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A S Gordon

Publications and source records attributed to A S Gordon.

At least 37 records · Page 2Linked to original sources
PubMed1996

The role of protein kinase C in cellular tolerance to ethanol.

We have shown that ethanol inhibits uptake of adenosine by a specific nucleoside transporter in NG108-15 neuroblastoma x glioma cells and that cAMP-dependent protein kinase (PKA) activity is required for this inhibition. After chronic exposure to ethanol, adenosine uptake is no longer inhibited on rechallenge with ethanol, i.e. transport has become tolerant to ethanol. Here we show that protein kinase C (PKC) contributes to ethanol-induced tolerance of adenosine transport. Activation of PKC by phorbol esters in control cells results in an ethanol-tolerant phenotype, similar to that produced by chronic ethanol exposure. In addition, chronic exposure to ethanol increases the amounts of alpha, delta, and epsilon PKC. However, reducing PKC activity by inhibition with chelerythrine during chronic exposure to ethanol or down-regulation by phorbol esters prevents the development of ethanol-induced tolerance of adenosine transport. By contrast, the inhibition of PKA activity produces tolerance to ethanol inhibition of adenosine uptake. When protein phosphatase inhibitors are present, inhibiting PKA activity has no effect on ethanol sensitivity of adenosine uptake, suggesting a role for protein phosphatases in the regulation of ethanol sensitivity of uptake. Taken together, our results suggest that PKA and PKC have opposing effects on the ethanol sensitivity of adenosine transport; PKA activity is required for ethanol sensitivity, and PKC activation produces tolerance. Based on these data, we propose that chronic ethanol exposure increases PKC activity, leading to the activation of a protein phosphatase (1 or 2A). This phosphatase then dephosphorylates a PKA-phosphorylated site, which is required for ethanol to inhibit adenosine uptake. Therefore, the sensitivity of adenosine transport to ethanol appears to be maintained by a balance of PKA and protein phosphatase activities, and PKC may regulate phosphatase activity.

Adenosine↗
PubMed1996

Ethanol causes translocation of cAMP-dependent protein kinase catalytic subunit to the nucleus.

Short- and long-term ethanol exposures have been shown to alter cellular levels of cAMP, but little is known about the effects of ethanol on cAMP-dependent protein kinase (PKA). When cAMP levels increase, the catalytic subunit of PKA (C alpha) is released from the regulatory subunit, phosphorylates nearby proteins, and then translocates to the nucleus, where it regulates gene expression. Altered localization of C alpha would have profound effects on multiple cellular functions. Therefore, we investigated whether ethanol alters intracellular localization of C alpha. NG108-15 cells were incubated in the presence or absence of ethanol for as long as 48 h, and localization of PKA subunits was determined by immunocytochemistry. We found that ethanol exposure produced a significant translocation of C alpha from the Golgi area to the nucleus. C alpha remained in the nucleus as long as ethanol was present. There was no effect of ethanol on localization of the type I regulatory subunit of PKA. Ethanol also caused a 43% decrease in the amount of type I regulatory subunit but had no effect on the amount of C alpha as determined by Western blot. These data suggest that ethanol-induced translocation of C alpha to the nucleus may account, in part, for diverse changes in cellular function and gene expression produced by alcohol.

Animals↗
PubMed1996

Activation of cyclic AMP-dependent protein kinase reverses tolerance of a nucleoside transporter to ethanol.

Adenosine mediates some of the acute and chronic effects of ethanol in neural cells. In cultured NG108-15 cells, ethanol inhibits adenosine uptake via a specific facilitative nucleoside transporter leading to an increase in extracellular adenosine, activation of adenosine A2 receptors and increases in intracellular cyclic AMP (cAMP). After chronic ethanol exposure, an adaptive decrease in receptor-stimulated cAMP levels occurs. Additionally, the transporter becomes insensitive to rechallenge with ethanol and adenosine uptake is not inhibited. cAMP levels are decreased in cells chronically exposed to ethanol and we show here that cAMP-dependent kinase (PKA) activity in cellular homogenates also is decreased. Therefore, decreased cAMP-dependent phosphorylation may be responsible for loss of ethanol sensitivity. To test this hypothesis, NG108-15 cells were treated with agents that alter PKA activity and the ethanol sensitivity of adenosine transport was measured. In naive cells, decreasing PKA activity with the cAMP antagonist, Rp-adenosine-3',5'-cyclic phosphorothioate, resulted in ethanol-insensitive adenosine uptake. This effect was blocked by the phosphatase inhibitor, okadaic acid. These results suggest that loss of ethanol sensitivity is correlated with decreased PKA activity. Therefore, stimulating PKA activity in chronically treated cells should restore sensitivity of adenosine uptake to inhibition by ethanol. Indeed, the cAMP agonist, Sp-adenosine-3',5'-cyclic phosphorothioate, restored ethanol sensitivity of transport in cells treated chronically with ethanol. Our results suggest that ethanol sensitivity of adenosine transport is regulated by PKA and protein phosphatase activities in NG108-15 cells. Moreover, the effects of chronic ethanol exposure on adenosine transport can be reversed by activating PKA.

Adenosine↗
PubMed1994

The role of adenosine in mediating cellular and molecular responses to ethanol.

We have found that ethanol-induced increases in extracellular adenosine activate adenosine receptors which, in turn, mediate many of the acute and chronic effects of ethanol in the nervous system. Several laboratories have demonstrated the importance of adenosine in mediating the acute and chronic effects of ethanol at multiple levels of investigation in the nervous system. These include genetic selection for ethanol sensitivity in mice, behavioral responses to ethanol in naive and tolerant animals, neurophysiologic responses in hippocampal slices, and at the level of cAMP signal transduction and gene expression in cultured neural cells. In this review we present results from our laboratory which document the role of adenosine in mediating ethanol-induced changes in neural function at a cellular and molecular level. A schematic summary of our findings is: Etoh-->decreases Ado uptake-->increases Extracellular Ado-->Activation of Adenosine A2 receptor-->increases cAMP-->increases PKA-->-->-->Heterologous Desensitization (decreases cAMP)-->-->-->insensitivity of adenosine uptake to ETOH

Adenosine↗
PubMed1994

A mutation in Arabidopsis that leads to constitutive expression of systemic acquired resistance.

Systemic acquired resistance (SAR) is a nonspecific defense response in plants that is associated with an increase in the endogenous level of salicylic acid (SA) and elevated expression of pathogenesis-related (PR) genes. To identify mutants involved in the regulation of PR genes and the onset of SAR, we transformed Arabidopsis with a reporter gene containing the promoter of a beta-1,3-glucanase-encoding PR gene (BGL2) and the coding region of beta-glucuronidase (GUS). The resulting transgenic line (BGL2-GUS) was mutagenized, and the M2 progeny were scored for constitutive GUS activity. We report the characterization of one mutant, cpr1 (constitutive expressor of PR genes), that was identified in this screen and shown by RNA gel blot analysis also to have elevated expression of the endogenous PR genes BGL2, PR-1, and PR-5. Genetic analyses indicated that the phenotype conferred by cpr1 is caused by a single, recessive nuclear mutation and is suppressed in plants producing a bacterial salicylate hydroxylase, which inactivates SA. Furthermore, biochemical analysis showed that the endogenous level of SA is elevated in the mutant. Finally, the cpr1 plants were found to be resistant to the fungal pathogen Peronospora parasitica NOCO2 and the bacterial pathogen Pseudomonas syringae pv maculicola ES4326, which are virulent in wild-type BGL2-GUS plants. Because the cpr1 mutation is recessive and associated with an elevated endogenous level of SA, we propose that the CPR1 gene product acts upstream of SA as a negative regulator of SAR.

Arabidopsis↗
PubMed1994

Regulation of extracellular copper-binding proteins in copper-resistant and copper-sensitive mutants of Vibrio alginolyticus.

Extracellular proteins of wild-type Vibrio alginolyticus were compared with those of copper-resistant and copper-sensitive mutants. One copper-resistant mutant (Cu40B3) constitutively produced an extracellular protein with the same apparent molecular mass (21 kDa) and chromatographic behavior as copper-binding protein (CuBP), a copper-induced supernatant protein which has been implicated in copper detoxification in wild-type V. alginolyticus. Copper-sensitive V. alginolyticus mutants displayed a range of alterations in supernatant protein profiles. CuBP was not detected in supernatants of one copper-sensitive mutant after cultures had been stressed with 50 microM copper. Increased resistance to copper was not induced by preincubation with subinhibitory levels of copper in the wild type or in the copper-resistant mutant Cu40B3. Copper-resistant mutants maintained the ability to grow on copper-amended agar after 10 or more subcultures on nonselective agar, demonstrating the stability of the phenotype. A derivative of Cu40B3 with wild-type sensitivity to copper which no longer constitutively expressed CuBP was isolated. The simultaneous loss of both constitutive CuBP production and copper resistance in Cu40B3 indicates that constitutive CuBP production is necessary for copper resistance in this mutant. These data support the hypothesis that the extracellular, ca. 20-kDa protein(s) of V. alginolyticus is an important factor in survival and growth of the organism at elevated copper concentrations. The range of phenotypes observed in copper-resistant and copper-sensitive V. alginolyticus indicate that altered sensitivity to copper was mediated by a variety of physiological changes.

Carrier Proteins↗
PubMed1994

Responses of diverse heterotrophic bacteria to elevated copper concentrations.

The influence of copper on the growth of Escherichia coli, Pseudomonas aeruginosa, Bacillus cereus, Bacillus subtilis, Vibrio parahaemolyticus, Vibrio alginolyticus (three strains), and an unidentified Vibrio sp. was examined in batch cultures. The effects of copper at micromolar concentrations varied from undetectable to complete growth inhibition. Each strain was able to recover from a growth lag observed after copper addition at a characteristic concentration. Copper concentrations that allowed recovery ranged from 25 to 150 microM. Extracellular proteins in the medium of cultures that had recovered from copper stress were compared with those from control cultures. Protein profiles were analyzed for the presence of proteins similar to extracellular copper-binding proteins (CuBP) previously reported in V. alginolyticus. CuBP-like proteins were found in each Vibrio sp. examined. A protein of similar molecular mass was also detected in copper-stressed cultures of P. aeruginosa and not in control cultures. Escherichia coli and Bacillus spp. did not produce CuBP-like proteins. The data show that CuBP-like proteins are not produced by all bacteria in response to copper stress and indicate that such proteins are common in marine Vibrio spp.

Bacillus↗
PubMed1994

Adenosine receptors mediate cellular adaptation to ethanol in NG108-15 cells.

Acute ethanol treatment of NG108-15 neuroblastoma x glioma hybrid cells results in inhibition of adenosine uptake with consequent increases in extracellular adenosine and intracellular cAMP concentrations. Chronic exposure to ethanol, however, causes heterologous desensitization of receptors coupled to adenylyl cyclase via stimulatory guanine nucleotide regulatory protein. This heterologous desensitization is correlated with a decrease in the amount of protein and mRNA for the GTP-binding subunit of stimulatory guanine nucleotide regulatory protein. In addition, after chronic exposure to ethanol, the adenosine transporter becomes tolerant to acute ethanol inhibition of adenosine uptake, and there is no longer an increase in extracellular adenosine. We have previously shown that extracellular adenosine is required for the development of ethanol-induced heterologous desensitization. To examine the role of adenosine receptors in mediating these responses to ethanol, we used BW A1434U, an adenosine receptor antagonist that does not inhibit nucleoside transport. BW A1434U caused a concentration-dependent inhibition of (-)-N6-(R-phenyl-isopropyl)-adenosine-stimulated cAMP production in NG108-15 cells. BW A1434U also completely blocked acute ethanol-induced increases in intracellular cAMP levels and prevented the development of ethanol-induced heterologous desensitization and the reduction in the GTP-binding subunit of stimulatory guanine nucleotide regulatory protein. In addition, BW A1434U prevented the development of tolerance to ethanol-induced inhibition of adenosine transport. Our results indicate that in NG108-15 cells, adenosine receptors mediate ethanol-induced changed in cAMP signal transduction and adenosine transport and that an adenosine receptor antagonist can block both these acute and chronic affects of ethanol.

Adaptation, Physiological↗
PubMed1994

Selective inhibition by ethanol of the type 1 facilitative glucose transporter (GLUT1).

Ethanol appears to modulate the function of selective mammalian receptors and transporters by interacting with highly specific membrane protein sites. Of the multiple types of nucleoside transporters known to be present in mammalian cells, we observed that ethanol inhibits only one class of facilitative nucleoside transporters, that inhibited by nitrobenzylmercaptopurine riboside. Because there are biochemical similarities between facilitative glucose transporters and nitrobenzylmercaptopurine riboside-sensitive nucleoside transporters, we tested whether ethanol might selectively inhibit a unique class of facilitative glucose transporters. We report here that ethanol inhibits hexose uptake in human lymphocytes and several cell lines expressing the ubiquitous facilitative type 1 glucose transporter (GLUT1). Ethanol inhibition of hexose uptake by GLUT1 is independent of ethanol inhibition of facilitative nucleoside transport. We also determined the ethanol sensitivity of various cloned human facilitative glucose transporters expressed in Chinese hamster ovary cells and we found that ethanol inhibits hexose uptake by GLUT1 but not uptake by GLUT3 or GLUT4 transporters. Our results suggest that a protein motif or motifs present in the GLUT1 amino acid sequence but absent in GLUT3 or GLUT4 proteins may confer ethanol sensitivity.

Animals↗
PubMed1993

Growth, copper-tolerant cells, and extracellular protein production in copper-stressed chemostat cultures of Vibrio alginolyticus.

The influence of elevated copper concentrations on cell numbers and extracellular protein production was investigated in chemostat cultures of Vibrio alginolyticus. High (20 microM) copper in the medium reservoir resulted in a dramatic drop in cell numbers which was overcome with time. The copper-stressed cultures established a new equilibrium cell concentration slightly (ca. 20%) lower than control cultures. Copper-stressed chemostat populations contained an increased number of copper-resistant cells, but these averaged only 26% of the copper-adapted population. Previously copper-stressed populations exhibited resistance to a second challenge with copper. Proteins with properties identical to those of copper-induced, copper-binding proteins (CuBPs) observed in batch cultures of V. alginolyticus were observed in the supernatants of copper-stressed chemostat cultures and not in controls. CuBPs from batch and chemostat cultures were identical in terms of their induction by copper, molecular weight, and retention volumes on both immobilized copper ion-affinity chromatography and reverse-phase high-performance liquid chromatography columns. The concentration of CuBP in the chemostat was dependent on copper concentration in the medium reservoir. Either one or two forms of CuBP were observed in various analyses from both batch and chemostat cultures. Gel-to-gel variability was implicated as a factor determining whether one or two forms were resolved in a given analysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial Proteins↗
PubMed1993

Inhibition of adenosine uptake by ethanol is specific for one class of nucleoside transporters.

Adenosine uptake via nucleoside transporters is inhibited when S49 and NG108-15 cell lines cells are exposed to ethanol. This inhibition leads to an accumulation of extracellular adenosine that binds to adenosine A2 receptors and increases cAMP production. Subsequently, there is a heterologous desensitization of receptors coupled to adenylyl cyclase for which adenosine also is required. There are multiple classes of facilitative and concentrative nucleoside transporters that could be inhibited by ethanol to initiate this cascade of events. In this paper, we establish that adenosine uptake by only one type of nucleoside transporter, an NBMPR-sensitive facilitative transporter, is inhibited by ethanol. There is no effect on other classes of nucleoside transporters even when present in the same cell. Thus, ethanol-induced extracellular accumulation of adenosine results specifically from inhibition of NBMPR-sensitive facilitative nucleoside transporters. We also find that human lymphocytes express only facilitative nucleoside transporters and that the NBMPR-sensitive type is predominant. Thus, inhibition of this type of transporter by ethanol may be related to the desensitization of cAMP signal transduction that we have reported in lymphocytes from alcoholics.

Adenosine↗
PubMed1992

HIV-associated pericardial effusions.

Following a case of cardiac tamponade in a patient with the acquired immunodeficiency syndrome (AIDS), we examined the frequency and clinical spectrum of pericardial effusions associated with human immunodeficiency virus infection (HIV) at our institution. Of 187 hospitalized patients documented to have pericardial effusions over a one-year period, 14 (7 percent) were known to be HIV-positive at the time of their echocardiograms. One patient presented with a large effusion and cardiac tamponade, three had moderate effusions, and ten had small effusions. The probable effusion etiology was established in four cases and included endocarditis (2), lymphoma (1), and myocardial infarction (1). In hospital mortality was 29 percent (4 of 14). From our study, as well as a growing number of reports in the literature, we conclude that HIV-associated pericardial effusions are frequently seen and that their clinical spectrum is broad.

Acquired Immunodeficiency Syndrome↗
PubMed1992

Neonatal erythropoiesis. II. Bone marrow and splenic erythropoietic activity: data suggest erythropoietin transfer via maternal milk.

In this study we examined erythropoiesis in the bone marrow and spleen of 9 to 12-day-old neonatal rats suckled by bleeding-induced anemic mothers. Quantitative evaluations of the spleen revealed a significant decrease in total numbers of nucleated RBC/mg spleen in 11-day-old pups nursed by the anemic mother which returned to control values by day 12. A significant reduction in total numbers of nucleated RBC/mg marrow was seen in both 11 and 12-day-old pups of anemic mothers when compared to control values. These results suggest that: 1. Ep is transmitted to suckling rats via the maternal milk; 2. Ep in the neonate exerts its influence predominantly at the level of the differentiated erythroid compartment probably by causing a shortening of the mean transit time of the proliferating erythroblast compartment and/or by decrease in the maturation time of the nonproliferating orthochromatic and reticulocyte compartments; 3. the regulation of erythropoiesis in the neonate differs from that in the adult.

Anemia↗
PubMed1992

Neonatal erythropoiesis. I. Peripheral blood erythropoietic parameters: data suggest erythropoietin transfer via maternal milk.

In this study peripheral blood erythropoietic parameters in 9 to 12-day-old neonatal rats suckled by experimentally-induced anemic mothers were examined. Stimulation of erythropoiesis in these pups was judged by increases in hematocrit, hemoglobin, mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration. Failure to observe increases in reticulocytes may be, in part, the result of decreased maturation time, as indicated by reticulocyte Heilmeyer maturation indices in peripheral blood. The reticulocyte maturation curve was shifted to the left in neonates nursing from anemic mothers. These results suggest that erythropoietin (Ep) is transmitted to suckling rats via maternal milk, and by escaping inactivation in their gastrointestinal tract stimulates their erythropoiesis. These findings, in agreement with our previous work, are supportive of studies by others and further indicate a maternal-neonatal erythropoietic relation in rats.

Anemia↗
PubMed1991

Sibling influences on adolescent drug use: older brothers on younger brothers.

This study was designed to examine sibling influences on adolescent drug use. The sample was composed of 278 white, predominantly middle-class male college students and their oldest brothers. Self-administered questionnaires were taken separately by the students and their brothers. The findings indicated that the oldest brother's personality and the sibling relationship had an influence on the younger brother's drug use. Three hypothesized mechanisms that supported the findings, the Personality Influence Mechanism, the Genetic Temperament Connection, and the Environmental Reactive Mechanism were discussed.

Adolescent↗
PubMed1991

cAMP-dependent protein kinase regulates inhibition of adenosine transport by ethanol.

Ethanol inhibits adenosine uptake, thereby increasing the concentration of extracellular adenosine. Elevation of extracellular adenosine increases intracellular cAMP concentration via activation of adenosine A2 receptors. Extracellular adenosine is also required for the subsequent development of ethanol-induced heterologous desensitization. Here we report that activation of cAMP-dependent protein kinase is necessary for inhibition of adenosine uptake by ethanol and for the consequent accumulation of extracellular adenosine. Ethanol does not inhibit adenosine uptake in mutants of the S49 cell line that lack receptor-stimulated cAMP production (unc cells) or cAMP-dependent protein kinase activity (kin- cells). Forskolin, which bypasses the receptor-coupling defect in unc cells to increase cAMP levels, restores inhibition of adenosine uptake by ethanol. In contrast, in kin- cells forskolin did not restore inhibition of adenosine uptake by ethanol, despite similar increases in cAMP levels. Taken together, these results suggest that cAMP-dependent protein kinase phosphorylates a component of the nucleoside transporter, thereby regulating the sensitivity of adenosine transport to ethanol.

Adenosine↗