ABSTRACT
The aim of this study was to comparatively study cyclin-dependent kinase 5 (CDK5) and c-Fos regulation by morphine in the brains of Lewis and Fischer 344 (F344) rats, which are known to differ in their behavioral sensitivities to several drugs of abuse. Two hours after an acute i.p. administration of morphine (10 mg kg(-1)) or saline (control), the animals were perfused and their brains prepared for immunohistochemistry. The number of CDK5 immunoreactive cells was significantly higher in the nucleus accumbens (NAC), the locus coeruleus (LC) and the nucleus tractus solitarius (NTS) of saline-injected F344 rats than in those of the Lewis rats. Morphine upregulated CDK5 with a varying pattern depending on the strain and brain area. The effect of the opioid was more marked in the NTS of the Lewis rats and the NAC of the F344 rats. Immunostaining of c-Fos was very low or absent in the control animals and was consistently up-regulated by morphine, especially in the LC and NTS of the F344 rats and the NAC of the Lewis rats. We propose that the acute morphine regulation of CDK5 expression in the NAC may predict the rate of drug intake and/or extinction of drug seeking, while the pattern of c-Fos activation may be more related to the differential acquisition of morphine-seeking behaviors.
Subject(s)
Brain/drug effects , Cyclin-Dependent Kinase 5/metabolism , Morphine/pharmacology , Narcotics/pharmacology , Proto-Oncogene Proteins c-fos/metabolism , Analysis of Variance , Animals , Brain/anatomy & histology , Brain/metabolism , Male , Rats , Rats, Inbred F344 , Rats, Inbred Lew , Species SpecificityABSTRACT
1. Angiotensin I-converting enzyme (EC 3.4.15.1) has been purified to electrophoretic homogeneity from chicken lung by using a facile two-step protocol which included affinity chromatography on Sepharose-bound captopril. 2. Captopril was a potent inhibitor of chicken lung angiotensin I-converting enzyme with Ki values of 2.0 nmol/l and 1.6 nmol/l for detergent-extracted and trypsin-extracted angiotensin I-converting enzymes, respectively. 3. Molecular weight comparison of trypsin-extracted (M(r)270,000) and detergent-extracted (M(r)690,000) angiotensin I-converting enzyme indicated that membrane-binding sequence contributed to a large extent to the enzyme molecule. 4. Kinetic properties of both forms of the enzyme suggested that the membrane-bound sequence contributed to an increase of the enzyme-substrate affinity.
Subject(s)
Lung/enzymology , Peptidyl-Dipeptidase A/isolation & purification , Animals , Captopril/pharmacology , Chickens , Chromatography, Affinity , In Vitro Techniques , Kinetics , Molecular Weight , Peptidyl-Dipeptidase A/chemistry , Peptidyl-Dipeptidase A/metabolismABSTRACT
Angiotensin I(AI)-converting enzyme (ACE) (EC 3.4.15.1) was solubilized from the membrane fraction of chicken lung using trypsin and nonidet P40 extraction, and then purified to homogeneity by captopril affinity chromatography. Comparison of trypsin-extracted and detergent-solubilized membrane-bound converting enzyme by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and isoelectric focusing indicated that the membrane-binding sequence contributed to a large extent to the size and charge of the enzyme. Both forms of the enzyme were glycoproteins but they differed in the glucidic content; 4.5% by weight of the enzyme in the trypsin-extracted ACE and 15% by weight of the enzyme in the detergent-solubilized ACE. In both cases hexoses were the most abundant residues. Both forms of the enzyme were found to contain 1 g-atom zinc/mol enzyme. The purified enzymes did not only split Hip-His-Leu but also AI and bradykinin. The Michaelis constant (Km) and maximum velocity (Vmax) values of the trypsin-extracted ACE for Hip-His-Leu were 52 x 10(-5) mol/l and 15.36 nmol/min respectively, and for AI they were 7.8 x 10(-5) mol/l and 0.45 nmol/min respectively. The Km and Vmax values of the detergent-solubilized ACE for Hip-His-Leu were 32 x 10(-5) mol/l and 11.75 nmol/min respectively, and for AI they were 6.5 x 10(-5) mol/l and 0.97 nmol/min.(ABSTRACT TRUNCATED AT 250 WORDS)
Subject(s)
Chickens/metabolism , Lung/chemistry , Peptidyl-Dipeptidase A/chemistry , Animals , Carbohydrates/analysis , Chromatography, Affinity , Electrophoresis, Polyacrylamide Gel , Isoelectric Focusing , Peptidyl-Dipeptidase A/analysis , Peptidyl-Dipeptidase A/metabolism , Zinc/analysisABSTRACT
Angiotensin I-converting enzyme activity was measured in homogenates of guinea pig (Cavia porcellus), chicken (Gallus domesticus), and carp (Cyprinus carpio) organs. The highest activity was found in guinea pig lung and chicken kidney. In carp the highest activity was found in heart and spleen, although gill arch also showed a high activity. Acute hypoxia decreased the angiotensin I-converting enzyme activity in guinea pig lung and carp gill arch, but the changes in chicken lung and kidney were considered nonsignificant.
Subject(s)
Hypoxia/metabolism , Peptidyl-Dipeptidase A/metabolism , Amino Acid Sequence , Animals , Carps , Chickens , Gills/enzymology , Guinea Pigs , Kidney/enzymology , Lung/enzymology , Molecular Sequence Data , Myocardium/enzymology , Species Specificity , Spleen/enzymology , Tissue DistributionABSTRACT
Angiotensin I converting enzyme activity was measured in homogenates of guinea pig and chicken organs (lung, kidney, heart, ileum, diaphragm and liver), using a spectrophotometric assay for hydrolysis of hippuryl-L-histidyl-L-leucine. High specific activities were found in lung, kidney and diaphragm, but the highest corresponded to guinea pig lung and chicken kidney. Acute hypoxia decreased angiotensin I converting enzyme activity in guinea pig lung and chicken diaphragm, but the changes in kidney were considered non-significant in both the guinea pig and chicken.
