Decrease of adenosine A-1 receptor gene expression in cerebral cortex of aged rats.

Changes of adenosine A-1 receptor (A1-AR) gene expression in aging were investigated in cerebral cortex using the rat aged from 2 months (adult) to 24 months (aged). Quantification of A1-AR protein level by immunoblotting analysis showed an age-related decrease of A1-AR in cerebral cortex of Wistar rats. Compared to the preparations from 2-month-old animals, the levels of A1-AR in the 6-, 12-, and 24-month-old rats were reduced by 14.3+/-5.2, 32.5+/-4.5 and 28.2+/-5.7%, respectively. Similar decrease of mRNA level in A1-AR was also obtained using Northern blotting analysis. Two representative spots of mRNA, a 3.4-kb transcript and a 5.6-kb transcript, were observed in X-ray film from cerebral cortex of rat hybridized with rat A1-AR cDNA probe. Compared to the 2 month-old rats, levels of the 5.6-kb transcript were decreased by 17.9+/-2.5, 27.4+/-3.2 and 23.1+/-2.1% in the 6-, 12- and 24-month-old rats, respectively. These results indicated an age-related decrease of A1-AR in cerebral cortex of the rat that seems responsible for the change of response to adenosine.

Changes of superoxide dismutase gene expression and activity in the brain of streptozotocin-induced diabetic rats.

In an attempt to discover the changes of superoxide dismutase (SOD) in the brain of diabetic state, we investigated the level of SOD in streptozotocin-induced diabetic rats (STZ-diabetic rats) using enzyme activity assay, Northern blotting analysis of mRNA levels and Western blotting of enzyme amount. Five discrete brain regions, cerebrocortex, hypothalamus, hippocampus, the remaining non-cortex cerebrum (NCC area) and cerebellum, were examined in STZ-diabetic rats to compare with age-matched normal rats. Higher levels of Mn-SOD including the activity, mRNA expression and immunoblot of enzyme were found in all areas of the brain from STZ-diabetic rats as compared with that in Wistar rats. Except in the cerebellum, similar changes of Cu,Zn-SOD were found in the brain of STZ-diabetic rats. These results indicate an increase of SOD both gene expression and activity in the brain of STZ-diabetic rats. This alteration of SOD may be one of the important factors for the vulnerability of the brain to oxygen free radicals or may be related to the pathophysiology of diabetes.

Decrease of nitric oxide synthase in the cerebrocortex of streptozotocin-induced diabetic rats.

In an attempt to know the role of nitric oxide in the disease of insulin-dependent diabetic mellitus (IDDM), the present study examined the change of nitric oxide synthase (NOS) both the activity and gene expression in cerebrocortex of streptozotocin-induced diabetic rats (STZ-diabetic rats). The activity of NOS determined by conversion of [3H] L-arginine to [3H] L-citrulline was markedly decreased in STZ-diabetic rats. Northern blot showed that STZ-diabetic rats expressed a lower mRNA level of neuronal NOS (nNOS). Western blot showed a similar decrease of nNOS in STZ-diabetic rats. However, the NOS activity was increased in rats receiving repeated supply of glucose named glucose-challenged rats. Although the mRNA level of nNOS was not changed in the glucose-challenged rats, the immunoblot of nNOS was also decreased in glucose-challenged rats. These findings suggested that NOS was lowered in the brain of STZ-diabetic rats in a way unrelated to the increase of glucose.

Insulin restores neuronal nitric oxide synthase expression in streptozotocin-induced diabetic rats.

Nitric oxide (NO) is known to play an important role in the pathophysiology of insulin-dependent diabetic mellitus (IDDM). In an attempt to investigate the relation between insulin and NO in IDDM, the present study employed male Wistar rats to induce IDDM by intravenous injection of streptozotocin (STZ). Four groups of rats were used; untreated normal control group, insulin treated STZ group, vehicle-treated STZ control, and one group of age-matched rats which were orally supplied with glucose to increase plasma glucose (glucose-challenged rats). Changes of the activity and gene expression of neuronal nitric oxide synthase (nNOS) were examined in cerebellum and kidney of these groups. The activity of nNOS in cerebellum, determined by conversion of [3H] L-arginine to [3H] L-citrulline, in STZ-induced diabetic rats was markedly lower than normal rats. Insulin treatment reversed the nNOS activity. Similar reversion by insulin treatment was also obtained in the gene expression of nNOS. However, the activity and gene expression of nNOS in glucose-challenged rats were not different from those in normal rats. The role of hyperglycemia can thus be ruled out. These findings indicated that an impairment of nNOS in the brain of rats with IDDM is mainly due to the absence of insulin.

Stimulatory effect of D-ephedrine on beta3-adrenoceptors in adipose tissue of rats.

The effect of ephedrine on beta3-adrenoceptos (beta3-AR) was studied in the isolated adipose tissue of Wistar rat. Incubation with D-ephedrine (0.1-10 microM) induced a concentration-dependent decrease of uptake of [14C]-deoxy-D-glucose into white adipose tissues (WAT). The inhibitory effect of D-ephedrine was potentiated by BRL 37344, the agonist of beta3-AR and concentration-dependently inhibited by SR 59230A, the selective antagonist of beta3-AR. The action of D-ephedrine on beta3-AR was further blocked by the antibodies for beta3-AR, but not the immunoglobulin. in a concentration-dependent manner. Moreover, D-ephedrine increased glycerol release from the isolated brown adipose tissues (BAT) and this action was also abolished by SR 59230A at concentration sufficient to block beta3-AR. Thus, these results suggest that D-ephedrine has the ability to activate beta3-AR both in WAT and BAT of Wistar rats in vitro.