NIH RELAIS Document Delivery NIK -- W1 AL309R PAOLO DEPETRILLO NIAAA, NIH-10083731 10 CENTER DR. MSC 1256/NIH BLDG. 10 ROOM 3C103 BETHESDA, MD 20892-1256 ATIN: PHONE: 301-496-9420 FAX 301-402-0445 E-MAIL: SUBMITTED: 2001-10-26 15:46:39 PRINTED: 2001-10-29 08:50:42 REQUEST NO.: — NIH-10053731 SENT VIA: LoaN Doc 4807591 NIH TITLE PUBLISHER/PLACE: VOLUME /ISSUE/PAGES : DATE: AUTHOR OF ARTICLE TITLE OF ARTICLE: ISSN: OTHER NOS/LETTERS: SOURCE: CALL NUMBER: REQUESTER INFO: DELIVERY: REPLY: Fiche to Paper Journal ALCOHOLISM, CLINICAL AND EXPERIMENTAL RESEARCH Williams And Wilkins, Baltimore, MD 1992 Apr;16(2):290-4 290-4 1992 DePetrillo PB; Swift RM: Ethanol exposure results in a transient decrease i 0145-6008 Library reports holding volume or year 707242 1317134 PubMed WL AL309R BW986 E-mail: pbdp@helix.nih.gov Mail: NOTICE: THIS MATERIAL MAY BE PROTECTED BY COPYRIGHT LaW (TITLE 17. U.S. CODE) ‘Mational-Institutes-of -Health,-Bethesda ,-MD-0145.5008/92/1602 029083.00/0 ‘ALCOMOUSH: CUNICAL AND ENPERMANTAL RESEARCH Ethanol Exposure Results in a Transient Decrease in Human Platelet cAMP Levels: Evidence for a Protein Kinase C Mediated Process P.B. DePetrito and R.M. Swat ‘At concentrations betweon 2 and 82 mw, ethanol is shown to dapross hhuman platolet cAMP levels. Tho effect ie biphasic, maximal at 30 ‘ec, with platelet concentrations of cAMP rotuming to baseline values at higher ethanol concentrations and at longer incubation times. The CAMP loworing effect of ethenol can be blocked by @ Phosphodiesterase (PPDE) inhibitor, S-leobuty!-1-methylxanthine (BMX), at concentration of 2 ms, suggesting that an increase in PPDE activity may be responsible for this ettoct. Exposure of plate- lets to 1(S-isoquinctinyiscifony)-2-methyipiporazine (M7), a protein kinase C (PKC) inhibitor, blocks the ethanol-induced decrease in platolt eAMP, suggesting ethanol may be acting through activation or PKC. Key Words: Alcohol, Eth, Blood Platelets, Protein Kinase C, H7 'HE EFFECT OF ethanol on intracellular cyclic AMP (cAMP) is complex, reflecting the diversity of mem- brane protein interactions that are involved in the regu- lation of this second messenger. Ethanol may interact with several components of the G-protein-adenylate cyclase complex to affect adenylate cyclase (AC) activity Ethanol has three classes of effects on the cAMP system of cells. First, ethanol appears to have a stimulatory effect on AC activity in most tissue systems that have been studied, including rat myocardium,’ mouse brain,” smooth muscle cells,” and Iymphocytes.* Ethanol facili tates adenosine-stimulated cAMP accumulation in cul- tured hybrid neuroblastoma-glioma cells,’ and beta-ago- nist stimulated AC in $49 lymphoma cells.* In contrast, ethanol is reported to exert an inhibitory effect on AC activity in platelet-derived membranes at ethanol concen- trations of $70 and 860 mu Second, chronic exposure to ethanol results in reduced AC activity with increased AC activity observed upon withdrawal.’ Third, several studies suggest that altered intracellular cAMP turnover is associated with alcoholism in humans. Lymphocytes isolated from abstinent alcohol- ics have reduced basal and adenosine stimulated cAMP ‘From the Division of Clinical Pharmacology and the Department of Medicine (P.B.D): and the Depanmenss of Psychiairy and Human Behavior and Psychiatry, Brown University, Roger Willams General Hospital, Providence, Rhode Island ‘Received for publication October 18, 190; accepted October 18, 1991 This research was supported in part by a grant frm the Pharmaceu tical Manufacturers Association Foundation. ‘Reprint requests: Department of Medicine, Roger Willams General Hospital, 825 Chatkstone Ave, Providence, RI 02008, Copyright © 1992 by The Research Society on Alcoholic. 290 accumulation as compared with normal subjects.* Leu- kocytes isolated from human alcoholics and grown in culture for several generations show decreases in cAMP levels” A persistent reduction in AC activity after stimu- lation with guanine nucleotide, cesium floride, or PGE, ‘was found in platelets isolated from abstinent alcoholics. Cyclic AMP plays a key role in modulating extracellular signal transductions in platelets. Patelet activation is pro- moted by decreased cAMP levels and is antagonized by increased cAMP levels." In the present study we report that at physiologically refevant concentrations, ethanol causes a transient depression in platelet cAMP levels, METHODS ‘Apyrase, bovine serum albumin, creatine phosphokinase (Type D, forskolin, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 3:isobuty/-1-methylsanthine (BMX), phorbol 12-myrstate ‘acetate diester (PMA) phosphocreatine, -).isoquinolinlsulfony])-2 imethylpiperazne (HT), sodium-cAMP, and were obtained from Sigma ‘Chemical Co, St Louis, MO, The 5,7 dimethy! 1.24 triazolo (1 S.ha) pyrimidine (DMTP) and chloraacetaldehyde were purchased from Ald- rich Chemical Co, Milvaukee, WI, Hank's Balanced Salt Solution’ (HBSS) and calcium deficient Eagle's medium” were prepared and ‘modified to include HEPES at $ my. MgCl: was substituted for MeSOx and titrated to pH 7.42. Platelets were supplied by the Rhode Island Blood Cente Providence, RI. These platelets were sored in acid-itrate- dextrose (ACD)" at room temperature, and were used prior to the expiration date. Fach bag of plateetrich plasma contained a total of | {05 x 10" platelets suspended in approximately 30 ml of ACD. Apyrase was added at this step to achieve a concentration of 20 wg/ee and the platelet suspension was spun on a table top centrifuge at 200 X g to Drecpitate erythrocytes. The platele-rich supernatant was + un at 800, x g for 19 min at room temperature and the platele-rich let resus- pended in 48 ml of Eagles meciur, This platelet suspension was spun 2 800 X g for 10 min at room temperature and the plateletrch pellet as again resuspended in an amount of modified Eagle's medium ‘calculated to result ina final platelet concentration of approximately § x 10" platelets/m. An aliquot of patelet suspension was counted using a hemocytometer. Clumping of platelets was less than 1%% of total counted, and erythrocyte contamination was on average less than 2 cells per high power field of radius {5 mm and depth of O.{ mm st & magnification x400. ‘This suspension was stored at room temperature and used for al subsequent experiments within 60 min of preparation, Prior to use, the suspension was incubated for 1S min at 37°C. ‘Standard curves of cAMP in platelet suspension were determined for cach asay. A 100s aliquot of cell suspension containing approximately 5 x 10" platelets was added to each of 15 1 Sl polyethylene mcrocen tcfuge tubes and immersed in boing water for 2 min. To cach set of thee tubes in the secs, Known amounts of @AMP in HBSS were added ‘corresponding to 0, 8,16, 32, and 40 pa. Alcohol Cn Exp Res, Vok 16, No 2, 192: pp 290-238 e Pccccccrediermpertermmrrrre1 4 4 + [ETHANOL AND PLATELET cAMP For platclet cAMP analysis, all incubations were initiated by the addition of 100 yl of platelet suspension containing 5 x 10" platelets to 100 ul of modified HBSS containing the appropriate reagents for the «experiment, as well as creatine phosphokinase, 100 units/e, 0.1% bovine serum albumin, and 5 mv phosphocreatine. The calcium concentration of the HBSS was adjusted so a5 to result ina final concentration of Ca fof 3.5 ma afer the addition of platelet suspension, Incubations were performed ina waterbath at 37°C forthe appropriate times, and reactions ‘terminated by immersing the tubes in boiling water for 2 min, ‘The final volume was adjusted to 0.2 ml with HSS. Subsequently, 50 al of DMTP internal standard (2x 10"* u) was added to each tube, Samples were deproteinized and noncycic nucleotides sorbed by add ing 75 ul each of Ba(OH); (0.25 m) and Zn80, (0.25 s) and the sample ‘was spun for 4 min at 14,000 x g on a table top ultracentrifuge, The sample was then derivatized by reacting with chloroacetaldehyde for 15, min at 100°C in a waterbath. A 10- aliquot ofthe final solution was injected into the column, Cyclic AMP was quantitated by high-performance liguid chromatog: raphy using fuorescent detection of etheno-cAMP.! A linear regresion ofthe peak height ratio of cAMP to DMTP on the amount of added cAMP was caleulated, using the derived parameters to calculate the ‘cAMP content of unknown samples. R-squared forthe linear repression ‘was alvays 20.98. All points for each experiment are the means of triplicate determinations, and the results ate expressed in terms of the ‘mean sen, Data was tested for normality and found tobe suitable for ANOVA. Statistical analysis of the data was performed by ANOVA using the derived parameters 10 compare points of interest on the curves. Fisher's least significant difference (sd) test was used to determine the significance level forthe comparisons, RESULTS. The basal level of platelet cAMP was found to be 50.7 + 5.4 (SEM) ps/10? cells for n = 14 different determina- tions. This compares favorably with human platelet cAMP determinations obtained by other workers."* In order to assess the viability of platelets and whether cAMP gener- ation would be limited by the availability of substrate, AC was maximally stimulated by addition of forskolin and 2 my IBMX to the incubation medium. Forskolin produces an increase in platelet cAMP that continues up to 20 min as shown in Fig. | Incubation of platelets with ethanol at concentrations up to 128 mM causes a significant decrease in platelet cAMP concentration at 30 se. The decrease is statistically significant at ethanol concentrations of 8 and 32 mM as ‘ofan ‘teste 13 ‘era 00 404 See aslo Seam vou Teg rin) Fig. 1. lncbaton of pales win forskolin a deect etator of AC, cnuaes niproasa lal: CAN concentration. Each po represent th mean © eh oln= Sexpertent (Material may be protected by copyright law (Title 17, U,$. Coded 2 can be seen in Fig 2. At higher concentrations of ethanol (512 my) no significant difference between ethanol and control are observed but a large increase in variance is seen, The decrease in platelet cAMP is shor lived as levels, return to baseline at 1 min, Incubation for time periods up to 20 min does not result in significant changes in platelet cAMP levels. ‘The cAMP lowering effect of ethanol can be blocked by IBMX, a PPDE inhibitor, at concentrations of 2 mM in the incubation medium, as shown in Fig. 3. Platelet cAMP levels are significantly decreased after a 30-sec incubation with 8 and 32 mM ethanol. The addition of ethanol to the ‘medium at concentrations up to 128 mx results in no statistically significant difference in platelet cAMP from baseline in the presence of 2 mat IBMX. In order to test the hypothesis that ethanol may exert its effects on platelet content of cAMP by interacting wiih PKC, the effects of H7, an inhibitor of PKC," were studied in this experiment. Fig. 3 illustrates that platelets incu- bated for IS min with H7 100 at and then exposed to cethano! for 30 sec did not respond with decreases in eAMP levels. Exposure of platelets to H7 alone appears to increase platclet cAMP levels in a dose dependent fashion as shown in Fig. 4 Stimulation of PKC with the phorbol ester PMA results, in dose dependent decreases in platelet suspensions with PMA at concentrations up to 10~” preincubation of the platelet cAMP as shown in Fig. 5. Exposure of platelets to varying concentrations of ethanol for 30 see at concentra- tions up to 160 ma did not cause further decreases in platelet cAMP. DISCUSSION ‘There have been conflicting reports of the effects of ethanol on human platelet cAMP levels. In one study, Effect of ethanot on platetet cAMP 2s, 20 is © 30 second incubation 10) 1 mfnute incubation percent enange | z 7 than nt Fig 2 Platoats wore hexoatad fr 20 seo of 1 mina vats conentratone cf thnol and cAMP lovee Geterined Each pot represents ean eo ‘n= 3 experiments, Satscalostng was prtered Using @ ctr ANOVA p ‘<0085 sgntzanty arent compured wth O ranean’ and tm at B me ‘shana. "p< 001 seat erent compared wh O ms ethan an min 132 methanol. Tho elt ofthe two baton es on pat eels of cAMP ‘ae sigicanty erent at p< 0.0001 En Tie