Biomedical subjects

E Castanas

Publications and source records attributed to E Castanas.

At least 19 recordsLinked to original sources

PubMed1992

Protein measurement of particulate and solubilized ovine liver membranes.

Protein concentration measurements are critical in biochemical work with cellular membranes, including the determination of cell surface receptor concentration in human malignant tissues obtained at surgery or after biopsy. In this study we compared the results of protein concentration measurements in ovine liver cellular membranes using either particulate preparations or membranes solubilized with four different detergents. In all cases protein was determined by two different indirect methods (Lowry's Folin phenol method and Bradford's Coomassie Brilliant Blue dye binding method) and compared to the direct biuret method. Our results indicate that the direct biuret method gives the highest protein concentrations followed by the method of Lowry. Maximal concentrations (approaching those obtained by the direct biuret method) were obtained after membrane solubilization with Triton X-100 (3-5%). It is suggested that either the direct biuret method (whenever protein concentrations permit it) or the method of Lowry after solubilization of membranes with Triton X-100 (3-5%) should be used preferentially for the determination of membrane protein samples.

Animals↗

PubMed1987

Evidence for high peptide alpha-amidating activity in the pancrease from neonatal rats.

A high peptidylglycine alpha-amidating mono-oxygenase (PAMase) activity has been measured in the pancreas of neonatal rats. A significant fraction of this activity is contained in the beta cells of the islets of Langerhans and is colocalized with thyrotropin-releasing hormone (TRH) and its precursor in secretory granules. The ontogenetic variation of PAMase activity in the pancrease parallels that of TRH concentrations, suggesting that this enzymatic activity is directly related to TRH biosynthesis. In addition, PAMase activity is able to generate TRH when incubated with less than Glu-His-Pro-Gly, a tetrapeptide present as a repetitive sequence in the TRH precursor. The perinatal evolution of the TRH precursor levels in the pancreas is similar to that of PAMase activity (unpublished results). Thus, the neonatal rat pancreas offers an endocrine model in which the levels of a neuropeptide precursor and an enzyme activity, involved in the posttranslational modification of this precursor, are similarly regulated. Our results suggest also that a fraction of PAMase activity may be produced outside of the beta cells and related to the biosynthesis of COOH-terminally amidated peptide(s) other than TRH. The ontogenetic changes in PAMase activity imply that the synthesis of this peptide(s) is high during the neonatal period, decreasing thereafter.

Aging↗

PubMed1987

Effect of passive immunization against corticotropin-releasing factor (CRF) on the postadrenalectomy changes of CRF binding sites in the rat anterior pituitary gland.

The concentration of corticotropin-releasing factor (CRF)-binding sites decreases in the rat anterior pituitary after adrenalectomy; this change may be related either to a direct effect of the circulating glucocorticoids at the pituitary level or to a desensitization of CRF receptors through an increased CRG release in hypophysial portal blood. In order to examine the latter possibility we have measured plasma adrenocorticotropin hormone (ACTH) levels and the number of anterior pituitary CRF binding sites in sham-operated and 24-hour adrenalectomized rats after blockade of endogenous CRF by passive immunization with an antiserum anti-rat CRF (CRF-AS), or after injection of normal rabbit serum (NRS). In NRS-injected rats, after sham operation, plasma ACTH concentration increased (227 +/- 34 vs. 118 +/- 19 pg/ml in controls) without change in CRF-binding sites capacity (20.7 +/- 2.6 vs. 24.6 +/- 3.5 fmol/mg protein in controls). Adrenalectomy induced a large rise in plasma ACTH (785 +/- 89 pg/ml) and a decrease in the number of CRF-binding sites (12.2 +/- 1.7 fmol/mg protein). After CRF-AS injection, plasma ACTH was normalized in sham-operated animals (149 +/- 24 pg/ml) and significantly reduced in adrenalectomized rats (472 +/- 76 pg/ml); the adrenalectomy-induced decrease in the number of CRF-binding sites was unaffected by the CRF-AS administration (12.2 +/- 1.7 fmol/mg protein). The administration of dexamethasone to adrenalectomized rats significantly reduced plasma ACTH concentrations (23.3 +/- 10.6 pg/ml) and prevented the loss in CRF-binding sites capacity (20.7 +/- 1.3 fmol/mg protein).(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenalectomy↗

PubMed1987

Photoaffinity crosslinking of etorphine with opioid binding sites in the bovine adrenal medulla.

The covalent crosslinking of [3H]etorphine with opioid binding sites in the bovine adrenal medulla is reported. Of all the radiolabeled opiates tested (ethylketocyclazocine, etorphine, [D-Ala2, D-Leu5]enkephalin, [D-Ala2, Me-Phe4, Gly5-ol]enkephalin only etorphine could be crosslinked under uv irradiation. In our conditions (black uv lamp, 160 W, peak mean 360 nm, from a distance of 10 cm) maximum covalent binding was observed after a 10-min irradiation. Protein concentration was a crucial factor for the irreversible/total binding ratio. A good ratio (50%) was obtained at protein concentrations of about 1.0 mg/ml. Covalent binding of nonmodified opiates could be of interest for the biochemical characterization of their binding sites.

Adrenal Medulla↗

PubMed1986

Evidence for high peptide alpha-amidation activity in the neonatal rat pancreas.

A high peptide alpha-amidating activity is present in a mitochondrial/secretory granules preparation from 3-day old rat pancreas. It is dependent on copper, ascorbate and molecular oxygen. This preparation is able to generate TRH when incubated with Pyroglu-His-Pro-Gly, a sequence present in the TRH precursor molecular. The peptide alpha-amidating activity may be involved in the high rate of TRH biosynthesis in the pancreas during the neonatal period. In the pancreas of adult rats which contain low levels of TRH, the peptide alpha-amidating activity is barely detectable.

Amidohydrolases↗

PubMed1986

Are opioid peptides co-localized with vasopressin or oxytocin in the neural lobe of the rat?

The content of vasopressin, oxytocin, neurophysin, leucine-enkephalin, methionine-enkephalin, dynorphin-(1-13), and alpha-neoendorphin in the rat neurohypophysis was measured after different periods of dehydration and after depolarisation of isolated neural lobes and of neurosecretory nerve endings. The rates at which the amount of neurohypophysial hormone and opioid peptides decreased, and the changes in the ratios between the amount of vasopressin or oxytocin and opioid peptide in the neurohypophysis after dehydration and in the incubation medium after depolarization in vitro cast some doubt on, and can be explained by mechanisms other than co-localisation of the different peptides.

Animals↗

PubMed1986

[Direct interaction of tricyclic antidepressants with opiate binding sites in the bovine adrenal medulla].

This note reports the interaction of three currently used tricyclic antidepressant drugs (clomipramine, imipramine and amitriptyline) with delta, mu and kappa opioid binding sites in the bovine adrenal medulla. Clomipramine was the only drug interacting with delta and mu sites. On the contrary, all three drugs showed a significant interactions with subtypes of the kappa binding site. Clomipramine was the most active on the kappa 2 and kappa 3 subtypes while amitriptyline showed the highest interaction with the kappa 1 subtype. On the contrary the tricyclic cyproheptadine did not present any interaction with opioid binding sites in our system. This interaction between tricyclic antidepressants and opioid binding sites might be the origin of their analgesic action.

Adrenal Medulla↗

PubMed1986

[Regulation of enkephalin biosynthesis in chromaffin cells].

Enkephalin peptides (ENK) are co-released with catecholamines from bovine chromaffin cells in culture. Drugs mimicking the effects of c-AMP increase ENK biosynthesis by increasing ENK mRNA, but are uneffective on ENK secretion. Nicotine, which causes a rapid release of ENK from these cells, also induces an increase in ENK biosynthesis and ENK mRNA. Reserpine enhance ENK precursor processing. The actions of these different pharmacological agents show that ENK biosynthesis is regulated at different levels in chromaffin cells.

Adrenal Medulla↗

PubMed1986

[Characterization and modulation of anterior pituitary binding sites for rat corticotropin releasing factor (r-CRF)].

Specific binding sites for rat CRF (r-CRF) have been characterized on rat anterior pituitary membranes. The binding of the radioiodinated analog of r-CRF (125I Tyr-r-CRF) was time, temperature, pH and protein dependent. No interaction was found with other neurohormones except with Arginine Vasopressin, but at supra physiological levels. Two classes of specific binding sites (high affinity and low affinity) for r-CRF were identified. Bilateral adrenalectomy provoked, since the 24th hour and up to 7 days, in addition of an increase of ACTH plasmatic levels, an abolition of the high affinity binding site; corticosterone treatment reversed these changes. This finding suggests that circulating glucocorticoids may control the anterior pituitary binding sites for CRF, either by a direct action on the anterior pituitary, or by a modulatory effect on hypothalamic CRF secretion.

Adrenalectomy↗

PubMed1986

[Demonstration of a TRH precursor in the pancreas of the newborn rat].

This study allows the indirect demonstration of a precursor for TRH in pancreatic extracts of 2-days old rats. The sequential treatment of these extracts with trypsin and carboxypeptidase A is followed by a large increase in Pyroglutamyl Histidine Proline (TRH-OH). The molecular weight of the protein that gives rise to TRH-OH after enzymatic treatment ranges between 30,000 and 40,000 daltons. During ontogenesis. TRH levels decrease earlier and more rapidly than that of TRH-precursor levels. These data suggest that changes in the processing of TRH-precursor play a role in the diminution of TRH concentrations that is observed during the first two weeks of life.

Animals↗

PubMed1985

TRH and TRH-OH in the pancreas of adult and newborn rats.

TRH and its metabolite TRH-OH have been measured by specific radioimmunoassays in acid extracts of pancreas in adults and developing rats. TRH and TRH-OH immunoreactivity had the same ontogenic pattern with a maximal concentration on day 4 followed by a progressive return towards adult levels on day 20. A significant linear correlation was found between TRH levels and the TRH/TRH-OH ratio. The range of TRH/TRH-OH ratio varied from 136 +/- 1.6, at the peak of concentrations of both peptides, to 18 +/- 3.9 on day 20. Pancreatic TRH and TRH-OH had the same elution pattern as corresponding synthetic peptides both on Biogel P2 and high-pressure liquid chromatography. The origin of TRH-OH as well as its potential function need further investigations.

Age Factors↗

PubMed1985

Modification of opioid ligand binding in the central and the peripheral nervous system by different buffers.

The modification of binding parameters (equilibrium dissociation constant and binding capacity) of three opioid ligands (DADLE, Etorphine and EKC) on bovine adrenal medulla and rat brain membranes have been examined in three buffer systems: Tris-HCl 50 mM, Hepes-NaOH 10 mM and Tes-KOH 10 mM. Major differences of these parameters have been found: Hepes-NaOH provoked a diminution of the apparent number of binding sites, while a concomitant diminution of the KD and Bmax was observed in Tes-KOH buffer. Substitution of counterions in these two buffers produced further changes of binding characteristics: in Hepes buffer we have observed an abolition of 3H DADLE binding, an enhancement of 3H EKC binding and no modification of 3H etorphine binding characteristics. On the contrary an abolition of the specific binding of all three ligands in Tes buffer was found in the bovine adrenal medulla while minor changes were observed in rat brain. It is concluded that, inspite same disadvantages (substitution for bivalent cations and temperature dependence), Tris-HCl is the buffer of choice for the analysis of opioid binding site interactions.

Adrenal Medulla↗

PubMed1985

Corticoliberin, somatocrinin and amine contents in normal and parkinsonian human hypothalamus.

We have compared hypothalamic contents of various neurotransmitters (dopamine (DA), norepinephrine and serotonin) and their metabolites (dihydroxyphenyl acetic acid, homovanilic acid, 5-hydroxyindoleacetic acid) in post-mortem human controls and parkinsonian hypothalami. Neurotransmitters and their metabolites were measured in 0.1 N HCl hypothalami extracts using electrochemical detection after high performance liquid chromatography. Using specific radioimmunoassays we have also measured corticoliberin and somatocrinin contents in these hypothalami. Despite a 50% decrease of DA contents in parkinsonian hypothalami, no variations of corticoliberin and somatocrinin contents were found: 16.6 +/- 1.78 pg/mg tissue in Parkinson disease vs 16.71 +/- 1.89 in controls for human corticotropin-releasing factor (hCRF 1-41) and 37.38 +/- 11 vs 45.16 for human growth-hormone-releasing factor (hGRF 1-44).

3,4-Dihydroxyphenylacetic Acid↗

PubMed1985

Evidence for a precursor for TRH in the neonatal rat pancreas.

Immunoreactive TRH-OH is present at low concentrations in acid extracts from 2- days old rat pancreas. The sequential treatment of these extracts with trypsin and carboxypeptidase A is followed by a three- and ten-fold increase in TRH-OH IR respectively. The molecular weight of the protein that gives rise to TRH-OH after enzymatic treatment ranges between 30000 and 40000 daltons. The appearance of TRH-OH in the tryptic digest suggests that TRH-OH is the COOH-terminal sequence of this protein. These results are the first evidence that TRH biosynthesis occurs through a large molecule precursor. However, this is an indirect demonstration since TRH cannot be generated under these conditions due to the lack of enzymatic amidation activity.

Animals↗

PubMed1985

[Corticotropin releasing factor].

The search for a neurohormone specifically controlling ACTH secretion resulted in the discovery of the corticotropin-releasing factor (CRF). This factor, located mainly in a paraventricular-infundibular hypothalamic tract, stimulates ACTH synthesis and secretion through a cAMP-dependent mechanism. The corticotropin-releasing factor is the predominant component of a complex control system of adrenal cortex secretion, which also includes catecholamines and the antidiuretic hormone. Its specificity as stimulant of the corticotropic function makes it an extremely useful tool for physiological and physiopathological studies of the hypothalamus-pituitary-adrenal cortex axis regulation.

Adrenocorticotropic Hormone↗

PubMed1985

Influence of acute, subchronic and chronic treatment with neuroleptic (haloperidol) on enkephalins and their precursors in the striatum of rat brain.

We examined the effects of chronic, subchronic and acute treatment with haloperidol on the ME, the MERGL and enkephalin precursor concentrations in rat brain. The changes affected primarily the striatum. The ME content was greatly increased by the treatment, the precursor level was decreased by the haloperidol treatment. The specific mRNA for proenkephalin A increased. For these reasons, we conclude that the effect of haloperidol increase both the biosynthesis and the processing of precursors of enkephalins in the striatum.

Animals↗

PubMed1985

Interaction of opiates with opioid binding sites in the bovine adrenal medulla: I. Interaction with delta and mu sites.

In the present study we examined the interaction of opiates with the delta and mu opioid binding sites in the bovine adrenal medulla. [3H][D-Ala2, D-Leu5]-enkephalin ( [3H]DADLE) in the presence of saturating concentrations of morphiceptin was used to analyze delta site interactions, whereas either [3H]DADLE in the presence of saturation concentrations of [D-Ser2, Leu5]-enkephalin-Thr6 (DSLET) or [3H][D-Ala2, Me-Phe4, Gly5-ol]-enkephalin ( [3H]DAGO) was used for the determination of mu sites. Both binding sites were found to interact stereoselectively with opiates. The binding was affected differentially by proteolytic enzymes (trypsin, alpha-chymotrypsin, pepsin), N-ethylmaleimide, and A2-phospholipase. Kinetic and equilibrium binding studies revealed that in each case radiolabeled opiates interact with one class of binding sites, following simple second-order bimolecular kinetics. Competition for binding by opiates and opioid peptides confirmed the delta and mu selectivity of these sites. Monovalent (Na+, Li+, K+) and divalent (Mg2+, Mn2+, Ca2+) ions interacted differentially with these two binding sites: In general, monovalent cations affected preferentially the apparent number of binding sites, whereas divalent ions modified the equilibrium dissociation constant. Furthermore, positive or negative cooperativity and an apparent heterogeneity of binding sites were detected under some ionic conditions.

Adrenal Medulla↗

PubMed1985

Interaction of opiates with opioid binding sites in the bovine adrenal medulla: II. Interaction with kappa sites.

In this study we examined the interaction of opiates with kappa binding sites in the bovine adrenal medulla. [3H]Ethylketocyclazocine (EKC), [3H]etorphine, and [3H]bremazocine stereoselective bindings were used to assay these interactions. The kappa sites were found to be heterogeneous: [3H]bremazocine identified with high affinity all subtypes of these sites. [3H]EKC, in the presence of saturating concentrations of [D-Ala2, D-Leu5]-enkephalin (DADLE) (5 microM), was used to identify kappa 1 sites, on which dynorphin A (1-13) bound with high affinity. Either [3H]EKC or [3H]etorphine in the presence of 5 microM DADLE identified the kappa 2 subtype. This subtype was found to interact with beta-endorphin and especially with the octapeptide Met5-enkephalyl-Arg6-Gly7-Leu8. Furthermore, [3H]etorphine identified in the bovine adrenal medulla a third high-affinity component, in the presence of 5 microM DADLE. This residual interaction was found to be equally stereoselective and presenting kappa selectivity. Met5-enkephalyl-Arg6-Phe7 interacted preferentially with this site. The three kappa subtypes interacted differentially with monovalent (Na+, K+, and Li+) and divalent (Ca2+, Mg2+, and Mn2+) ions by modification of the apparent concentration of the accessible sites and/or by changes of the apparent KD for radioligands. Modifying agents (proteolytic enzymes, thiol-modifying reagents, and A2-phospholipase) produced different effects on each subtype of the kappa site, suggesting a different protein (or protein-lipid?) composition.

Adrenal Medulla↗