Effect of S(−)- and R(+)-salsolinol on the POMC gene expression and ACTH release of an anterior pituitary cell line

Alcohol,Vol. 12, No. 5, 447--452,1995 Copyright©1995ElsevierScienceLtd Printedin the USA.All fightsreserved 0741-8329/95$9.50 + .00

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Effect of S ( - )- and R(+)-Salsolinol on the POMC Gene Expression and ACTH Release of an Anterior Pituitary Cell Line INGO PUTSCHER, HANKA HABER, ANETT WINKLER, J t ) R N S F I C K E L A N D M A T T H I A S F. M E L Z I G l

Research Institute f o r Molecular Pharmacology, Department o f Cellular & Biochemical Pharmacology, Alfred-Kowalke-Str. 4, 10315 Berlin, Germany Received 29 N o v e m b e r 1994; Accepted 16 M a r c h 1995 PUTSCHER, I., H. HABER, A. WINKLER, J. FICKEL AND M. F. MELZIG. Effect of S(-)-andR(+)-salsolinol on the POMC gene expression and ACTH release of an anterior pituitary cell line. ALCOHOL 12(5) 447-452, 1995.-Tc~rahydroisoquinolines(TIQs) are thought to play an important role in the process of development of alcohol dependence. Being a condensation product between the alcohol metabolite acetaldehyde and dopamine they might be involved in the balance of the opioid system as well as the reward system. Therefore, the influence of the TIQ salsolinol (SAL) on the pro-opiomelanocortin (POMC) gene expression was investigated using the ART-20mouse anterior pituitary tumor cell line. Our results show a significant decrease in the POMC gene expression by the S( - )-enantiumer of SAL. The basal secretion of adrenocorticotropin (ACTH) as well as the corticotropin-releasing factor (CRF)-stimulated ACTH release remained unchanged after R(+)- and S ( - )-SAL treatment. Interestingly, it was clearly shown that a reduction of intraceUularcAMP level occurred after the treatment of the cells with S(-)-SAL whereas R(+)-SAL did not affect the cAMP production. The obtained results suggest that S( - )-SAL is possibly involved in the establishment of the opioid deficiencyin alcoholics. Salsolinol

Tetrahydroisoquinolines

Pro-opiomelanocortin

POMC

AtT-20 cells

ACTH secretion

effect of SAL on the dopamine O-methylation by catechol-Omethyltransferase (COMT) (4). These findings suggest that TIQs, especially SAL and THP, are related to the etiology of alcoholism. It has been reported that chronic ethanol treatment leads to a decrease in the 8-endorphin (~-END) release in the intermediate lobe of the rat pituitary (19). Furthermore, the ~END levels measured in the CSF of alcohol addicts were significantly lower than those of controls whereas the adrenocorticotropin (ACTH) levels were five times higher than those of the control values (9). Investigations in rats showed a decrease in Pr0-opiomelanocortin (POMC) mRNA in the anterior as well as in the neurointermediate lobe after chronic ethanol exposure (3,24). However, it has not been tested if SAL can alter the POMC gene expression and the ACTH secretion of corticotrope cells. For this reason we investigated the influence of SAL on both the POMC mRNA content and the ACTH release in AtT-20 cells.

THE PROPERTIES of tel~rahydroisoquinofines (TIQs) have been discussed for several years, especially in regard to Parkinson's disease and their possible role in the development of alcoholism. Both salsolinol (SAL) and tetrahydropapaveroline (THP) induce voluntary alcohol drinking after chronic as well as after acute intracerebroventricular application in rats. This effect can be inhibited by the opiate antagonists naloxone and naltrexone (16), therefore showing possible connections with the opioid system. Furthermore, SAL was detected in brain, cerebrospinal fluid (CSF), and urine both in healthy and alcoholic subjects. Significantly higher values of the alkaloid were found in the urine and CSF of alcoholic patients compared to controls (5). Addition~[y, investigations of blood samples showed an increased level of SAL in the plasma of chronic alcoholics compared to nonalcoholics (7). A modulating influence of SAL on the dopamine metabolism is assumed due to the inhibition of human brain type A monoamine oxidase by SAL enantiomers (15) as well as the competitive inhibitory

' To whom requests for reprints should be addressed. 447

448

PUTSCHER ET AL.

Because of the existence of two enantiomers of SAL we used R ( + ) - S A L and S ( - )-SAL in our experiments to determine possible differences of their actions. METHOD

Cell Culture The ART-20 (clone D16v) mouse anterior pituitary tumor cell line was routinely grown in Dulbecco's modified Eagle medium (DMEM, Sigma, Germany) supplemented with 10% fetal calf serum (Gibco, UK) in a humidified atmosphere of 5O7o CO 2 at 37°C, The AtT-20 cells were adapted to a serumreduced medium (further AtT-20/DI cells) composed of DMEM/F12 (Nutrient mixture F-12 Ham's, Sigma, Germany) with the addition of 1 mg/ml of bovine albumin (low endotoxin, fatty acid free, Serva, Germany), 260/zg/ml linoleic acid albumin from bovine serum (Sigma, Germany), serumfree medium (SFM) supplement (Boehringer Mannbeim, Germany), and 0.2o70 fetal calf serum (8,13). S ( - ) - and R ( + ) SAL were synthesized in our laboratory as previously described (11), human corticotropin-releasing factor (CRF) was provided by Sigma, and all other chemicals were of analytical grade. Cell counting was performed microscopically in Fuchs-Rosenthal chambers after addition of Trypan blue for recognition of dead cells or by analysis with the Cell Analyzer System CASY1 (Schiixfe, Germany).

Cytotoxicity of TIQs The cytotoxicity of SAL was estimated by counting all cells after the addition of various amounts of the SAL enantiomers. The substances were added 24 h after plating the cells and then the cells were further cultivated for 3 days. Six determinations in three independent experiments were carried out for each SAL concentration. Mean + SD was calculated from the obtained data (n = 18).

Effect of TIQs on the A C T H Release ACTH was determined by a two-step competitive ELISA as described by Fickel et al. (8) using A C T H rabbit antibody serum R02 (kind gift from Dr. Rohde, Berlin, Germany) in a 1 : 15,000 dilution and peroxidase-conjugated goat antibodies to rabbit IgG (Sigma, Germany) at a dilution of 1 : 2000. Orthophenylenediamine solution was added and the resulting colour was measured at a wave length of 490 nm using a microplate reader. The experiments were carried out with a short-term as well as a long-term incubation of the ceils with the SAL enantiomers. In short-term experiments the cells were cultivated as described above for 4 days before the substances were added. The incubation time with R- and S-SAL was 4 h. The longterm experiments were performed by a 72-h incubation time with the TIQs, which was started 24 h after seeding the cells. The TIQ concentrations used were 10, 1, and 0.1 /~M. In all experiments the basal and the CRF (0.1 /zM)-stimulated ACTH secretion was measured in comparison to untreated controls. Data were obtained from two independent experiments with six parallel probes. Each probe was measured twice. The data were calculated as mean + SD (n = 24).

Effect of TIQs on the POMC mRNA Levels The POMC mRNA level of the cells was estimated after a short-term incubation with R- and S-SAL at a concentration of 1/zM.

Total cellular RNA from the cultivated cell samples was extracted as described by Chomczynski and Sacchi (2) and determined photometrically. Defined amounts (1, 2, 3/~g and 10 /~g for the DotBlot and Northern Blot analyses, respectively) of total cellular RNA were blotted on Hybond N + membranes (Amersham, UK) using a DotBlot apparatus (BioRad, USA), and Pressure blot station (Stratagene, USA), respectively. The membranes were fixed 5 min with 0.05 N NaOH and washed three times in 2 x SSC (0.3 M NaC1, 0.03 M Na-citrate). The RNA was hybridized with a digoxigenin (DIG)-labeled antisence RNA obtained by in vitro transcription of a 150-bp fragment of POMC exon 3 (gift from Prof. V. H611t, Munich, Germany), cloned into the transcription vector pBS (Stratagene, USA). The labeling reaction, prehybridization, and hybridization were carried out accordingly to the standard procedures described in the manuals (DIG RNA Labeling Kit, DIG Nucleic Acid Detection Kit; Boehringer Mannheim, Germany). Following washing the DIG-labeled RNA was detected by an enzyme-catalyzed chemiluminescence reaction as described in the DIG Luminescent Detection Kit (Boehringer Mannheim, Germany). Then the filter was exposed to ORWO X-ray film (ORWO, Germany) for 30 rain at room temperature. The resulting autoradiograms were quantified using a video-based image analysis system (Molecular Dynamics, Germany). After removal of the hybridization signal accordingly to Winkler et al. (23), the membranes were rehybridized with a DIG-labeled oligo-dTo2_~8) probe to standardize the results. The labeling reaction and the hybridization were carried out following the manufacturer's instructions (DIG Oligo 3 'end Labeling Kit, DIG Nucleic Acid Detection Kit; Boehringer Mannheim, Germany). Chemiluminography and quantification were carried out as described above, The data were calculated as the ratio of values of the POMC signal to the signal of the standard probe oligo-dT(~2_~s).The results shown from the POMC measurement are the means + SD of four independent experiments, each one consisting of four replications. The significance of the differences between the means was evaluated by Student's t-test after analysis of the variance (p < 0.05).

Effect of TIQs on the Cellular cAMP Content cAMP accumulation was measured in intact cells that were plated at a density of 100,000 cells/well in a 24-w¢11 plate. The cells were cultivated for 4 days as described above. Following washing with DMEM (Sigma, Germany) supplemented with 25 mM HEPES, 0.1 o7o BSA (Serva, Germany), and 0.365 mg/ ml glutamine the cells were preincubated for 30 rain with 500 /~1 of the same medium, additionally containing 10/LM isobutylmethyl-xanthine. Then the TIQs (1/~M) were added to each well and cAMP was allowed to accumulate for 30 min at 37°C. The supernatants were removed and the reactions were stopped by the addition of 1 ml/well ice-cold 80% ethanol. The plates were stored for 2 h at - 20°C followed by scraping of the cells and rinsing each well with a further 1 ml of ice-cold 80% ethanol. Then the cells were centrifuged and the resulting supernatant was lyophilized and stored at - 2 0 ° C until assayed, cAMP levels were determined by the method of cAMP binding protein using a commercial available kit and according to the instructions of the manufacturer (Amersham, UK). The cAMP determination was carried out with two AtT-20 cell cultures. Six incubations with and without the SAL enantiomers were performed from each culture and then the cAMP content was estimated for each incubation in duplicate (n = 24).

SALSOLINOL A N D POMC GENE EXPRESSION

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to a reduction of intracellular cAMP whereas the R ( + ) enantiomer did not influence the formation o f this second messenger. In correlation with the CRF-stimulated A C T H secretion of our cell model a significant increase in cAMP induced by CRF was detectable (control = 27 pmol/mg protein; 0.1 #M CRH = 548 pmol/mg protein). However, it is noteworthy that the drastical increase in the second messenger level leads only to a twofold elevation o f the A C T H release. Changes o f the CRF-induced cAMP values were not observed by either S( - )- or R(+)-SAL (data not shown).

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FIG. 1. Inhibition of the proliferation of AtT-20 cells caused by different concentrations of S( - )- and R(+ )-SAL. Incubation of the cells with the TIQs was performed for 3 days followed by counting all cells. The following IC5o values were estimated: S(-)-SAL = 32 #M and R(+)-SAL = 86 #M. Each point of the curve is a result of six determinations in three independent experiments. F~SULTS TO exclude nonspecific effects in all further pharmacological experiments we tested the cytotoxicity of R ( + ) - S A L as well as S ( - ) - S A L . Figure 1 shows the different inhibitory effects o f the optical antipodes on the proliferation of AtT-20 cells. The following IC5o values were determined: R ( + )-SAL (ICs0 = 86/~mol/l) and S ( - ) - S A L (ICs0 -- 32 #tool/l). Inhibition of cell proliferation was only observed at concentrations higher than 4 #mol/l; therefore, most of the experiments were carried out with the final concentration of 1/zmol/1. In a second step the influence of SAL on the POMC gene expression was examined. Therefore, the cell cultures were treated with the drugs for 4 h. As shown in Fig. 2, the S ( - ) enantiomer caused a siguificant decrease in the POMC mRNA level whereas no changes were detectable after incubating the cells with R ( + ) - S A L . The result of a Northern blot analysis is illustrated in Fig. 3, which emphasizes the fact that S ( - ) - S A L inhibits the POMC gene expression. In further investigations we were interested in studying the influence of SAL on the basal A C T H secretion o f AtT-20 cells, because it might have been possible that the POMC gene expression inhibited by S( - )-SAL is followed by an inhibition of the A C T H secretion. The cells were incubated both for a short time (4 h) and a long time period (3 days) with the enantiomers of SAL. However, the expected S ( - ) - S A L induced decrease in the A C T H release could not be found. In contrast, it was found that neither S ( - )- nor R ( + ) - S A L produce any alteration of the basal A C T H secretion in short-term (Fig. 4) or long-term experiments (data not shown). For this reason we tested the effect o f SAL on the CRFstimulated A C T H release. In AtT-20/D1 cells treated with 0.1 #M CRF we found a twofold increase in the A C T H secretion but no influence o f the SAL-enantiomers on the CRF-induced elevation after 4 h or after 72 h (data not shown). A C T H secretion can be induced by stimulation of adenylate cyclase and therefore increases the intracellular cAMP level. To investigate a possible effect o f SAL on the adenylate cyclase, cAMP levels in untreated as well as CRF-stimulated AtT-20 cells were determined. Figure 5 shows the results o f the cAMP assays. Treatment of the cells with S ( - ) - S A L leads

Our results reveal different toxic properties of the enantiomers o f SAL (Fig. 1) shown by the different IC5o values measured for R ( + ) - and S ( - ) - S A L . The dose-response curves are nearly parallel, indicating a similar mechanism o f the cytotoxicity but different effectiveness o f the substances. Investigations done with isolated mitochondria showed an inhibitory influence of SAL on the mitochondrial respiration (21). Furthermore, SAL caused an alteration o f several cellular structures, especially the mitochondria in endothelial cells (14). Probably the inhibited cell proliferation caused by SAL in an enantioselective manner is a result of damaged mitochondria as well as decreased energy consumption. However, we do not know for certain the mechanism responsible for the inhibited cell proliferation of ART-20 cells. Figures 2 and 3 indicate that SAL enantioselectively and specifically affects the POMC gene expression in pituitary cells. The downregulation was selectively induced by the S ( - )enantiomer of SAL. Interestingly, it was described that ethanol exposure leads to a decrease of the POMC mRNA level in the anterior pituitary accompanied by an inhibition of the adenylate cyclase activity (3,12). Ethanol itself has neither an affinity nor selectivity for pre- or postsyaaptic receptor subtypes in neurons of the brain (16). Our results, illustrating the influence of SAL on the transcription of the POMC gene as well as the decrease in cAMP levels caused by S ( - ) - S A L , could indicate a possible link to the effects of ethanol described above. The second messenger cAMP is a regulatory element of the POMC gene expression. Its effect is mediated via the protein kinase A (PK A)-dependent cAMP response

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FIG. 2. Influence of the TIQs on the POMC gene expression of ART-20 cells. The cells were incubated with R(+)- and S ( - )-SAL at a concentration of 1 #M for 4 h. Values over the bars represent SD. *Significant effect of S( - )-SAL vs. the control (t7 < 0.05).

450

PUTSCHER ET AL.

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FIG. 3. Chemiluminogram of Northern blot hybridized with the DIG-labeled POMC exon 3 probe. Lane 1: molecular weight marker )~Bst/EII. Lane 2:10/~g total RNA of AtT-20 cells (control). Lane 3: 10 #g total RNA of ART-20cells treated with S( - )-SAL (1 gM) for 4 h. Lane 4: 10 gg total RNA of AtT-20 cells treated with R(+)-SAL (1/~M) for 4 h.

element binding protein (CREB) and subsequently binding to the transcription factor I I D (TF I I D ) . Thus, the decreased level of the POMC mRNA presumably is the result of the reduced cAMP formation. These results suggest that SAL, the condensation product of acetaldehyde (the first metabolite of ethanol) with dopamine, might be involved in the establishment of the opioid

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deficiency in alcoholics, which was reported by several authors (22) via inhibition of the POMC gene expression. Experimental evidence suggests that brain dopamine plays an important role in mediating the rewarding effect of ethanol (1,16). Dopaminergic compounds also have effects on the POMC mRNA levels in the rat pituitary. Administration of haloperidol, a D2 receptor antagonist, led to an increase in POMC mRNA, and the D2 receptor agonist bromocriptine caused a decrease in the POMC gene expression in the intermediate lobe (6). Regarding the chemical structure, biogene-

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concentration [Izmol/l] FIG. 4. Infiucnce of R(+)- and S(-)-SAL (I0, I, and 0.I # M ) on the basal secretion (without CRF) of A C T H . A C T H levelswcrc measured by E L I S A after incubation of the cellswith the TIQs for 4 h. The data represent the means S D (n = 24).

FIG. 5. Effect ef R(+)- and S(-)-SAL on the cAMP accumulation of AtT-20 cells without stimulation with CRF. The enantiomers were used at a concentration of 1 pM. The data represent the means + SD (n = 24). *Significanteffect of S(- )-SAL vs. the control (p < 0.05).

SALSOLINOL AND POIVIC GENE EXPRESSION

451

sis, and the inhibiting effect on the adenylate cyclase, we suppose that S ( - ) - S A L mediates its effect by a dopaminergic receptor, probably the D2 subtype. We expected a change of the ACTH secretion in correlation to the observed decrease in the POMC transcript, but no alteration of the ACTH level was detectable in our cell system by treatment with SAL. The contribution of newly synthesized and stored proteins has to be considered in regard to the basal secretion. It is possible that the secretory granules of corticotrope anterior pituitary cells contain enough ACTH to keep the basal level unchanged over the investigated period of time. Therefore, we examined the influence of SAL on the stimulated ACTH release. CRF, as the most potent activator of the POMC-derived peptide secretion, was used in these experiments. It is known that CRF is released in response to various stressors and regulates ACTH secretion and glucocorticoid production via the hypothalanlic-pituitary-adrenal (HPA) axis. Acute ethanol exposure increased the release of ACTH from the pituitary, supporting the hypothesis that acute ethanol treatment stimulates the HPA system (10). There is evidence that this effect is possibly mediated by hypothalamic CRF and so may reflect the stress response of the HPA axis to ethanol (17). The SALenantiomers did not significmltlyaffect both the CRF-stimulated ACTH secretion and the cAMP accumulation. Because the downregulation of the POMC gene expression measured in the shortterm experiments did not yield to a decrease in the ACTH secretion, further investigations axe necessary for the estimation of the cellular content of ACTH'.

Despite this, the effect of SAL on the /~-END secretion should be determined. Although both ACTH and/3-END are coded by the POMC gene in equal relation, the secretory granules of anterior pituitary cells contain more ACTH than/5END. This indicates the dependence of granule contents from secretory pathway, especially from activity pattern and occurrence of protein convertases (18), which are responsible for the processing of the POMC precursor protein (20). The discrepancy between the POMC gene expression downregulation and the relatively unaffected peptide (ACTH) release shows the regulatory independence of those two systems and needs to be investigated further. In summary, our results show specific and stereoselective effects of SAL on the AtT-20 mouse anterior pituitary tumor cells. The downregulation of the POMC gene expression as well as the decreased cAMP formation caused by S ( - ) - S A L support the concept of a deficiency of endogenous opioids linked to ethanol intake. Thus, it can be assumed that TIQs are probably involved in regulating mechanisms that lead to the establishment of addiction. Although neither S ( - ) - nor R(+)-SAL affect both the adrenocorticotropin release and the cAMP production stimulated by CRF, other pathways like those of the dopaminergic system could be influenced. ACKNOWLEDGEMENT This study was supported by the Bundesministerium fuer Forschung und Technologie (BMFT 07NBL07/6).

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