RESUMO
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.
Assuntos
Diabetes Mellitus Experimental/enzimologia , Hipoglicemiantes/farmacologia , Insulina/farmacologia , Óxido Nítrico Sintase/biossíntese , Animais , Arginina/metabolismo , Glicemia/metabolismo , Northern Blotting , Western Blotting , Citrulina/metabolismo , Masculino , Óxido Nítrico Sintase Tipo I , Óxido Nítrico Sintase Tipo II , Ratos , Ratos WistarRESUMO
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.
Assuntos
Córtex Cerebral/enzimologia , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Óxido Nítrico Sintase/metabolismo , Animais , Northern Blotting , Diabetes Mellitus Experimental/genética , Regulação Enzimológica da Expressão Gênica , Masculino , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase Tipo I , Óxido Nítrico Sintase Tipo II , RNA Mensageiro , Ratos , Ratos WistarRESUMO
It has been documented that C6 glioma cells can be changed into normal glial cells when they were cultured in serum free medium. In the present study, the expressions of inducible nitric oxide synthase (iNOS) and superoxide dismutase (SOD) were investigated. The mRNA level of iNOS was markedly increased by lipopolysaccharide (LPS) in cultured rat brain astrocytes (RBA-1) but not in C6 glioma cells. However, increase of mRNA for iNOS by LPS can be obtained in C6 glioma cells when they were cultured in serum free medium. The mRNA level of magnesium-SOD (Mn-SOD) was increased by LPS in both cells while the expression of constituted SOD (Cu,Zn-SOD) was not stimulated by LPS. Western blotting analysis indicating the amount of protein showed a similar change. After serum deprivation, the protein of iNOS or Mn-SOD was increased by LPS in C6 glioma cells in a way similar as that in RBA-1 cells. These results suggest that serum free conditioned C6 glioma cells were adapted to astroglial cell-like properties which may express more iNOS and Mn-SOD mRNA in the presence of LPS.
Assuntos
Proteínas Sanguíneas/farmacologia , Óxido Nítrico Sintase/metabolismo , Superóxido Dismutase/metabolismo , Animais , Astrócitos/citologia , Astrócitos/enzimologia , Northern Blotting , Western Blotting , Encéfalo/citologia , Encéfalo/enzimologia , Meios de Cultura , Meios de Cultura Livres de Soro/farmacologia , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/fisiologia , Glioma , Lipopolissacarídeos/farmacologia , Óxido Nítrico Sintase/análise , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase Tipo II , RNA Mensageiro/análise , Ratos , Superóxido Dismutase/análise , Superóxido Dismutase/genética , Células Tumorais CultivadasRESUMO
The M2 receptor (M2-mAChR) is quantitatively the dominant muscarinic subtype in animal bladders. The alterations in its protein quantity and biosynthesis during diabetic cystopathy were investigated. Three-month-old male Wistar rats were divided into two groups: (1) 2-week-old diabetics; and (2) normoglycemic control rats. Diabetes was induced by single intravenous injection of 60 mg/kg streptozotocin. The amount of M2 receptor protein in the rat bladder body tissue was measured by Western immunoblotting using monoclonal antibodies. For determination of M2 muscarinic receptor mRNA in the bladder tissue, the method of Northern blotting was employed. The results of the Western immunoblotting showed that the amount of M2-mAChR protein in the diabetic bladder was significantly increased by 40.0 +/- 6.2% when compared with the control bladder (P < 0.05, n = 8). The Northern blotting demonstrated a 69.3 +/- 8.5% increase of the M2-mAChR mRNA in the diabetic bladder (P < 0.05, n = 8). The findings of the present study demonstrated an up-regulation of M2-mAChR biosynthesis in the diabetic urinary bladder. This phenomenon could lead to increased reactivity to acetylcholine and thus results in detrusor instability.
