Biomedical subjects

T Costa

Publications and source records attributed to T Costa.

At least 109 records · Page 6Linked to original sources

PubMed1987

Antisera against the 3-17 sequence of rat G alpha i recognize only a 40 kDa G-protein in brain.

To obtain antisera specific for the GTP-binding protein Gi alpha we immunized rabbits against a synthetic peptide derived from the N-terminal (3-17) sequence predicted from the rat Gi alpha cDNA clone published by Itoh et al. (1986) (Proc. Natl. Acad. Sci. USA 83, 3776-3780). Western-blot analysis of bovine brain G-proteins purified and resolved by hydrophobic chromatography and of rat striatal membranes, indicate that this antiserum does not recognize 41 kDa alpha i or 39 kDa alpha o. However, it reacts with a 40 kDa alpha-subunit. The data suggest that the sequence deduced from the rat G alpha i cDNA corresponds to a G40 alpha protein and that N-terminus directed antisera are useful tools to discriminate between two different G alpha i-like types of G-proteins present in mammalian brain.

Amino Acid Sequence↗

PubMed1987

delta EPhe4-enkephalin analogs. Delta receptors in rat brain are different from those in mouse vas deferens.

Conformationally restricted enkephalin analogs containing E-cyclopropylphenylalanine (delta EPhe), [D-Ala2, (2R,3S)-delta EPhe4,Leu5]enkephalin and its (2S,3R) isomer, were evaluated in receptor-binding assays using rat brain and in assays using muscle preparations. The (2S,3R) isomer was almost completely inactive in all assays. In contrast, the (2R,3S) isomer showed a very high affinity for the delta and a very weak affinity for the mu receptors in rat brain. The extent of delta affinity and the selectivity of this isomer were almost equal to those of [D-Pen2,D-Pen5]enkephalin. However, the (2R,3S) isomer was inactive in both the mouse vas deferens and guinea pig ileum assays, and showed no antagonistic activity in these tissues. These results indicate that the (2R,3S) isomer interacts with the delta receptors in rat brain, but not with those in the mouse vas deferens, and they suggest that the delta receptors in the central and peripheral nervous systems are different from each other.

Animals↗

PubMed1987

Opioid receptor desensitization in NG 108-15 cells. Differential effects of a full and a partial agonist on the opioid-dependent GTPase.

Opioid-receptor binding and the opioid-mediated stimulation of low Km GTPase and inhibition of adenylate cyclase were studied in membranes derived from NG 108-15 cells pretreated with either the opioid peptide [D-Ala2, D-Leu5]enkephalin (DADLE) or morphine. Pretreatment with DADLE resulted in a concentration-dependent loss of responsiveness of GTPase to the peptide; this effect was entirely accounted for by a reduction in the maximal stimulation produced acutely by DADLE, without changes in the EC50 of the peptide, indicating a non-competitive type of desensitization. The degree of desensitization of GTPase was similar after one and 24 hr of pretreatment with DADLE, indicating that the process occurs rapidly. In contrast, morphine, which was 70-80% as potent as DADLE in stimulating GTPase and inhibiting adenylate cyclase in acute conditions, induced only a minimal desensitization of the opioid-GTPase system and, in contrast to DADLE, did not desensitize adenylate cyclase. Pretreatment with DADLE for one hour led to a decrease in opioid receptor density which was quantitatively similar to the degree of desensitization of GTPase: both these effects of DADLE were antagonized to a similar extent when morphine was also present in the pretreatment. Thus, desensitization of the opioid-stimulated GTPase appears to be correlated with down-regulation of the opioid receptor. Moreover, these findings suggest that partial agonists cannot induce this process.

Adenylyl Cyclase Inhibitors↗

PubMed1987

Radiologic findings in the Proteus syndrome.

The radiological findings in two patients with the Proteus syndrome are described. Features in our two cases not previously mentioned or stressed include vertebral dysplasia and enlargement (megaspondylodysplasia), bilateral genu valgum, recurrent after surgery and intraabdominal and mesenteric lipomatosis. Emergency laparotomy was performed on the first patient who had a twisted necrotic portion of mesenteric fat. Macrodactyly, skeletal muscle atrophy and subcutaneous fat accumulation in the abdominal wall were present in both. In addition the second patient was mentally retarded and had frontal bony prominence of skull. Computed tomography was used for the specific diagnosis of the lipomatous tissues in both patients.

Bone and Bones↗

PubMed1987

Synthesis, receptor binding activity and fluorescence property of fluorescent enkephalin analogs containing L-1-pyrenylalanine.

The novel fluorescent amino acid, L-1-pyrenylalanine (L-Pya), was prepared by the asymmetric hydrogenation of cyclic dehydrodipeptide. Fluorescent enkephalins containing one or two Pya residues at position 1,4 or 5 of [D-Ala2, Leu5]enkephalin were synthesized by the solution method. Mono-Pya-enkephalins showed strong fluorescence intensities and potent binding affinities with specificity and selectivity for opiate receptors. However, di-Pya-enkephalins showed markedly decreased receptor binding affinities. These results indicate that the incorporation of two Pya residues into enkephalin makes the peptide unable to interact with the opiate receptors, although introduction of one Pya residue is effective to elicit a specific receptor interaction. Di-Pya-enkephalins showed intramolecular excimer spectra, indicating that the peptides are able to take possible folded conformations.

Animals↗

PubMed1987

GTPase and adenylate cyclase desensitize at different rates in NG108-15 cells.

The time course of opioid receptor binding disappearance and loss of responsiveness of the opioid-controlled GTPase and adenylate cyclase were compared in membranes derived from NG108-15 cells pretreated with the opioid peptide agonist [D-Ala2,D-Leu5]enkephalin (DADLE). Upon pretreatment with DADLE, a rapid desensitization of the opioid-stimulated GTPase occurred with a time course distinguishable as two exponential components having respective half-lives of 5-9 and 60-80 min. Opioid receptor binding activity, as assessed using [3H]diprenorphine, also decayed as two exponential components whose half-lives were similar to those for GTPase desensitization (7 and 120 min). However, when [3H]diprenorphine binding was measured in the presence of sodium and GTP, only the second, slow component was apparent. In contrast, desensitization of the opioid-controlled adenylate cyclase occurred as only one exponential decaying process, displaying a half-life of 57 min. Whereas the loss of responsiveness of GTPase to DADLE was entirely accounted for by a reduction in the maximal stimulation produced acutely by DADLE, desensitization of adenylate cyclase was characterized by both a decrease in maximal inhibition and a shift to the right of the EC50 of the agonist in inhibiting acutely the enzyme. In addition, after 1 hr of pretreatment with DADLE, the opioid-stimulated GTPase was desensitized by 65%, whereas 80% of maximal inhibition of adenylate cyclase could still be achieved. We suggest that: the rapid loss of responsiveness of the opioid-GTPase system results from an uncoupling between the receptor and the nucleotide-binding regulatory protein (N); the fast decaying GTPase activity appears to be not directly related to the opioid-mediated inhibition of adenylate cyclase; and the slow decaying GTPase activity, as well as the desensitization of the opioid-adenylate cyclase, is most likely accounted for by down-regulation of the opioid receptor. These findings may indicate that part of the opioid-stimulated GTPase in the membrane is not involved in inhibition of the cyclase and could reflect the activity of a regulatory protein which couples opioid receptors to another membrane effector. Alternatively, they might be interpreted on the basis of a model which involves a tight coupling between receptor activation and N protein and a large amplification mechanism between N protein and adenylate cyclase.

Adenylyl Cyclase Inhibitors↗

PubMed1986

Tyr1-substituted and fluorescent Pya1-enkephalins bind strongly and selectively to mu and delta opiate receptors.

The fluorescent enkephalins in which an essential Tyr1 residue is replaced by L-1-pyrenylalanine (Pya) were synthesized and examined in the receptor binding assays. [Pya1, Leu5]Enkephalin and its methyl ester showed binding characteristics specific for the opiate receptors, exhibiting a potent inhibition of Tyr1-containing enkephalins. Surprisingly, the methyl ester displayed almost the same potencies to those of DAGO-enkephalin. This analog bound 24-fold more strongly to mu than to delta-receptors. C-terminal free analog Pya1-Enk-OH was delta-preferential with a fairly good affinity. These results indicate that Tyr1 in enkephalin is not necessary to recognition of the opiate receptors.

Alanine↗

PubMed1986

Target size analysis of opioid receptors. No difference between receptor types, but discrimination between two receptor states.

Target size analysis of opioid receptor is complicated by the presence of multi-exponential inactivation curves. Irradiation of intact frozen tissue proved essential to eliminate such artifacts, due to indirect irradiation effects. Upon irradiation condition, opioid binding activity was inactivated in a single mono-exponential manner. Identical inactivation curves were obtained for mu, delta and kappa binding activities in brain membranes from rat, guinea-pig and frog and in NG 108-15 cells: the molecular mass obtained was 98 +/- 2 kDa. However, when opioid binding was assayed in the presence of Na+, Mg2+ and GTP, the molecular mass was found to be only 56 +/- 4.4 kDa. We suggest that the opioid recognition site comprises a unit of 56 kDa and that in the absence of Na+, Mg2+ and GTP an additional membrane component of 40-44 kDa is necessary for high-affinity opioid binding.

Animals↗

PubMed1986

H1-histaminergic activation stimulates inositol-1-phosphate accumulation in chromaffin cells.

Adrenal medullary chromaffin cells maintained in vitro were prelabeled with [3H]inositol and the accumulation of [3H]inositol-1-phosphate, was determined following stimulation with a variety of pharmacological agents. Carbachol, bradykinin, and histamine produced significantly greater accumulation of [3H] inositol-1-phosphate over basal levels, with histamine producing the greatest effect. H1-histamine receptor antagonists, mepyramine, pyrilamine, tripelennamine and clemastine were all able to reduce or completely block the histamine response. The two specific H2-histamine receptor antagonists, cimetidine and ranitidine, had no effect on this response. Histamine dose-response characteristics in the presence of mepyramine and clemastine suggest the H1 antagonism to be competitive in nature.

Adrenal Medulla↗

PubMed1986

Association of amyoplasia with gastroschisis, bowel atresia, and defects of the muscular layer of the trunk.

We reviewed 225 cases of amyoplasia, and the association of amyoplasia with gastroschisis and with monozygotic twinning was confirmed. In addition, an apparently increased association of bowel atresia and defects in the muscular layer of the trunk wall with amyoplasia was observed. The association of amyoplasia, monozygotic twinning, and these trunk wall defects strongly suggests that the pathogenesis of amyoplasia is linked to some type of vascular compromise.

Abdominal Muscles↗

PubMed1986

Effects of sodium and GTP on the binding kinetics of [3H]diprenorphine in NG 108-15 cell membranes.

Equilibrium binding isotherms of [3H]diprenorphine in membranes from NG 108-15 cells are consistent with a homogeneous population of binding sites. Upon addition of Na+, Mg2+ and GTP, only a 2-fold reduction in affinity with a minor decrease in the number of sites is observed. Dissociation curves of [3H]diprenorphine, however, are clearly biphasic: in the absence of Na+, Mg2+ and GTP, 80% of the bound ligand dissociates slowly with a t1/2 of 100 min, and only 20% rapidly (t1/2 4.5 min). In the presence of Mg2+, nearly all the binding is found in the slowly dissociating form. Upon the addition of either Na+ or GTP, 20-30% of the binding dissociates more rapidly. The rate constant of the rapidly dissociating form generated by Na+, however, is 2.5 times greater than that induced by the presence of GTP. Thus, the addition of both, Na+ and GTP, converts about 80% of the receptor into a very fast dissociating form (t1/2 1.7 min). Exposure of intact cells to pertussis toxin (10 ng/ml) or treatment of membranes with N-ethyl maleimide (500 microM), strongly reduces the proportion of the slowly dissociating component. Following these treatments, the effect of GTP is reduced or abolished, but that of Na+ remains unaffected. We conclude from these data that the effects of Na+ and GTP are not only distinct in site but also in mechanism of action and that there are three forms of opioid receptors that can be differentiated by their kinetic properties. The slowly dissociating receptor form requires a functional N unit.

Animals↗

PubMed1986

Interaction of dimeric and monomeric enkephalins with NG108-15 hybrid cells. A kinetic analysis.

The binding of the enkephalin dimer [D-Ala2, Leu5-NH-CH2-]2 (DPE2) is characterized by its high affinity for receptors on NG108-15 hybrid cells, the affinity constant K = 4.7 X 10(9) M-1 is up to 8-fold that of monomers (0.6 to 1.0 X 10(9) M-1), and a maximal binding capacity equal to one half that of the monomers. Kinetic studies showed that DPE2 binds with a 2-fold higher rate, k1 = 6.3 X 10(7) M-1min-1, than monomers (2.4 to 3.8 X 10(7) M-1min-1), and dissociates at a slower rate than monomers. Dissociation of DPE2 was consistently bi- or multiphasic but increased about 12% only after 3 hr of dissociation in the presence of a large excess of unlabeled enkephalin. The dissociation kinetics of monomers varied with enkephalin and experimental conditions used. Consistent with the value for the maximal binding capacity, the kinetic studies are interpreted in support of the hypothesis that DPE2 binds by cross-linking two subunits of one receptor.

Animals↗

PubMed1986

Differential sensitivity of basal and opioid-stimulated low Km GTPase to guanine nucleotide analogs.

In membranes derived from NG108-15 cells, the opioid peptide [D-Ala2,D-Leu5]enkephalin (DADLE) stimulates a low Km GTPase. The nucleotide analogs guanosine 5'-O-(2-thio)diphosphate (GDP beta S), guanosine 5'-(beta,gamma-imido)triphosphate [Gpp(NH)p] and guanosine 5'-O-(3-thio)-triphosphate (GTP gamma S) inhibit the basal enzymatic activity with the order of potency GTP gamma S greater than Gpp (NH)p greater than GDP beta S. In the presence of DADLE, the inhibition isotherms of GDP beta S and Gpp(NH)p are shifted to the right five- and fourfold, respectively, compared to the inhibition observed in the absence of DADLE. In contrast, the IC50 of GTP gamma S for inhibiting the enzyme is reduced by 55% in the presence of the opioid. Both Gpp(NH)p and GTP gamma S produce a concentration-dependent increase in the Km(app) of GTPase, without affecting its Vmax, indicating a competitive inhibition. However, the replots of Km(app) versus inhibitor concentration are hyperbolic, suggesting a partial type of inhibition. Both Gpp(NH)p and GTP gamma S, but not GTP, induce an increase in the EC50 of DADLE for stimulating GTPase. These findings indicate that the basal and the opioid-stimulated low Km GTPase differ in their respective sensitivities to inhibition by guanine nucleotide analogs.

Animals↗

PubMed1986

Cystamine-enkephalin dimer. Syntheses and biological activities of enkephalin analogs containing cystamine and cysteamine.

A cystamine-enkephalin dimer, containing two molecules of [D-Ala2, Leu5] enkephalin cross-linked at the COOH-terminal leucine residue with cystamine, (NH2-CH2-CH2-S-)2, has been synthesized in order to examine directly the dimerization effect of an enkephalin molecule on the opiate receptor interactions. In a comparison of potencies against [3H]-[D-Ala2,D-Leu5] enkephalin (3H-DADLE) and [3H]-[D-Ala2,MePhe4,Gly-ol5] enkephalin (3H-DAGO) as delta and mu tracers, respectively, enkephalin dimer showed a very high affinity, especially for the delta opiate receptors. Dimer was almost threefold more potent than DADLE, which is one of the most utilized delta ligand to date. When the binding affinity of cystamine-dimer was compared with that of its reduced thiol-monomer, namely [D-Ala2,Leu5,cysteamine6] enkephalin, the increment in affinity was four to fivefold for both delta and mu receptors. The results strongly indicate that the dimeric enkephalin is more potent presumably due to the simultaneous interaction with the two binding sites of the opiate receptors.

Animals↗

PubMed1986

Synthesis and receptor binding affinity of both E- and Z-dehydrophenylalanine4 enkephalins.

The dehydrophenylalanine4-enkephalin having the E-configuration (delta EPhe; phenyl and C = 0, cis) was prepared by photoisomerization of the Z-isomer with 3100 A light, followed by reversed-phase HPLC separation of the resulting mixture of the Z- and E-isomers. In the radioligand receptor binding assays, the E-isomer of [D-Ala2, delta Phe4, Leu5]enkephalin exhibited an extremely diminished affinity as compared with the Z-isomer, namely 150-260-fold loss of affinity for the delta and mu opiate receptors. The results indicate that the interrelationship of the Tyr1 and Phe4 residues in the enkephalin molecule seems to be of great importance in receptor recognition.

Animals↗

PubMed1986

Opiate receptor binding characteristics of dimeric analogues of mu-selective DAGO-enkephalin.

DAGO-enkephalin ([ D-Ala2, MePhe4, Gly-ol5]enkephalin), a highly selective ligand for mu opiate receptors, was dimerized with a series of alpha,omega-alkanedioic acids (n = 2-12) at the OH-terminus. In the radioligand receptor binding assays with rat brain, most of the DAGO-enkephalin dimers with cross-linking methylene chain n (DEDn) were more potent than DAGO monomer. For delta receptors, affinity of DEDn was maximized with n = 8, which might be related to an optimal distance between two binding sites. For mu receptors, an increase in chain length resulted in a progressive loss of potency. Although all of DEDn are considerably mu-selective, with a mu/delta ratio of 15-50, DEDn exhibited fairly flat binding curves with 15-50% smaller sloped than that of DAGO, suggesting that the dimers interact more strongly with one of the possible two mu binding sites.

Animals↗

PubMed1986

New disorder of vitamin B12 metabolism (cobalamin F) presenting as methylmalonic aciduria.

An infant with vitamin B12-responsive methylmalonic aciduria and no homocystinuria or megaloblastic anemia presented with stomatitis, glossitis, convulsions, and developmental delay. Cultured fibroblasts showed defective incorporation of both [14C]5-methyltetrahydrofolate and [14C]propionate into protein by whole cells and a decrease of methionine synthase activity in cell extracts. Despite excessive incorporation of [57Co]cyano-B12 by fibroblasts from the patient, free vitamin B12 was unable to efflux from lysosomes, and, therefore, synthesis of both adenosyl-B12 and methyl-B12 was impaired.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗

PubMed1985

Delta and mu opiate receptor probes: fluorescent enkephalins with high receptor affinity and specificity.

The fluorescent amino acid, L-1-pyrenylalanine (Pya) was incorporated into [D-Ala2,Leu5]enkephalin and its methyl ester at position 4 or 5. Pya-enkephalins showed strong fluorescent intensity and displayed high binding affinity for opiate receptors. Pya4-enkephalins showed high specificity for the mu receptors, while Pya5-enkephalins showed high specificity and selectivity for the delta receptors. Particularly, [D-Ala2,Pya5]enkephalin was as potent as the most utilized delta-specific ligand of [D-Ala2,D-Leu5]enkephalin (DADLE), and yet its delta-selectivity was about 5-times greater than that of DADLE. Thus, Pya-enkephalins per se can be utilized as a fluorescent probe or tracer for the opiate receptor-binding assays.

Alanine↗