Dopaminergic system modulation of nociceptive response in long-term diabetic rats.

The present study examines the effects of dopaminergic system modulation on nociceptive response time in male diabetic rats. In this study, diabetes was induced by streptozotocin (STZ, 45 mg/kg) in adult male Sprague-Dawley rats. Insulin replacement therapy was initiated 6 weeks after the induction of diabetes for one-half of the diabetic group (1.5-2.5 IU/12 h/rat) and was continued throughout the duration of the study (up to 14 weeks). After 6 weeks of daily insulin replacement therapy, eight rats from each experimental group (STZ-diabetic, STZ-diabetic+insulin and nondiabetic control) were injected with either bromocriptine (BROM, 3 mg/kg/12 h), haloperidol (HALO, 1.5 mg/kg/12 h) or vehicle. Nociceptive response was measured by the hot plate (HP) latency test before the induction of diabetes (baseline), every 3 weeks for the first 12 weeks and then on days 5, 9 and 14 of treatment with dopaminergic agents. Animals were sacrificed 3 or 4 days after the last HP test and the brain, blood, spinal cord (SC), pituitary and adrenal glands (AD) were dissected for Met-enkephalin (ME) assay. The results show that nociceptive response of untreated diabetic animals increased gradually and significantly over the duration of this study. Administration of BROM and HALO significantly decreased and increased the nociceptive response, respectively, in all groups. However, the response of the diabetic group was more pronounced than that of the other two groups, especially for those treated with BROM. Daily insulin administration normalized nociceptive response to that of the nondiabetic controls. Diabetic animals receiving insulin replacement+BROM also showed normalized nociceptive response while the diabetic animals+HALO did not. Moreover, the administration of HALO and BROM resulted in an increase and decrease ME concentrations, respectively, in most tissues and brain regions examined. The effect of these dopaminergic agents on ME levels was greater in brain regions and tissues of the diabetic rats than in the diabetic groups receiving vehicle or in the nondiabetic control receiving these two agents. These data suggest that diabetes alters the sensitivity of the dopaminergic receptors and that altered response of the dopaminergic system could be indirectly involved in the modulation of nociception in diabetic rats possibly through the enhancement and/or deactivation of the endogenous Met-enkephalinergic system.

The analgesic effects of tryptophan and its metabolites in the rat.

Male Sprague-Dawley rats weighing 150-200 g were given doses of tryptophan methyl ester or its metabolites; kynurenine sulphate, kynurenic acid, xanthurenic acid, quinolinic acid, anthranilic acid methyl ester or picolinic acid methyl ester. Doses administered intraperitoneally were 50, 100, 200, 300, 400 and 600 mg kg-1. Pain sensitivity was assessed using the hotplate and tailflick methods at 30 min before and at 30-min interval after the injection of test compounds. The administrations of tryptophan, kynurenic acid, quinolinic acid, anthranilic acid, xanthurenic acid, picolinic acid, and kynurenine were associated with analgesia. Animals given 300 or 600 mg kg-1 of tryptophan exhibited a significant decrease (P<0.05; P<0.01, respectively) in pain sensitivity with the hotplate test. l-Kynurenic acid (300 mg kg-1) produced analgesia (P<0.01) 30 min after drug administration. Quinolinic and anthranilic acids both produced prolonged decrease in pain sensitivity (P<0.05) using the tailflick test. These results indicate that tryptophan and some of its metabolites possess analgesic properties.

The thymolytic effect of cocaine and monoaminergic drugs in the mouse.

Cocaine abuse is known to be associated with suppression of the immune system. In this experiment, animals treated with cocaine (CC) in their drinking water (200 mg/L) for 10 days resulted in a significant decrease in thymus weight in HSD-NDA male Swiss mice. A subsequent in-vitro experiment was designed to investigate the direct effect of cocaine and other dopaminergic agents on thymocyte proliferation. Thymus glands were aseptically removed from mice and thymocytes were isolated and then incubated in microtiter plates with various concentrations (10[-8]-10[-4] M) of CC, apomorphine (AM), haloperidol (HP), dopamine (DA) and epinephrine (EN) for 18 hrs. The results revealed that cocaine inhibits [3H]-thymidine uptake into DNA in a dose-dependent manner. Apomorphine, haloperidol and dopamine also exhibited a similar dose dependent inhibition of thymocyte proliferation. The IC25s for DNA synthesis inhibition were 8.2 x 10(-6) M, 4.3 x 10(-7) M, 2.5 x 10(-7) M, and 1.1 x 10(-7) M for CC, HP, DA and AM, respectively. EN was found not to have any significant effect on DNA synthesis. The results suggest that thymus gland atrophy, associated with use of CC may be related to the inhibition of thymocyte proliferation.

Endogenous opioid peptide mediation of hypoalgesic response in long-term diabetic rats.

This study was designed to examine the role of endogenous opioid peptide mediation of elevated pain threshold in adult male Sprague-Dawley rats with long-term diabetes mellitus induced by streptozotocin (STZ). Diabetes resulted in a significant elevation in pain threshold as measured by the tail-flick and/or hotplate latency tests. The hypoalgesic response in diabetic rats to hotplate testing developed gradually over a 4-6 week period after a transient hyperalgesia during the first two weeks of diabetes. The elevation of pain threshold achieved peak level by the fourth week after STZ administration, and remained at that level throughout the experimental period (up to 13 weeks). This hypoalgesic state in diabetic animals is thought to be mediated by opioid receptors (i.e. mu and delta). The involvement of the mu receptor is supported by the effect of naltrexone on the STZ-diabetic rats; naltrexone significantly attenuated the increase in tail-flick and hotplate latencies, compared to that of the non-diabetic controls. Furthermore, the concentration of native (free) Met-enkephalin in the spinal cord of STZ-diabetic rats was about 5-fold higher than that of non-diabetic animals. Such high levels of Met-enkephalin suggest the involvement of delta opioid receptors in the hypoalgesic response observed in STZ-diabetic rats. Seven weeks of insulin treatment, initiated after development of the hypoalgesic response, normalized not only plasma glucose level and body weight of diabetic rats, but also returned their antinociceptive latency toward normal. The results of this study showed that long-term diabetes is associated with altered pain threshold and further support the hypothesis for endogenous opioid peptide mediation of hypoalgesia in chronically diabetic rats that can be prevented by insulin treatment.

Sulphoxidation and sulphation capacity in patients with primary biliary cirrhosis.

We have previously reported an association of impaired S-oxidation with primary biliary cirrhosis. In order to confirm and further define this relationship, we retested S-oxidation capacity via three metabolic pathways and sulphation capacity via a fourth pathway. Metabolism of S-carboxymethyl-L-cysteine is polymorphic -20% of healthy individuals being poor S-oxidisers. We found 26% with primary biliary cirrhosis were poor S-oxidisers, compared with 36% with other liver disease and 25% of healthy controls. Differences were not statistically significant. S-oxidation of ranitidine is dependent upon flavin mono-oxygenases. We showed a non-significant trend toward less S-oxide in primary biliary cirrhosis and other liver disease, compared with healthy controls, with no significant difference between disease groups. Conversion of cysteine to sulphate depends predominantly on cysteine dioxygenase. Impaired activity may be reflected by decreased plasma sulphate and elevated cysteine. We found that the plasma cysteine: sulphate ratio was significantly elevated not only in primary biliary cirrhosis (p < 0.0001), but also in other liver disease (p < 0.0001), compared with healthy individuals. Sulphation capacity was studied by analysing paracetamol metabolism. Paracetamol sulphate and sulphate: glucuronide ratio were reduced in primary biliary cirrhosis compared with normal individuals, (p < 0.05). A trend towards less sulphate in primary biliary cirrhosis compared other liver disease was not significant (p = 0.42). We conclude that although sulphation and some sulphoxidation pathways are impaired in primary biliary cirrhosis, we can currently find no evidence to substantiate the hypothesis that primary biliary cirrhosis is a disease specifically associated with poor S-oxidation, as assessed via these metabolic pathways.

The adverse influence of pregnancy upon sulphation: a clue to the pathogenesis of intrahepatic cholestasis of pregnancy?

Sulphation of oestrogens and monohydroxy bile acids is important in attenuating their cholestatic potential. Thus, impairment of sulphation could lead to retention of cholestatic compounds and precipitate intrahepatic cholestasis in susceptible individuals. We tested the hypothesis that such a mechanism may be involved in the pathogenesis of intrahepatic cholestasis of pregnancy. In vivo and in vitro assessment of sulphation capacity was performed in patients with cholestasis of pregnancy, compared with control females on and off the oestrogen-containing oral contraceptive pill and control individuals during normal pregnancy and post partum, to assess the influence of high oestrogen states upon this metabolic pathway. During in vivo studies utilising paracetamol as a metabolic probe, the proportion of paracetamol sulphate and sulphate: glucuronide ratio were decreased in those with elevated oestrogens, whether the rise in oestrogens was endogenous, in pregnancy (paracetamol sulphate p < 0.05; paracetamol sulphate:glucuronide ratio p < 0.01), or exogenous, with the contraceptive pill (paracetamol sulphate p = 0.2; paracetamol sulphate:glucuronide ratio p < 0.001). In vitro, platelet sulphotransferase activity was measured, utilising phenol as substrate. Sulphotransferase activity decreased during pregnancy compared with repeat measurements post partum (p < 0.005) and compared with non-pregnant individuals (p < 0.05). In conclusion, we have shown that elevated oestrogens are associated with significant impairment in sulphation capacity. An imbalance of sulphation with glucuronidation provoked by high circulating oestrogen levels may be contributory in the pathogenesis of cholestasis of pregnancy.

Circadian rhythms of paracetamol metabolism in healthy subjects; a preliminary report.

Paracetamol was used as a "probe" drug to study the circadian rhythms of metabolite ratios in man. Paracetamol was orally administered to six volunteers at different times of day, 0-8 h and 8-24 h urine samples being measured for sulphate and glucuronide formation. Results showed a wide interindividual variation in paracetamol metabolite excretion among the six subjects. However, when a 500 mg dose was administered, free paracetamol excretion was minimal when the dose was given at 12.00 h and maximal when given at 20.00 h for the 0-8 h collection period. Sulphate excretion rose slightly at night and decreased gradually during the day. Glucuronide excretion was greatest with drug administration at 16.00 h and least if paracetamol was ingested at 08.00 h. The 8-24 h profiles were roughly similar. At a higher dose (1500 mg), free paracetamol excretion showed a minimum from dosing at 20.00 h and a maximum from dosing at 24.00 h in both 0-8 h and 8-24 h collections, while the sulphate conjugate peaked for doses at 20.00 h and 8.00 h with collections at 0-8 h and 8-24 h respectively. The glucuronide conjugate was maximal for paracetamol administration at 16.00 h for both 0-8 h and 8-24 h collections. There appears to be a 12 hour phase variation in excretion; this may result from circadian rhythms in absorption and enzyme activities. These parameters may also affect metabolism at higher dose levels, so that the hepatotoxicity of paracetamol could vary with the time of dose.