calpain and Substance-Withdrawal-Syndrome

Chronic alcohol consumption causes multifaceted damage to the central nervous system (CNS), underlying mechanisms of which are gradually being unraveled. In our previous studies, activation of calpain, a calcium-activated neutral protease has been found to cause detrimental alterations in spinal motor neurons following ethanol (EtOH) exposure in vitro. However, it is not known whether calpain plays a pivotal role in chronic EtOH exposure-induced structural damage to CNS in vivo. To test the possible involvement of calpain in EtOH-associated neurodegenerative mechanisms the present investigation was conducted in a well-established mouse model of alcohol dependence - chronic intermittent EtOH (CIE) exposure and withdrawal. Our studies indicated significant loss of axonal proteins (neurofilament light and heavy, 50-60%), myelin proteins (myelin basic protein, 20-40% proteolipid protein, 25%) and enzyme (2', 3'-cyclic-nucleotide 3'-phosphodiesterase, 21-55%) following CIE in multiple regions of brain including hippocampus, corpus callosum, cerebellum, and importantly in spinal cord. These CIE-induced deleterious effects escalated after withdrawal in each CNS region tested. Increased expression and activity of calpain along with enhanced ratio of active calpain to calpastatin (sole endogenous inhibitor) was observed after withdrawal compared to EtOH exposure. Pharmacological inhibition of calpain with calpeptin (25 μg/kg) prior to each EtOH vapor inhalation significantly attenuated damage to axons and myelin as demonstrated by immuno-profiles of axonal and myelin proteins, and Luxol Fast Blue staining. Calpain inhibition significantly protected the ultrastructural integrity of axons and myelin compared to control as confirmed by electron microscopy. Together, these findings confirm CIE exposure and withdrawal induced structural alterations in axons and myelin, predominantly after withdrawal and corroborate calpain inhibition as a potential protective strategy against EtOH associated CNS degeneration.

Topics: Administration, Inhalation; Alcoholism; Animals; Axons; Brain; Calpain; Central Nervous System Depressants; Dipeptides; Disease Models, Animal; Ethanol; Glycoproteins; Male; Mice, Inbred C57BL; Myelin Sheath; Nerve Degeneration; Neuroprotective Agents; Spinal Cord; Substance Withdrawal Syndrome

Heat shock protein (Hsp27) renders cardioprotection from stress situations but little is known about its role in myofilaments. In this study we have evaluated the relationship between Hsp27 and troponin response after naloxone-induced morphine withdrawal. Rats were treated with two morphine (75 mg) pellets during six days. Precipitated withdrawal was induced by naloxone on day seven. Hsp27 expression, Hsp27 phosphorylated at serine 82 (Ser82), cardiac troponin T (cTnT), cardiac troponin I (cTnI) and µ-calpain were evaluated by immunoblotting in left ventricle. Hsp, cTnT and cTnI was also evaluated by immunofluorescence procedure. Our results show that enhancement in Hsp27 expression and phosphorylation induced by naloxone-precipitated morphine withdrawal occurs with concomitant increases of cTnT and µ-calpain expression, whereas cTnI was decreased. We also observed co-localization of Hsp27 with cTnT in cardiac tissues. These findings provide new information into the possible role of Hsp27 in the protection of cTnT degradation by µ-calpain (a protease mediating proteolysis of cTnT and cTnI) after morphine withdrawal.

Topics: Analgesics, Opioid; Animals; Arterial Pressure; Calpain; Heart Rate; Heart Ventricles; HSP27 Heat-Shock Proteins; Male; Morphine; Morphine Dependence; Naloxone; Narcotic Antagonists; Phosphorylation; Rats, Sprague-Dawley; Substance Withdrawal Syndrome; Troponin I; Troponin T

The present study was designed to determine the effect of N'-[6,7-dichloro-4-(4-methoxy-phenyl)-3-oxo-3,4-dihydroquinoxalin-2-yl] hydrazide (SJA 7019), a selective nonpeptide inhibitor of calpain, and sodium orthovanadate, a selective inhibitor of tyrosine phosphatase, on the development of physiological dependence, as assessed by precipitated morphine withdrawal behavior in mice. Subchronic morphine administration (5 mg/kg, intraperitoneally, twice daily for 5 days), followed by a single injection of naloxone was used to precipitate the opioid withdrawal syndrome in mice. Behavioral observations were made immediately after naloxone treatment. Withdrawal syndrome was assessed quantitatively in terms of the withdrawal severity score and the frequency of jumping, rearing, forepaw licking, and circling. Daily single administration of SJA 7019 (1.5, 3, and 6 mg/kg, intraperitoneally) or sodium orthovanadate (5, 10, and 20 mg/kg, intraperitoneally) was continued during the morphine treatment procedure. Administration of SJA 7019 as well as the sodium orthovanadate dose-dependently attenuated the naloxone-induced morphine withdrawal syndrome. Neither SJA 7019 nor sodium orthovanadate significantly affected locomotor activity or morphine-induced antinociception. Therefore, it may be concluded that treatment with SJA 7019 or sodium orthovanadate during the morphine exposure period attenuated the development of physiological dependence on morphine, possibly through mechanisms linked to activation of tyrosine phosphatase and calpain.

Topics: Analgesics, Opioid; Animals; Calpain; Female; Male; Mice; Morphine; Morphine Dependence; Motor Activity; Naloxone; Protein Tyrosine Phosphatases; Stereotyped Behavior; Substance Withdrawal Syndrome; Vanadates