Equilibrative Adenosine Transport in Rat Hepatocytes after Chronic Ethanol Feeding

Acute treatment of cells with ethanol in vitro inhibits adenosine uptake via equilibrative nucleoside transporters. After longer periods of exposure to ethanol in culture, rechallenge with ethanol no longer inhibits adenosine uptake. Herein, we have investigated the long-term effects of ethanol consumption in vivo on equilibrative nucleoside transport. Rats were fed a liquid diet containing 35% of calories as ethanol (ethanol-fed). Control rats were pair-fed a liquid diet that isocalorically substituted maltose dextrins for ethanol. After 4 weeks of ethanol consumption, nucleoside transport was measured in isolated hepatocytes. Uptake of [³H]adenosine was lower in ethanol-fed rats compared with control. Influx of the nonmetabolizable nucleoside analog, [³H]formycin B, was also decreased after ethanol feeding. However, neither the number of nitrobenzylthioinosine (NBMPR) binding sites or inhibition of adenosine uptake by NBMPR were affected by ethanol feeding. In controls, acute treatment of isolated hepatocytes with 100 itim ethanol inhibited [³H]adenosine uptake by 30–40%. However, in ethanol-fed rats, acute challenge with ethanol did not inhibit [³H]adenosine uptake. These data demonstrate that long-term ethanol feeding decreases equilibrative nucleoside transport in hepatocytes independent of a change in the number of nucleoside transporters and renders adenosine uptake insensitive to inhibition by ethanol.     

Effect of Ethanol on [3H]Nitrendipine Binding to Calcium Channels in Brain Membranes

We examined the effect of ethanol on [3H]nitrendipine binding to rat brain membranes, to investigate the possibility that voltage-dependent calcium channels are involved in synaptic actions of ethanol. Ethanol inhibited specific [3H]nitrendipine binding with Ki = 460 mM, by decreasing binding affinity without altering the maximal number of sites labeled. At a lower concentration (100 mM), ethanol had no effect on inhibition of binding by verapamil or diltiazem. The high concentrations of ethanol required to produce alterations in [3H]nitrendipine binding suggest that ethanol and classical calcium channel antagonists influence calcium-mediated synaptic processes by separate mechanisms.     

Dibutyryl cyclic AMP, adenosine and guanosine blockade of the dopamine, ergocryptine and apomorphine inhibition of prolactin release in vitro.

Rat anterior hemipituitaries were incubated in Krebs-Ringer bicarbonate containing [3H]leucine. Newly synthesized [3H]prolactin and [3H]GH in the pituitary and incubation medium were assayed, as was the radioimmunoassayable prolactin released into the medium during a 5-h incubation. Dopamine (7.5 X 10(-8)M), ergocryptine (4 X 10(-10) M) and apomorphine (6 X 10(-8)M) all significantly inhibited both radioimmunoassayable prolactin release and newly-labeled [3H]prolactin release without affecting [3H]GH release. Conversely, dibutyryl cyclic AMP (2.5 mM) stimulated radioimmunoassayable prolactin release as well as [3H]prolactin and [3H]GH release. The addition of 2.5 mM dibutyryl cyclic AMP to media containing dopamine, ergocryptine or apomorphine completely restored both radioimmunoassayable prolactin release and [3H]prolactin release to at least control levels. Dopamine, ergocryptine and apomorphine all inhibited incorporation of [3H]leucine into prolactin but not into GH, whereas 2.5 mM dibltyryl cyclic AMP with any one of the inhibitors restored total incorporation into [3H]prolactin to levels insignificantly lower than the nucleotide-stimulated incorporation. Adenosine and guanosine at 2.5 mM also stimulated incorporation into [3H]prolactin and blocked the inhibitory effects of apomorphine upon [3H]prolactin synthesis and release. These nucleosides also stimulated [3H]GH release; and guanosine, but not adenosine, stimulated incorporation into [3H]GH. The ability of dibutryl cyclic AMP to block the effects of dopamine, ergocryptine and apomorphine upon prolactin release is consistent with these three inhibitors acting by a common mechanism. Cyclic AMP could be hypothesized as a second messenger for prolactin release, but the ability of adenosine and guanosine to mimic almost perfectly the effects of this cyclic nucleotide does not allow any conclusive interpretation.     

Combined Inhibitory Effects of Ethanol and Adenosine on the Responses of Rabbit Platelets to Thrombin

We have previously shown that acutely administered ethanol, resulting in blood alcohol concentrations of 40-00 mM, inhibits experimentally induced arterial thrombosis in rabbits. This inhibition by ethanol in vivo is more pronounced than that observed on stimulated platelets in vitro, when a similar concentration of ethanol is added before an aggregating agent. It may be, then, that ethanol has combined effects in vivo with other inhibitors of platelet function. Adenosine has been found to be an important mediator of some of the in vivo effects of ethanol, and we investigated whether ethanol has combined inhibitory effects with adenosine on thrombin-stimulated platelet responses in vitro. Aggregation and secretion of [¹⁴C]serotonin from washed, prelabeled rabbit platelets, pretreated with aspirin, were studied. Maximal aggregation induced by 0.15 units thrombin/ml was slightly inhibited by 87 mM ethanol; secretion of serotonin was reduced from 24% to 12%. However, when thrombin-induced aggregation was significantly reduced by 1 μM adenosine, ethanol, at 44 and 87 mM, further inhibited aggregation. Secretion of [¹⁴C]serotonin was reduced to <3%, with the combination of adenosine and the higher concentration of ethanol. Ethanol did not increase platelet cyclic AMP (cAMP) above basal levels, nor did it affect the increase in cAMP caused by adenosine. The adenosine receptor antagonist, 8-phenyltheophylline, at 1 μM, blocked the inhibitory effects of adenosine on platelet responses and prevented the adenosine-induced increase in cAMP. Unexpectedly, however, 8-phenyltheophylline (1–2 μM) did not completely block the combined inhibitory effects of ethanol and adenosine; this incomplete reversal was not associated with increases in cAMP over basal levels. 8-Phenyltheophylline may by useful in vivo in determining whether ethanol has combined inhibitory effects with adenosine on experimentally induced arterial thrombosis.     

Myocardial glucose utilization. Failure of adenosine to alter it and inhibition by the adenosine analogue N6-(L-2-phenylisopropyl)adenosine

The effects of adenosine and the nonmetabolizable adenosine analogue N6-(L-2-phenylisopropyl)adenosine (PIA) on glucose transport or metabolism were determined in purified myocardial sarcolemmal vesicles, isolated cardiocytes, and perfused hearts. Adenosine (100 microM) did not affect hexose transport in myocytes. Also, adenosine deaminase, added to metabolize adenosine to inosine, did not alter transport of hexose into myocytes regardless of whether or not insulin was present. In contrast, PIA effectively inhibited 3-O-methyl-D-glucose uptake in myocytes even during insulin stimulation. PIA inhibited D-glucose-specific transport in both rat and bovine cardiac sarcolemmal vesicles (Ki = 26 microM at [D-glucose] = 5 mM). However, insulin did not affect glucose transport in sarcolemmal vesicles, which implies that receptor-coupled processes probably are not intact in this preparation. Thus, inhibition of PIA may not be receptor mediated. Also, PIA inhibited binding of cytochalasin B to bovine cardiac sarcolemmal vesicles, which supports the idea that PIA inhibits glucose flux by binding to the glucose transporter. To determine if adenosine altered glucose metabolism rather than transport, we measured the rate of 3H2O production from metabolism of D-[2-3H]glucose in paced rat hearts ([D-glucose] = 5.5 mM, [pyruvate] = 0.2 mM) perfused with a range of PIA or adenosine concentrations with or without 0.01 microM insulin. Adenosine (0.01-100 microM) in the presence or absence of insulin increased coronary flow but did not change glycolytic rates. Similar results were obtained with PIA (no insulin) rather than adenosine in the perfusate. However, with glucose as the only exogenous substrate, 100 microM PIA inhibited glycolysis by approximately 50%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Incorporation of adenosine into ATP: formation of compartmentalized ATP.

The incorporation of [3H]adenosine, [3H]adenine, and [3H]hypoxanthine into adenine nucleotides of nude (athymic) mouse liver and human hepatoma grown subcutaneously in nude mice was studied. 3H and 32P radioactive labeling in vivo of acid-soluble nucleotides followed by chromatographic procedures indicated that, in contrast to [3H]adenine and [3H]hypoxanthine, [3H]adenosine is preferentially incorporated into ATP in comparison with its incorporation into AMP and ADP. This phenomenon, as well as complementing the recently reported 3-fold increase in total cellular ATP upon treatmen- with 0.5-1.0 mM concentrations of adenosine, indicates the formation from adenosine of compartmentalized ATP that is not produced from either adenine or hypoxanthine. The observed effect is of larger magnitude in the growth-arrested normal liver than in the actively growing tumor.     

Evaluation of the effect of ethanol's toxic metabolite acetaldehyde on the gastrointestinal oligopeptide transporter, PEPT1: in vitro and in vivo studies

Background: The effects of alcohol consumption and its subsequent metabolism on drug transport, absorption and pharmacokinetics are poorly understood. This study examines the effects of the ethanol metabolite, acetaldehyde, on the clinically relevant drug transporter, PEPT1. The metabolism of ethanol and the following acetaldehyde formation is thought to modulate the uptake capacity of PEPT1 within the gastrointestinal tract for a variety of clinically important peptidomimetic drug compounds. Methods: Glycylsarcosine ([³H]‐GlySar), a nonhydrolysable PEPT1 specific substrate was used in our studies. In vitro uptake studies were performed in the Caco‐2 and Chinese hamster ovary (CHO)‐hPEPT1 cell models, measuring cellular uptake of labeled compound against increasing levels of unlabeled compound in the presence of acetaldehyde. In vivo absorption of [³H]‐GlySar was measured in male Sprague–Dawley rats that were treated with oral dose of ethanol/disulfiram (5 g/kg / 100 mg/kg) for 6 days. These results were compared to control rats treated with saline, ethanol alone or disulfiram alone. Results: In vitro uptake of [³H]‐GlySar in CHO‐hPEPT1 cells treated with 1 mM acetaldehyde was significantly decreased (p < 0.05) as compared to untreated controls. The uptake of [³H]‐GlySar in Caco‐2 cell monolayers treated with 1 mM acetaldehyde was also significantly decreased as compared to the untreated control cells. In vivo absorption of [³H]‐GlySar in ethanol treated rats, as measured by AUC_{0–12 hours} were decreased by approximately 50% versus the control rat group. Conclusion: The effects of acetaldehyde due to consumption of ethanol on the uptake and bioavailability of therapeutic drug compounds transported by the PEPT1 oligopeptide transporter have not been documented. In the present studies, we demonstrate that acetaldehyde significantly modulates PEPT1 function and, thereby, affects drug bioavailability. To our best knowledge, this is the first report on the effects of an ethanol metabolite on substrate absorption in the gastrointestinal tract, rather than interactions in the liver, which is an under‐represented area of research in alcohol pathophysiology.     

Effects of H2-receptor antagonists on gastric alcohol dehydrogenase activity

Inhibition of gastric alcohol dehydrogenase (ADH) activity by cimetidine results in elevated blood levels of ethanol after moderate consumption. To search for alternative H2-blockers lacking such an effect, we compared cimetidine, ranitidine, nizatidine, and famotidine. They inhibited rat gastric ADH noncompetitively, with a Ki for ethanol oxidation of 0.68 mM for cimetidine, 0.5 mM for ranitidine, 1 mM for nizatidine, and 4.5 mM for famotidine. These concentrations are higher than therapeutic plasma levels, but intracellular concentrations in the gastric mucosa (assessed with [3H]cimetidine and [14C]famotidine) were at least 10- and 2-fold greater than in the blood, respectively. These results suggests that, given at therapeutic doses in vivo, the degree of inhibition by cimetidine and ranitidine should be significant and comparable, that by nizatidine should be smaller, and that by famotidine should be negligible. These drugs also exerted either mixed or competitive inhibition of rat hepatic ADH, but the effects of cimetidine and famotidine were observed at concentrations unlikely to occur in vivo. Thus, in alcoholics and in social drinkers who require treatment with H2-receptor antagonists, famotidine might be preferable to the other H2 blockers tested.     

Tolerance to Adenosine''s Accentuation of Ethanol-Induced Motor Incoordination in Ethanol-Tolerant Mice

Our previously published reports have provided data that have supported a functional correlation between ethanol-induced changes in the characteristics of adenosine receptor, adenosine uptake and release in the brain, and ethanol-induced motor incoordination. The present data demonstrated a cross-tolerance between ethanol and adenosine further supporting the hypothesis that brain adenosine modulates the motor impairing effects of ethanol. Mice that received (-)-N6-cyclohexyladenosine (CHA) [0.25 mg/kg/day, intraperitoneally (ip)] for 10 days exhibited marked attenuation (cross-tolerance) to acute ethanol-induced motor incoordination compared with chronic saline (ip) controls. The attenuation of acute ethanol-induced motor incoordination was essentially same in animals that received CHA (25 ng/5 microliters/day for 10 days) by the intracerebroventricular (icv) route as opposed to the controls that chronically received artificial cerebral spinal fluid by the same route. Similarly, tolerance was exhibited to acute CHA (0.125 mg/kg ip and 12.5 ng/5 microliters icv) by animals fed liquid ethanol (19.5 g/kg/24 hr) for 10 days compared with none in the pair-fed sucrose controls. Scatchard plots using cerebellar tissue homogenates from animals given chronic CHA or chronic ethanol indicated no change in Bmax and/or Kd values for CHA binding when compared with CHA binding in tissues from their respective controls. However, a lack of any change in the binding characteristics cannot rule out the involvement of adenosine receptors in the observed cross-tolerance between ethanol and CHA. The results may suggest desensitization of adenosine A1 receptors due to chronic CHA and ethanol as an alternate possible explanation in the development of cross-tolerance between adenosine (CHA) and ethanol.     

Ethanol Fails to Modify [3H]GR65630 Binding to 5-HT3 Receptors in NCB-20 Cells and in Rat Cerebral Membranes

Low concentrations of ethanol have been found to enhance the electrophysiologic effect of serotonin (5-HT) acting at 5-HT3 receptors on NCB-20 cells. To determine whether this action of ethanol reflects a change in the agonist-receptor interaction, the effect of ethanol (100 mM) on agonist and antagonist binding to 5-HT3 receptor was studied in vitro in membrane from NCB-20 cells and from cortex plus hippocampus of rat. The antagonist [3H]GR65630 was used to label 5-HT3 recognition sites. Ethanol did not change the characteristics of saturable [3H]GR65630 binding in either membrane preparation. In competition studies, the agonists 5-HT and 2-methyl-5-HT completely inhibited the binding of [3H]GR65630 to NCB-20 cell membranes, while in brain membranes the maximum displacement of specific [3H]GR65630 binding by 5-HT was approximately 30%. Ethanol decreased the affinity of the receptor for 2-methyl-5-HT, but not to 5-HT in NCB-20 cells, and had no effect on agonist binding in brain membranes. The results indicate that enhancement of 5-HT responses at 5-HT3 receptors by ethanol is not a result of changes in the equilibrium binding characteristics of the agonist.     

Effects of proglumide on pancreatic acinar cell function

Proglumide, a putative gastrin receptor antagonist, inhibited cholecystokinin (CCK)-stimulated amylase release and [3H]-2-deoxy-D-glucose uptake by isolated mouse pancreatic acini. Inhibition was reversible and competitive in nature with a KI of 0.7 mM. Proglumide also competitively inhibited the binding of 125I-CCK to its receptor in pancreas and brain; the KI for this interaction was 1.0 mM. In contrast, proglumide did not inhibit carbachol-stimulated amylase release, insulin-stimulated glucose transport and protein synthesis, or the binding of insulin to its receptors. Proglumide at 10 mM did, however, reduce both basal [3H]-2-deoxy-D-glucose uptake and [3H]-leucine incorporation into protein. We conclude that proglumide is a competitive and specific, albeit weak antagonist of CCK receptors. Higher concentrations of the drug may have other more nonspecific effects.     

Acute ethanol-induced adenosine diphosphate ribosylation regulates the functional activity of rat striatal pertussis toxin-sensitive g proteins

BACKGROUND: We demonstrated previously that striatal adenosine modulates ethanol-induced motor incoordination (EIMI) via adenosine A1 receptors coupled to pertussis toxin (PT)-sensitive G protein and adenylyl cyclase-cyclic adenosine monophosphate (cAMP). Additionally, intrastriatal (IST) PT antagonizes EIMI and its potentiation by the adenosine A1 agonist N-cyclohexyladenosine; it also inhibits cAMP concentration. METHODS: Guide cannulas were stereotaxically implanted for IST pretreatment with PT followed 5 days later by IST of N-cyclohexyladenosine and intraperitoneal ethanol. The adenosine diphosphate (ADP) ribosylation reaction involved PT-catalyzed [P]nicotinamide adenine dinucleotide (NAD) labeling of rat striatal membranes. Antagonism of EIMI (Rotorod method) after IST microinfusion of PT was investigated to determine whether it was due to a decrease in the functional activity of G proteins due to ADP ribosylation of the Gialpha subunit caused it. RESULTS: Striatal membranes from IST PT (0.5 microg)-treated animals exhibited significantly attenuated (up to 90%) in vitro ADP ribosylation with [P]NAD. Striatal membranes from animals injected with ethanol (1.5 g/kg intraperitoneally) exhibited statistically significant increase (11%) in in vitro ADP ribosylation. Similarly, ethanol (50 mM) added to striatal membranes from untreated animals produced significant stimulation of in vitro ADP ribosylation. The decrease in the functional activity of G proteins due to ADP ribosylation of the Gialpha subunit after IST PT was functionally correlated with marked attenuation in EIMI, as observed previously. This finding suggests a blockade of functional activity of PT-sensitive striatal Gi/Go proteins (i.e., fewer available sites for labeled NAD incorporation). The in vivo ethanol results indicate that it must have caused an increase in the ribosylation capacity of Gialpha in vivo (i.e., increased Gi activity). Increased ADP ribosylation by in vitro ethanol increases Gi/Go activity, consistent with EIMI, as previously reported. CONCLUSIONS: The results provide biochemical evidence of an ethanol-induced increase in ADP ribosylation of Gialpha causing a decrease in the functional activity of G proteins coupled via Gi/Go to adenylyl cyclase-cAMP. These results confirm the previously observed antagonism of EIMI by PT (IST).     

Gonadotropin-stimulated uptake of steroid hormones by Rana pipiens ovarian follicles incubated in vitro

The uptake of [³H]pregnenolone by frog ovarian follicles incubated in vitro is stimulated 50–60% by frog pituitary homogenate. This effect is mimicked by mammalian LH, but not by other pituitary or placental hormones. The uptake of progesterone is also stimulated by pituitary hormone, whereas, 17β-estradiol uptake is not. Incubation of follicles for less than an hour with gonadotropic hormones is sufficient to stimulate steroid uptake, and this process is fully inhibited by puromycin and partially inhibited by actinomycin D. Removal of follicular envelope interferes with the action of the pituitary hormone. Increasing concentrations of steroid hormone (pregnenolone) in the incubation medium stimulated [³H]pregnenolone uptake to an even greater extent than pituitary hormones. The possibility that gonadotropin-stimulated uptake of progestins is mediated by synthesis of a pregnenolone-like steroid is discussed.     

Ethanol exposure decreases glutamate uptake in the nucleus accumbens

BACKGROUND: An increased level of extracellular glutamate is a key neurochemical feature associated with ethanol exposure and withdrawal. METHODS: In the current study, extracellular levels of glutamate and glutamate transport in the nucleus accumbens were assayed 24 hr after repeated ethanol exposure (1 g/kg ip daily for 7 days) with use of in vivo no-net-flux microdialysis and in vitro [(3)H]glutamate uptake, respectively. RESULTS: Microdialysis revealed higher extracellular glutamate concentrations in the nucleus accumbens of rats that were given ethanol. The increase in basal extracellular glutamate levels was accounted for in part by a decrease in the in vivo probe recovery of glutamate. Moreover, an in vitro accumbens slice preparation measuring [(3)H]glutamate uptake revealed that Na(+)-dependent [(3)H]glutamate uptake was significantly reduced 24 hr after 7 days of repeated ethanol exposure. The ethanol-induced deficit in glutamate uptake was not associated with decreased total tissue levels of the transporters GLAST or GLT1. The in vivo and in vitro ethanol-induced changes in glutamate levels and uptake returned to control levels 14 days after discontinuing 7 days of repeated ethanol exposure. CONCLUSIONS: These results suggest that the previously reported increases in extracellular glutamate induced by ethanol exposure may be due in part to deficits in glutamate transport.     

Alcohol-induced reductions in cardiac protein synthesis in vivo are not ameliorated by treatment with the dihydropyridine calcium channel blocker amlodipine

BACKGROUND: Various studies have indicated that acute ethanol dosage perturbs cardiac function and/or structure with concomitant reductions in protein synthesis. Cellular calcium homeostasis is also perturbed, which may contribute to altered protein synthesis. This is supported by the observation that calcium channel blockers can prevent numerous features of alcohol-induced pathology. However, many of these studies have been carried out in vitro, employing supraphysiological levels of alcohol, or have failed to address whether their results obtained in isolated systems have direct relevance in vivo. The aim of the present investigation was to examine the response of cardiac protein synthesis in vivo due to a physiologically relevant dose of ethanol and determine whether a calcium channel antagonist could prevent these effects. METHODS: Changes in cardiac protein synthesis rates in vivo were assessed by measuring the fractional rates of protein synthesis (i.e., ks) using a "flooding dose" of [3H]phenylalanine. Rats were treated either acutely (10 mg/kg body weight, 3 hr) or chronically (10 mg/kg body weight/day, 30 days) with amlodipine, a dihydropyridine-type calcium channel blocker, before dosing with ethanol (75 mmol/kg body weight, 2.5 hr). RESULTS: Ethanol (75 mmol/kg body weight) inhibited cardiac protein synthesis after 1 hr. Similar responses were recorded at 2.5 and 6 hr after ethanol dosage. At 24 hr, ethanol decreased food intakes. However, a direct comparison between pair-fed controls and alcohol-dosed rats also showed a decrease in cardiac protein synthesis after 24 hr. Acute alcohol dosage reduced cardiac protein synthesis in mixed, myofibrillary, and sarcoplasmic protein fractions. Similar results were obtained when data were expressed relative to ribonucleic acid (i.e., kRNA). Neither acute nor chronic treatments with the calcium antagonist amlodipine ameliorated the deleterious actions of ethanol on protein synthesis. CONCLUSIONS: Ethanol may affect cardiac protein synthesis independently of altered calcium entry.     

Pyrophosphate, phosphate ion interaction: effects on calcium pyrophosphate and calcium hydroxyapatite crystal formation in aqueous solutions

The relationship between ambient ionic conditions that favor pyrophosphate (PPi) versus phosphate (Pi) biomineralization is important to understanding the pathogenesis of chondrocalcinosis. We studied aqueous solutions at pH 7.4, 37 degrees C, [Na+] = 140 mM, [Mg+ +] = 0.5 mM, [Ca+ +] = 1.0 or 1.5 mM over a range of pyrophosphate and phosphate concentrations to determine the effect of different ambient concentrations and ratios of Pi/PPi on calcium pyrophosphate dihydrate (CPPD) and calcium hydroxyapatite (HA) crystal formation. We found that the Pi/PPi ratio is an extremely important determinant of the crystal product formed. At low [Pi], CPPD crystal formation is partially inhibited by Pi; at higher [Pi], calcium pyrophosphate, calcium phosphate and calcium pyrophosphate-phosphate complexes amorphous to x-ray diffraction are formed; whereas at still higher [Pi], HA crystal formation partially inhibited by PPi. We conclude that CPPD forms when the ratio [Pi]/[PPi] less than 3 and HA forms when [Pi]/[PPi] greater than 100.     

Somatostatin inhibits insulin-stimulated amino acid uptake into cultured rat myoblasts

The effects of somatostatin on the acute metabolic actions of insulin on newborn rat myoblasts in culture has been examined during monolayer culture. Somatostatin significantly inhibited the insulin-stimulated uptake of [3H]leucine and [3H]amino-isobutyric acid into myoblasts but had no effect on basal (unstimulated) uptake of these two substances. The lowest concentration of somatostatin to have a significant effect was 10 micrograms/l, and this was apparent in all the experiments undertaken. The inhibitory effect of somatostatin was seen at all effective concentrations of insulin used (0.3-1 U/l). These findings lend support to the concept of an endocrine role for somatostatin in vivo and suggest that a peripheral antagonism may exist between circulating insulin and somatostatin on anabolic processes such as nutrient uptake into cells.     

Inhibition of an equilibrative nucleoside transporter by cannabidiol: a mechanism of cannabinoid immunosuppression

The plant-derived cannabinoids delta9-tetrahydrocannabinol (THC) and cannabidiol (CBD) both have immunosuppressive effects; although some effects of THC are mediated by the CB2 receptor, CB2 binds CBD weakly. In examining the effects of THC and CBD on microglial proliferation, we found that these compounds potently inhibit [3H]thymidine incorporation into a murine microglial cell line with no effect on cell cycle. Treatment with THC and CBD decreased [3H]thymidine uptake into microglia, with IC50 values that match inhibition of [3H]thymidine incorporation into DNA. CBD and, less potently, THC decreased uptake of [3H]adenosine to a similar extent as [3H]thymidine in both murine microglia and RAW264.7 macrophages. Binding studies confirm that CBD binds to the equilibrative nucleoside transporter 1 with a Ki < 250 nM. Because adenosine agonists have antiinflammatory effects, and because uptake of adenosine is a primary mechanism of terminating adenosine signaling, we tested the hypothesis that CBD is immunosuppressive because it enhances endogenous adenosine signaling. In vivo treatment with a low dose of CBD decreases TNFalpha production in lipopolysaccharide-treated mice; this effect is reversed with an A2A adenosine receptor antagonist and abolished in A2A receptor knockout mice. These studies demonstrate that CBD has the ability to enhance adenosine signaling through inhibition of uptake and provide a non-cannabinoid receptor mechanism by which CBD can decrease inflammation.     

Measurement of Protein Synthetic Activity by Determination of Peptidyl[3H]puromycin Formation in Liver Slices after Ethanol Administration

We investigated the utility of [3H]puromycin as an alternate and adjunct precursor to amino acids for measuring protein synthetic activity in rat liver slices. Slices were incubated in the presence of either [3H]puromycin or radiolabeled valine to compare the incorporation of these isotopic precursors into nascent hepatocellular proteins. Compared to liver slices from controls, comparable decreases in the incorporation of both [3H]puromycin and labeled valine were observed in experiments using slices from fasted rats and in slices preincubated with 25 mM ethanol. Radiolabeling of nascent polypeptides with either [3H]puromycin or labeled valine in liver slices from rats fed a liquid diet containing ethanol was also decreased compared to slices from pair-fed control and chow-fed animals. Our results demonstrated the validity of using [3H]puromycin to detect changes in protein synthetic activity under these conditions. The potential advantage of using [3H]puromycin for in vivo studies is discussed.