8-Br-cAMP. These results demonstrate that, as observed in epithelially-derived cancers, a nocturnal physiological melatonin concentration acutely suppress the proliferative activity of mesenchymal human LMS xenografts while long-term treatment of established tumors with a pharmacological dose of melatonin induced tumor regression via a melatonin receptor-mediated signal transduction mechanism involving the inhibition of tumor LA uptake and metabolism.

Melatonin decreases cell proliferation and transformation in a melatonin receptor-dependent manner

There are conflicting claims for the role of melatonin in oncogenesis. In addition, the mechanism(s) underlying melatonin's effects in oncogenic processes is (are) unknown. In this study, the effects of melatonin exposure on cell proliferation and transformation were assessed in NIH3T3 cells transfected with either the human mt 1 (NIH-mt1) or MT 2 (NIH-MT2) melatonin receptors. The effects of melatonin exposure on proliferation was assessed by direct cell counts and [ 3H]thymidine uptake assays. The effect of chronic melatonin pretreatment on transformation was assessed by focus assays. In both NIH-mt1 and NIH-MT2 cells, melatonin pretreatment decreased cell proliferation and transformation. Control (NIH-neo) cells did not show this effect. However, as revealed by the [ 3H]thymidine uptake assays, an increase in DNA synthesis occurred in NIH-mt1 cells, whereas no increase occurred in the NIH-MT2 or NIH-neo cells. Upon examination of melatonin receptors, a decrease in the function of both mt 1 and MT 2 receptors occurred. These data suggest that perhaps an attenuation of receptor-mediated processes are involved in the anti-proliferative and anti-transformation capabilities of melatonin in NIH3T3 cells. In addition, based on the [ 3H]thymidine assays, receptor mediated signal transduction mechanisms may slow the growth of cells via actions on the cell cycle. The results from this study shed new insight on the putative mechanisms underlying melatonin's effects on cell proliferation and transformation and lends support for a protective role of melatonin in oncogenesis.

Regulation of prostanoid synthesis in microglial cells and effects of prostaglandin E 2 on microglial functions

Brain prostanoid levels are normally low but can increase after ischemia and during inflammatory and infectious diseases. High prostanoid levels can affect brain function in several ways. In particular, prostaglandin E 2 (PGE 2) might exert both immunodepressive and proinflammatory actions. The present short review focuses on the regulation of prostanoid synthesis in microglial cultures and on the possible role of PGE 2 in the down-regulation of microglial activation induced by lipopolysaccharide (LPS). Our studies were carried out using purified mouse or rat microglial cultures. LPS induced a dose-dependent expression of the inducible isoform of cyclooxygenase (COX-2), both in neonatal and adult microglial cultures. In the latter, the inducibility of COX-2 increased with time in culture, paralleling the acquisition of a more ‘activated’ microglial phenotype, and appeared to account for the time-dependent increase in the PGE 2/TXB 2 production ratio. The LPS-induced COX-2 expression and prostanoid production were down-regulated by potentially neurotoxic agents, such as nitric oxide (NO), the proinflammatory cytokine IFN-γ (which acted both directly and indirectly, through its NO-inducing activity) and the HIV regulatory protein tat. On the other hand, COX-2 expression was up-regulated by the macrophage-deactivating cytokine TGF-β1, by exogenous PGE 2 itself, which acted through EP2 receptors linked to cyclic AMP generation, and by non steroidal anti-inflammatory drugs. Interestingly, PGE 2 utilized the same EP2 receptor-mediated signal transduction mechanism to down-regulate the expression of the inducible NO synthase and the production of NO. Largely, but not exclusively, through its effect on cyclic AMP, PGE 2 can also: i) depress the expression of major histocompatibility complex class II antigens and of the costimulatory molecule B7-2; ii) down-regulate TNF and up-regulate IL-10 microglial production; iii) inhibit microglial IL-12 secretion. These observations, together with literature data on in vivo models of central nervous system (CNS) diseases, suggest a neuroprotective role of PGE 2 in pathological conditions.

Neuropeptide Y inhibits ciliary beat frequency in human ciliated cells via nPKC, independently of PKA.

The intracellular mechanisms whereby the inhibitory neurotransmitter neuropeptide Y (NPY) decreases ciliary beat frequency (CBF) were investigated in cultured human tracheal and bronchial ciliated cells. CBF was measured by nonstationary analysis laser light scattering. NPY at 1 and 10 microM decreased CBF from a baseline of 6.7 +/- 0.5 (n = 12) to 6.1 +/- 0.5 (P < 0.05) and 5.8 +/- 0.4 (P < 0.01) Hz, respectively. Prior application of PYX-1, an NPY antagonist, prevented the decreases of CBF induced by both doses of NPY. Two broad protein kinase C (PKC) kinase inhibitors, staurosporine and calphostin C, also abolished the NPY-induced decrease in CBF. The NPY-induced decrease in CBF was abolished by GF 109203X, a novel PKC (nPKC) isoform inhibitor, whereas this decrease in CBF was not attenuated by Gö-6976, a specific inhibitor of conventional PKC isoforms. Because pretreatment with NPY did not block the stimulation of CBF by forskolin and pretreatment with forskolin did not abolish the NPY-induced inhibition of CBF, this NPY receptor-mediated signal transduction mechanism appears to be independent of the adenylate cyclase-protein kinase A (PKA) pathway. Inhibition of Ca2+-ATPase by thapsigargin also prevented the suppression of CBF induced by subsequent application of NPY. These novel data indicate that, in cultured human epithelia, NPY decreases CBF below its basal level via the activation of an nPKC isoform and Ca2+-ATPase, independent of the activity of PKA. This is consistent with the proposition that NPY is an autonomic efferent inhibitory neurotransmitter regulating mucociliary transport.

Alpha-adrenoceptor activation induces rhythmic contractile activity in carotid arteries from young, not adult, rats.

Rhythmic contractions are produced by small arteries, arterioles and veins in several vascular beds, but they are often absent in large arteries. However, under certain conditions, and in certain disease states large arteries may produce rhythmic contractions. For this reason, the present study was undertaken to test the hypothesis that rhythmic contractions may be a 'normal' response in large arteries at some stage in development. We investigated this hypothesis by examining contractions of carotid arteries in male and female rats aged 15, 25 and 30 days and in adult rats. Rhythmic contractions were produced by exposure to, or during washout of alpha-adrenoceptor agonists in young, but not adult, rats. In particular, rhythmic activity was identified in 40, 95 and 50% of the arteries from, respectively, 15, 25 and 30-day-old rats. No differences were found in rhythmic activity between female and male rats. Furthermore, the rhythmic activity was not inhibited by the K+ channel blocker TEA (20 mM) or the Na+/K(+)-ATPase inhibitor, ouabain (32 microM). Nor was it inhibited by endothelial denudation. However, the Ca2+ channel blocker, nifedipine (0.1 microM), completely eliminated rhythmic contraction. These results suggest that receptor-induced rhythmic contractile activity is a 'normal' characteristic at approximately 25 days of development in carotid arteries of rats, but that this activity declines with age until it is completely absent by adulthood. We proposed that this difference was due to unfused and fused Ca2+ channel activities in, respectively, young and adult rat arteries, to differential expression of Ca2+ channel isotypes, or to differences in receptor-mediated signal transduction mechanisms 'upstream' from Ca2+ channels.

Differential action of secreto-inhibitors on proopiomelanocortin biosynthesis in the intermediate pituitary of Xenopus laevis.

In the South African clawed toad Xenopus laevis, background adaptation is regulated by alpha MSH, a POMC-derived peptide. After transfer of the animal from a black to a white background, secretion of alpha MSH from the intermediate pituitary lobe is inhibited by the hypothalamic neurotransmitter neuropeptide Y (NPY). The neurointermediate lobe in vitro is also subject to inhibitory regulation by dopamine and gamma-aminobutyric acid (GABA). In the nerve terminals contacting the intermediate lobe of the pituitary, GABA is contained in electron-lucent vesicles, whereas dopamine and NPY coexist in electron-dense vesicles. To study the role of these secreto-inhibitors in the regulation of POMC biosynthesis, the rate of incorporation of radioactive amino acids into POMC protein was determined after in vitro treatment of the neurointermediate pituitary with NPY, apomorphine (dopamine D2 receptor agonist), isoguvacine (GABAA receptor agonist) and baclofen (GABAB receptor agonist). After 24 h of treatment, inhibition of POMC biosynthesis by NPY and apomorphine was 77% and 74%, respectively. Isoguvacine treatment resulted in an inhibition of 59%, whereas no significant effect of baclofen was observed. When neurointermediate lobes were treated for 3 days, inhibition of POMC biosynthesis by NPY was maintained, and inhibition by apomorphine was even stronger, whereas isoguvacine gave an inhibition of 52%, and baclofen produced 34% inhibition. Superfusion experiments on alpha MSH secretion showed that prolonged treatment with the GABA receptor agonists results in a desensitization of GABA receptor-mediated signal transduction mechanisms, whereas the NPY receptor does not show desensitization. The observations indicate differential actions of the secreto-inhibitors NPY, apomorphine, and GABA agonists on POMC biosynthesis in the Xenopus intermediate pituitary, suggesting a major role for dopamine and NPY, whereas GABA, acting via two receptor types, does not seem to have a major function in long term control of POMC biosynthesis.

Receptor-mediated signal transduction

The elucidation for the mechanism of receptor-mediated signal transduction has been the aim of our extensive studies. Cyclic AMP, which was controlled by membrane adenylyl cyclase, was an intracellular signal (the first second messenger in cells proposed by Sutherland) given by hormones and neurotransmitters. The GTP-binding (G) proteins play an important role in communication between membrane receptors and the adenylyl cyclase catalytic unit. One (Gs) of the G proteins is involved in the activation, while the other (Gi) is involved in the inhibition of adenylyl cyclase. Islet-activating protein (IAP, pertussis toxin, PTX) catalyses the transfer of the ADP-ribose moiety of NAD to the alpha subunit of Gi, resulting in a complete loss of the Gi functions. In some cases, arachidonic acid (AA) regulates cell functions as a second messenger. AA serves as a precursor to a number of biologically active lipids including prostaglandins and leukotrienes. Activation of cell surface receptors of many cell types results in the release of AA from membrane phospholipids by phospholipase A2 (PLA2). A new family of PLA2 has been discovered in the cytosol of various cells. The activation of receptor-mediated AA release by cytosolic PLA2 was also regulated by PTX-sensitive G proteins. PTX treatment inhibited cell growth of fibroblasts by serum and growth factors. G proteins have been involved in receptor-receptor interactions in neuronal cells. These findings suggest the regulatory roles of cell surface receptors-coupled G proteins in signal transductions and cell functions.

Epidermal Growth Factor Receptor Cytoplasmic Domain Mutations Trigger Ligand-Independent Transformation

The transforming gene product of avian erythroblastosis virus, v-erbB, is derived from the epidermal growth factor (EGF) receptor but has lost its extracellular ligand-binding domain and was mutated in its cytoplasmic portion, which is thought to be responsible for biological signal generation. We have repaired the deletion of extracellular EGF-binding sequences and investigated the functional consequences of cytoplasmic erbB mutations. Within the resulting EGF receptors, the autophosphorylation activities of the cytoplasmic domains of v-erbB-H and v-erbB-ES4 were fully ligand dependent in intact cells. However, the mitogenic and transforming signaling activities of an EGF receptor carrying v-erbB-ES4 (but not v-erbB-H) cytoplasmic sequences remained ligand independent, whereas those of a receptor with a v-erbB-H cytoplasmic domain were regulated by EGF or transforming growth factor alpha. Thus, structural alterations in the cytoplasmic domain of growth factor receptor tyrosine kinases may induce constitutive signaling activity without autophosphorylation. These findings provide new insight into the mechanism of receptor-mediated signal transduction and suggest a novel alternative for subversion of cellular control mechanisms and proto-oncogene activation.

Epidermal growth factor receptor cytoplasmic domain mutations trigger ligand-independent transformation.

The transforming gene product of avian erythroblastosis virus, v-erbB, is derived from the epidermal growth factor (EGF) receptor but has lost its extracellular ligand-binding domain and was mutated in its cytoplasmic portion, which is thought to be responsible for biological signal generation. We have repaired the deletion of extracellular EGF-binding sequences and investigated the functional consequences of cytoplasmic erbB mutations. Within the resulting EGF receptors, the autophosphorylation activities of the cytoplasmic domains of v-erbB-H and v-erbB-ES4 were fully ligand dependent in intact cells. However, the mitogenic and transforming signaling activities of an EGF receptor carrying v-erbB-ES4 (but not v-erbB-H) cytoplasmic sequences remained ligand independent, whereas those of a receptor with a v-erbB-H cytoplasmic domain were regulated by EGF or transforming growth factor alpha. Thus, structural alterations in the cytoplasmic domain of growth factor receptor tyrosine kinases may induce constitutive signaling activity without autophosphorylation. These findings provide new insight into the mechanism of receptor-mediated signal transduction and suggest a novel alternative for subversion of cellular control mechanisms and proto-oncogene activation.

Effects of pertussis toxin on the behavioural and ECoG spectrum changes induced by clonidine and yohimbine after their microinfusion into the locus coeruleus.

1. Pertussis toxin, a substance which interferes selectively with receptor-mediated signal transduction mechanisms, was injected into the locus coeruleus of rats 1, 2, 3, 6 or 10 days before the microinjection of clonidine or yohimbine into the same site. 2. Clonidine produced in control rats typical behavioural sedation and/or sleep and ECoG synchronization while yohimbine produced behavioural arousal and ECoG desynchronization. 3. The behavioural and ECoG effects of both compounds were blocked in animals pretreated with pertussis toxin. This activity was more marked from 2 to 6 days after pertussis toxin pretreatment and was restored 10 days after toxin administration. In addition, the behavioural and ECoG slow-wave sleep observed after intraperitoneal administration of clonidine (0.2 mumol kg-1) was significantly reduced by prior (3 days) microinfusion of pertussis toxin into the locus coeruleus. 4. These results are consistent with the hypothesis that the behavioural and ECoG effects of clonidine and yohimbine are mediated via a guanine regulatory protein which is affected by pertussis toxin.

Defective control of cytoplasmic calcium concentration in T lymphocytes from old mice.

Cytoplasmic calcium concentration ([Ca]i) rises within minutes of exposure of T lymphocytes to a mitogen. T cells from old mice are defective in this reaction, a defect that could reflect either altered signal transduction or instead a more general age-associated change in intracellular calcium regulation. We therefore tested the ability of T cells from old mice to regulate their [Ca]i concentration after exposure to low concentrations of ionomycin, an agent that raises [Ca]i but bypasses receptor-mediated signal transduction mechanisms. Exposure of T cells to ionomycin leads to an abrupt increase in [Ca]i followed by stabilization at a dose-dependent plateau level that is affected by extracellular EGTA, by calmodulin inhibitors, and by modulators of protein kinase C. Plateau levels of [Ca]i after ionomycin challenge were consistently lower in T cells from old mice than in T cells from young mice. Flow cytometric experiments showed that while essentially all T cells from both old and young mice responded to ionomycin, they did so to an extent that depended on donor age. The age-dependent increase in resistance to ionomycin-induced changes in [Ca]i cannot be attributed to diminished membrane permeability to the ionomycin-calcium complex. The data suggest that aging may lead, in T lymphocytes, to a relative resistance to increases in [Ca]i, a resistance that in turn prevents cell activation.

Role of inositol phospholipid hydrolysis in the initiation of agonist-induced contractions of rat uterus: effects of domination by 17 beta-estradiol and progesterone.

The role of inositol phospholipid (IP) hydrolysis in agonist-mediated contractility was examined in rat uterine smooth muscle by comparing carbachol-, oxytocin-, and PGF2 alpha-mediated [3H]IP accumulation and tension generation. In both estrogen- and progesterone-dominated uteri, all three agonists exhibited dose-dependent contractile responses. Agonist potencies (EC50 values) for eliciting [3H]IP accumulation or contractile responses were found to be very similar and did not change significantly between hormonal states. Maximal responses of agonist-mediated [3H]IP accumulation and tension generation were significantly affected by the endocrine state of the uterus and were dependent on the agonist examined. Maximal carbachol- and PGF2 alpha-induced [3H]IP accumulation were found to be elevated in estrogen-dominated relative to progesterone-dominated uteri, whereas maximal forces generated by these two agonists were smaller in progesterone-dominated relative to estrogen-dominated tissues. Oxytocin-induced responses did not differ between hormonal states. To determine whether these differences between [3H]IP accumulation and contractility responses could be attributed to changes in receptor-mediated signal transduction mechanisms, receptor expression and coupling to phospholipase C were studied. Myometrial muscarinic and oxytocin receptors assessed by radioligand binding were found to have three- to four-fold greater capacities in estrogen-dominated than in progesterone-dominated uteri without significant changes in agonist affinities. Agonist-mediated [3H]IP accumulation was potently inhibited by both pertussis and cholera toxins in both hormonal states. These experiments show that estrogen- and progesterone-dominated environments regulate both uterine excitability and contractility and that the mechanisms of this regulation are complex and dependent on the agonist system stimulated.

Corticotropin-releasing hormone (CRH) receptors in brain.

Studies with iodine-125-labeled analogues of CRH have identified, characterized, and localized binding sites for CRH in rat and human brain. In addition, we have demonstrated that CRH stimulates cAMP production in various regions of the rat CNS. The significant regional differences in the stoichiometric relationship between receptor number and receptor-mediated cAMP production suggests that some populations of CRH receptors in brain may be functionally coupled to alternative signal transduction mechanisms. The autoradiographic data demonstrate that the distribution of CRH binding sites in the rat CNS corresponds well with the immunohistochemical distribution of CRH pathways and pharmacological sites of action of CRH in some areas of brain but not in others. The demonstration of an upregulation of CRH receptors following a decrease in CRH-IR in the cerebral cortex in Alzheimer's disease indicates a physiological relevance of the receptor site and is consistent with the concept that CRH acts as a neurotransmitter in regulating normal cortical functions. In addition, the data suggest that disease of this peptidergic system may be important in certain clinical manifestations of Alzheimer's disease. The effects of chronic atropine treatment to selectively upregulate CRH receptors in the cerebral cortex suggests an interaction between CRH and acetylcholine. These data provide further support for the proposed role of CRH as a neurotransmitter in the CNS. Furthermore, these studies demonstrating the characteristics of CRH receptors and CRH receptor-mediated signal transduction mechanisms in brain provide a means for better understanding the various functions of this neuropeptide under physiological and pathological conditions.

Turnover of inositol phosphates in brain during ischemia-induced breakdown of polyphosphoinositides

Using either [ 32]ATP or [ 3H]inositol as precursors which were injected intraventricularly into rat brain, decapitative ischemic treatment resulted in a more rapid loss of labeled phosphatidylinositol 4,5-biphosphates than phosphatidylinositol 4-phosphates in the initial 30 s-1 min. When polyphosphoinositides were labeled with [ 3H]inositol, the breakdown of these compounds was accompanied by a time-dependent appearance of labeled inositol phosphates. Although the level of radioactivity of inositol trisphosphate was low, a peak labeling activity was shown at 30 s. The radioactivity of inositol bisphosphate showed an increase after a delay of 30 s, and reached a peak at 1 min before declining to the baseline level at 5 min. There was also a lag period of 30 s for the appearance of labeled inositol monophosphate, after which the radioactivity continued to increase in a biphasic manner for the entire 5 min period. Results indicate that decapitative ischemic treatment to rats can serve as an experimental model for assessing in vivo stimulation of the receptor-mediated signal transduction mechanism related to polyphosphoinositide breakdown and subsequent turnover of inositol phosphates in brain.

CHRONIC ETHANOL EFFECT ON THE ACIDIC PHOSPHOLIPIDS OF SYNAPTOSOMES ISOLATED FROM CEREBRAL CORTEX OF C57BL/6NNIA MICE—A COMPARISON WITH AGE

A total of six groups of C57BL/6NNIA male mice at 6, 18 and 28 months of age were administered a liquid diet containing either ethanol (8 g/kg body wt/day) or an isocaloric amount of glucose for three weeks. Sixteen hours after the last ethanol feeding, homogenates of cerebral cortices were prepared for isolation of the synaptosome-mitochondrial fraction. Phospholipids were separated by two-dimensional tlc and the level of individual phospholipids was determined by assaying the lipid phosphorus content. Based on protein content, there was a decrease (18%) in the level of phosphatidylserines between samples from 6 and 28 month-old mice; no age difference was observed in other phospholipids. Ethanol consumption resulted in a three-fold increase in poly-phosphoinositide level in the 6 month-old group, but the difference diminished considerably with age. The increase in poly-phosphoinositide level suggests a link between chronic ethanol administration and the receptor-mediated signal transduction mechanism involving phosphoinositide turnover. Results further indicate that animals in the younger age group are more readily influenced by the chronic effect of ethanol than those in the older age group.