MORPHINE INFLUENCE ON ADENYLATE CYCLASE ACTIVITY IN PERIPHERAL BLOOD LYMPHOCYTES OF ALCOHOLIC PATIENTS N. B. GAMALEYA,* N. L. VEKSHINA, T. P. NEBARAKOVA, S. I. TRONNIKOV and I. P. ANOKHINA All-Union Scientific Research Centre for Medical and Biological Problems of Narcology, Maly Mogilcevskii per. 3, 121921 Moscow, U.S.S.R. (Received 1 February 1991; accepted for publication \SJuly 1991) Abstract — The effect of morphine on adenylate cyclase activity in lymphocytes was tested in 20 normal controls, 16 alcoholics in withdrawal and 9 sober alcoholics. Alcoholics in withdrawal were characterized by a significantly increased stimulatory effect of morphine, whereas sober alcoholics showed an inhibitory effect. The morphine effect was abolished by naloxone and correlated with the severity of withdrawal and alcohol intoxication.
INTRODUCTION There is evidence for the existence of a heterogeneous population of binding sites for opioid ligands on immune cells (Ovadia et al., 1989). The data concerning the existence of such sites on circulating human T-lymphocytes were presented by Mehrishi and Mills (1983) and Madden et al. (1987), who described the sites of specific binding for [3H] naloxone, resembling opioid receptors of mu and delta types. It is also known that one of the mechanisms of opioid action on cell metabolism, including the immune cell, is the regulation of the activity of cyclase enzymes in the plasmatic membrane (Kost et al., 1983). This is why changes in the character of opioid modulation of the activity of these systems could serve as indirect evidence of changes in the appropriate binding sites. In previous work, opioid effects on cAMP levels were evaluated in lymphocytes from small groups of healthy people. There are no data in the literature on opiate-dependent adenylate cyclase (AC) in diseases in which
opiate system disturbances play a certain pathogenetic role, e.g. alcoholism (Hoffman et al., 1982; Anokhina etal., 1989a). The purpose of the present study was to test the functional activity of opiate-dependent adenylate cyclase in peripheral blood lymphocytes of alcoholics compared with normal controls by using morphine, naloxone and their combination.
'Author to whom correspondence should be addressed. 515
METHODS Lymphocytes were isolated from 15-20 ml of heparinized blood using Ficoll-Verograf in gradients. Cell suspensions contained usually not less than 5 X 106 lymphocytes. The incubation mixture contained 50 mM TrisHC1 buffer (pH 7.4), 10 mM KC1, 20 mM MgCl2, 6 mM theophylline, 1 mM ATP in a final volume of 0.5 ml (Anokina et al., 19896). The reaction started with the addition of the lymphocytes (0.5-1.0 million cells) and was stopped after 30 min of incubation at 37°C by addition of 1 ml of 96% ethanol followed by centrifugation at 16,000 g for 10 min. The cAMP containing supernatant was dried under vacuum and stored at —20°C until determination. The amounts of cAMP were determined with a commercial 'Cyclic AMP assay kit'
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Alcohol & Alcoholism, Vol 26, No 5/6, pp. 515-518.1991 Printed in Great Britain
N. B. GAMALEYA el al.
516
cal craving for alcohol. Neuroleptics were the drugs of choice. RESULTS
Basal and morphine-modulated activities of AC in lymphocytes of the groups studied are shown in Table 1. Morphine did not alter cyclase activity of control subjects significantly, but increased it by 50% in withdrawn alcoholics on admission (P < 0.01) and by 123% 1 week later (P < 0.001). The 49% increase at 3-5 weeks after admission was not significant. Morphine, on the other hand, decreased AC activity by 51% in sober alcoholics on admission (P < 0.001) and by 39% one week later (P < 0.02). Table 2 shows the effects of morphine, naloxone and their combination on the AC activity of lymphocytes from withdrawn alcoholic patients, taken on admission, 1 week later or 3-5 weeks later. The presented data show the stimulatory effect of morphine and its prevention by naloxone, thus suggesting the specificity of the phenomenon and its possible connection to opiate binding sites.
Table 1. Basal and morphine-modulated adenylate cyclase activity in lymphocytes (mean ± S.E.M.) Groups studied
1. Normal controls (/; = 20) 2. Alcoholics in withdrawal: a) on admission (n = 16) b) 1 week later (n = 16) c) 3-5 weeks later (n = 11) 3. Sober alcoholics a) on admission (/i = 9)
b) 1 week later (;; = 7)
Adenylate cyclase activity (pmol/min/10'' lymphocytes)
Comparison to basal activity (paired test)
Basal
Morphinemodulated
0.41 ± 0 1 1
0.40 ± 0.1
NS
0.563 ± 0.104
INTRODUCTION There is evidence for the existence of a heterogeneous population of binding sites for opioid ligands on immune cells (Ovadia et al., 1989). The data concerning the existence of such sites on circulating human T-lymphocytes were presented by Mehrishi and Mills (1983) and Madden et al. (1987), who described the sites of specific binding for [3H] naloxone, resembling opioid receptors of mu and delta types. It is also known that one of the mechanisms of opioid action on cell metabolism, including the immune cell, is the regulation of the activity of cyclase enzymes in the plasmatic membrane (Kost et al., 1983). This is why changes in the character of opioid modulation of the activity of these systems could serve as indirect evidence of changes in the appropriate binding sites. In previous work, opioid effects on cAMP levels were evaluated in lymphocytes from small groups of healthy people. There are no data in the literature on opiate-dependent adenylate cyclase (AC) in diseases in which
opiate system disturbances play a certain pathogenetic role, e.g. alcoholism (Hoffman et al., 1982; Anokhina etal., 1989a). The purpose of the present study was to test the functional activity of opiate-dependent adenylate cyclase in peripheral blood lymphocytes of alcoholics compared with normal controls by using morphine, naloxone and their combination.
'Author to whom correspondence should be addressed. 515
METHODS Lymphocytes were isolated from 15-20 ml of heparinized blood using Ficoll-Verograf in gradients. Cell suspensions contained usually not less than 5 X 106 lymphocytes. The incubation mixture contained 50 mM TrisHC1 buffer (pH 7.4), 10 mM KC1, 20 mM MgCl2, 6 mM theophylline, 1 mM ATP in a final volume of 0.5 ml (Anokina et al., 19896). The reaction started with the addition of the lymphocytes (0.5-1.0 million cells) and was stopped after 30 min of incubation at 37°C by addition of 1 ml of 96% ethanol followed by centrifugation at 16,000 g for 10 min. The cAMP containing supernatant was dried under vacuum and stored at —20°C until determination. The amounts of cAMP were determined with a commercial 'Cyclic AMP assay kit'
Downloaded from https://academic.oup.com/alcalc/article-abstract/26/5-6/515/138350 by Midwestern University - Downers Grove Campus user on 15 January 2019
0735-0414/91 $3.00+ 0 00 Pergamon Press pic © 1991 Medical Council on Alcoholism
Alcohol & Alcoholism, Vol 26, No 5/6, pp. 515-518.1991 Printed in Great Britain
N. B. GAMALEYA el al.
516
cal craving for alcohol. Neuroleptics were the drugs of choice. RESULTS
Basal and morphine-modulated activities of AC in lymphocytes of the groups studied are shown in Table 1. Morphine did not alter cyclase activity of control subjects significantly, but increased it by 50% in withdrawn alcoholics on admission (P < 0.01) and by 123% 1 week later (P < 0.001). The 49% increase at 3-5 weeks after admission was not significant. Morphine, on the other hand, decreased AC activity by 51% in sober alcoholics on admission (P < 0.001) and by 39% one week later (P < 0.02). Table 2 shows the effects of morphine, naloxone and their combination on the AC activity of lymphocytes from withdrawn alcoholic patients, taken on admission, 1 week later or 3-5 weeks later. The presented data show the stimulatory effect of morphine and its prevention by naloxone, thus suggesting the specificity of the phenomenon and its possible connection to opiate binding sites.
Table 1. Basal and morphine-modulated adenylate cyclase activity in lymphocytes (mean ± S.E.M.) Groups studied
1. Normal controls (/; = 20) 2. Alcoholics in withdrawal: a) on admission (n = 16) b) 1 week later (n = 16) c) 3-5 weeks later (n = 11) 3. Sober alcoholics a) on admission (/i = 9)
b) 1 week later (;; = 7)
Adenylate cyclase activity (pmol/min/10'' lymphocytes)
Comparison to basal activity (paired test)
Basal
Morphinemodulated
0.41 ± 0 1 1
0.40 ± 0.1
NS
0.563 ± 0.104
