Search PubMed⌕ Search

Biomedical subjects

T Costa

Publications and source records attributed to T Costa.

At least 91 records · Page 5Linked to original sources
PubMed1989

Binding characteristics of a series of dimeric tripeptide enkephalins for delta opiate receptors in rat brain and NG108-15 cells.

The N-terminal tripeptide enkephalin analogue, Tyr-D-Ala-Gly, was dimerized at the C-terminus systematically with a series of alpha,omega-diaminoalkanes, NH2-(CH2)n-NH2 (n = 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, and 22). The binding affinities of dimers for delta opiate receptors in rat brain were evaluated and compared with those for delta receptors in NG108-15 cells. Although the monomeric tripeptide amide was almost inactive, dimers showed a dramatic increase in binding affinity (8-900 times). The enhancement of affinity was apparently related to the number of methylene chains in the crosslinking spacer moiety, and it was maximal at n = 14-18 in the rat brain. In NG cells the activity increased progressively from n = 2 to n = 22 without reaching any apparent peak. These results suggest that delta receptors in rat brain and NG cells may have slight structural differences.

Animals↗
PubMed1989

Kluver-Bucy syndrome in systemic lupus erythematosus.

The Kluver-Bucy syndrome has not been previously reported as a complication of systemic lupus erythematosus (SLE). A 48-year-old female is described who sustained several cerebral infarcts, some of which were bitemporal, due to SLE vasculopathy. She developed a complex behavioural picture consisting of global aphasia, left-side neglect, hyperorality, hypermetamorphosis and hypersexuality. She displayed appropriate emotional reaction to visually presented objects, indicating that her Kluver-Bucy syndrome could not be explained by lack of visual recognition.

Aphasia↗
PubMed1989

Direct modulation of voltage-dependent calcium channels by muscarinic activation of a pertussis toxin-sensitive G-protein in hippocampal neurons.

Acetylcholine (Ach) reversibly reduces the high voltage-activated (HVA) calcium (Ca) current in hippocampal neurons. Pretreatment of the cells with pertussis toxin (PTX) abolishes the Ach effect, suggesting that PTX-sensitive GTP-binding regulatory proteins (G-proteins) are involved in the signal transduction mechanism that links Ach receptor activation to inhibition of Ca channel activity. This effect is mimicked by intracellular application of the nonhydrolyzable GTP analog GTP gamma S. Intracellular application of purified G-proteins restored the response to Ach in PTX-treated cells. Furthermore, Ach inhibits the Ca current independently of the presence of cyclic AMP and of the protein kinase C inhibitor H-7 and neither does the Ach effect on the Ca current seem to be correlated to a transient increase in intracellular Ca. Our results suggest that activation of the alpha-subunit of the PTX-sensitive G-protein could directly modulate the HVA Ca channel without involving second messenger systems.

Acetylcholine↗
PubMed1989

Antagonists with negative intrinsic activity at delta opioid receptors coupled to GTP-binding proteins.

According to classical models of drug-receptor interactions, competitive antagonists share with agonists the ability to bind to a common site on the receptor molecule. However, they are different from agonists, as they cannot trigger the "stimulus" that leads to biological responses--i.e., they lack intrinsic activity. For those receptors whose signals are transduced to effector systems by GTP-binding regulatory proteins (G proteins), a mechanistic equivalent of such a stimulus is an increased ability of agonist-bound receptor to accelerate nucleotide exchange and thus GTPase activity on the G-protein molecule. Here we show that for a member of this family of receptors (delta opioid receptors in membranes of NG108-15 neuroblastoma-glioma cells), two types of competitive antagonists can be distinguished. One type has no intrinsic activity, since it neither stimulates nor inhibits the GTPase activity of G proteins and its apparent affinity for the receptor is not altered by pertussis toxin-mediated uncoupling of receptor and G protein. The second type, however, can inhibit GTPase and thus exhibits negative intrinsic activity; its affinity for receptors is increased following uncoupling from G proteins. The existence of antagonists with negative intrinsic activity may be a general feature of several classes of neurotransmitters or hormone receptors and calls for a reevaluation of biological effects produced by competitive antagonists.

Animals↗
PubMed1989

Chronic exposure of NG 108-15 cells to opiate agonists does not alter the amount of the guanine nucleotide-binding proteins Gi and Go.

We have characterized the pertussis toxin substrate in NG 108-15 cell membranes using site-specific antisera and ADP-ribosylation. Cell membranes contain two pertussis toxin-sensitive guanine nucleotide-binding protein alpha-subunits (G alpha) whose Rf values in gel electrophoresis coincide with those of G alpha o and G alpha i2. The total quantity of Gi and Go immunoreactivity amounted to 24.3 +/- 2.8 pmol/mg, whereas only 1.5 +/- 0.2 pmol/mg are capable of undergoing ADP-ribosylation catalyzed by pertussis toxin. Pretreatment of cells with the agonist [D-Ala2,D-Leu2]-enkephalin (DADLE) for 24 h and DADLE or morphine for 72 h did not alter the incorporation of ADP-ribose or the immunoreactive amount of Gi and Go subunits. However, pretreatment for 72 h with naloxone increased the incorporation of ADP-ribose without an apparent change in affinity or in the immunochemically determined protein levels of Gi and Go. This indicates that the process of down-regulation and desensitization of the delta-opioid receptor neither requires quantitative alterations in the levels of Gi and Go nor changes in the degree of coupling among their subunits. In contrast, chronic exposure to antagonists seems to alter the degree of precoupling between alpha- and beta-subunits of Gi and/or Go.

Adenosine Diphosphate Ribose↗
PubMed1989

Opioid receptors of neuroblastoma cells are in two domains of the plasma membrane that differ in content of G proteins.

Opioid receptors of NG 108-15 cell membranes are distributed in two membrane fractions sedimenting at 20,000 g (P2) and 200,000 g(P3). The number of receptors is identical in P2 and P3, but in P2 all sites are present in one high-affinity state (2 nM), whereas in P3 60% of these receptors display lower affinity (150 nM). Upon addition of GTP or pretreatment with pertussis toxin, 80% of the sites exist in low affinity in both P2 and P3. Therefore, the effect of GTP and pertussis toxin on agonist binding appears to be smaller in P2 than in P3. In contrast, sodium inhibits agonist binding in P2 and P3 to the same extent and with identical potency. Opioid-mediated stimulation of GTPase is much greater in P2 than in P3, whereas inhibition of adenylate cyclase does not differ in the two fractions. Using site-specific antibodies and pertussis toxin-catalyzed ADP-ribosylation, we found that the amount of G proteins in P3 is only 30-50% of that in P2. Treatment of intact cells with the hydrophilic protein-modifying agent sulfosuccinimido-biotin results in biotinylation of proteins from both fractions and in a similar reduction of opioid binding in P2 and P3. Likewise, exposure of intact cells to the alkylating opioid antagonist, chlornaltrexamine, produces identical degrees of receptor inactivation in P2 and P3. The rate of in vivo pertussis toxin-mediated modification of G proteins is not different in the two fractions.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Diphosphate Ribose↗
PubMed1989

Enzymatic degradation of GTP and its "stable" analogues produce apparent isomerization of opioid receptors.

GTP reduces the affinity of agonists to opioid receptors in membranes from NG 108-15 cells. This can be demonstrated using a short incubation time in equilibrium binding and in dissociation experiments: GTP decreases equilibrium binding, increases the percentage of fast dissociating binding and produces a conversion of high into low affinity receptors. However, upon prolonged incubation time (more than 60 min) these effects of GTP are gradually lost. This loss of guanine nucleotide effect is also observed with other nucleotides, namely GDP, GTP gamma S and GppNHp. Incubation of radioactive GTP with membranes and subsequent analysis of the guanine nucleotides by TLC (thin layer chromatography) reveals that GTP is rapidly hydrolyzed to GDP and thereupon to the inactive nucleotide GMP and guanosine. The so-called "hydrolysis-resistant" analogues of GTP, GTP gamma S and GppNHp are also degraded under these experimental conditions. The hydrolysis is apparent with membranes from different tissues and occurs with half times of less than 5 min for GTP and of 10 - 30 min for GDP and "stable" GTP-analogues. Conditions that reduce the degradation of guanine nucleotides during incubation with membranes are described.

Animals↗
PubMed1989

Distribution of the alpha-subunit of the guanine nucleotide-binding protein Gi2 and its comparison to G alpha o.

Site specific antisera against a synthetic peptide corresponding to the sequence 3-17 of G alpha 12 have been raised and the specificity examined using purified homogeneous Go, Gi2 and Gi containing a 41 kDa alpha-subunit. The distribution of G alpha i2 was investigated in plasma membranes from different tissues and cells and compared to the distribution of G alpha o and other pertussis toxin sensitive G alpha. Considerable amounts of G alpha i2 were found in endocrine tissue especially in membranes from the adrenal and thyroid, in leucocytes and platelets where it constitutes the major, if not only, pertussis toxin-sensitive G alpha, as well as in some cell lines (C6, NG 108-15, S49 cyc-); erythrocytes contained a 41 kDa G alpha i which was different from G alpha i2. G alpha o was present abundantly in nervous tissue, adrenal medulla and cortex but also found in low amounts in other membranes except for lung, liver and blood cells. Subcellular fractionation of cardiac ventricular muscle demonstrated the presence of G alpha o and low amounts of G alpha i2 in sarcolemma, but only 41kDa G alpha i was present in sarcoplasmic reticulum. The importance of the distinct distribution in terms of signal transduction is discussed.

Animals↗
PubMed1989

Mu and delta opioid receptors inhibit serotonin release in rat hippocampus.

We studied the influence of opioid agonists on the release of serotonin (5-HT) elicited by K+ (20 mM) in superfused slices of rat hippocampus. K+-evoked outflow of serotonin was inhibited significantly up to 50% in the presence of the mu-selective agonist [D-Ala2,N-methyl-Phe4,Gly5-ol]enkephalin (DAGO) and of the delta-selective agonist [D-Pen2,D-Pen5]enkephalin (DPDPE). U50,488H a selective kappa agonist, at concentrations between 0.1 to 1 microM, produced an inhibition of 5-HT-release lower than that observed in the presence of mu and delta agonists. The delta antagonist ICI 174,864 (N,N-diallyl-Tyr1,Aib2,Aib3)Leu-enkephalin potently inhibited the effect of DPDPE but did not affect the inhibition produced by DAGO. In contrast, the mu-selective antagonist D-Phe-Cys-Tyr-D-Trp-Nle-Thr-Pen-Thr-NH2 at 1 microM significantly reversed the inhibitory effect produced by a maximal dose of DAGO (0.1 microM) but not the corresponding effect produced by a maximal dose of DPDPE (1 microM). Naloxone was a competitive antagonist of DAGO but noncompetitive antagonist of DPDPE. Treatment of hippocampal slices with pertussis toxin did not alter the K+-evoked release of 5-HT but abolished the inhibitory effect of both DAGO and DPDPE.

Animals↗
PubMed1988

Pertussis toxin inactivates the presynaptic serotonin autoreceptor in the hippocampus.

The serotonin (5-HT) agonists, D-lysergic acid diethylamide (LSD) and 5-methoxytryptamine (5-Me-O-T), dose dependently inhibited the K+-evoked outflow of [3H]5-HT from preloaded rat hippocampal slices in vitro, indicating activation of the 5-HT autoreceptor. However, this effect was abolished in slices pretreated with pertussis toxin. It is thus concluded that the 5-HT autoreceptor in the hippocampus is coupled to G proteins.

5-Methoxytryptamine↗
PubMed1988

Sodium modulates opioid receptors through a membrane component different from G-proteins. Demonstration by target size analysis.

The target size for opioid receptor binding was studied after manipulations known to affect the interactions between receptor and GTP-binding regulatory proteins (G-proteins). Addition of GTP or its analogs to the binding reaction, exposure of intact cells to pertussis toxin prior to irradiation, or treatment of irradiated membranes with N-ethylmaleimide did not change the target size (approximately equal to 100 kDa) for opioid receptors in NG 108-15 cells and rat brain. These data suggest that the 100-kDa species does not include an active subunit of a G-protein or alternatively that GTP does not promote the dissociation of the receptor-G-protein complex. The presence of Na+ (100 mM) in the radioligand binding assay induced a biphasic decay curve for agonist binding and a flattening of the monoexponential decay curve for a partial agonist. In both cases the effect was explained by an irradiation-induced loss of the low affinity state of the opioid receptor produced by the addition of Na+. This suggests that an allosteric inhibitor that mediates the effect of sodium on the receptor is destroyed at low doses of irradiation, leaving receptors which are no longer regulated by sodium. The effect of Na+ on target size was slightly increased by the simultaneous addition of GTP but was not altered by pertussis toxin treatment. Thus, the sodium unit is distinct from G-proteins and may represent a new component of the opioid receptor complex. Assuming a simple bimolecular model of one Na+ unit/receptor, the size of this inhibitor can be measured as 168 kDa.

Animals↗
PubMed1988

A highly selective ligand for brain delta opiate receptors, a cyclopropyl(E)Phe(4)-enkephalin analog, suppresses mu receptor-mediated thermal analgesia by morphine.

[D-Ala(2)(2R,3S)-delta(E)Phe(4)Leu(5)]enkephalin (CP-OH) [delta denoting cyclopropyl; superscript E indicating the E-configuration about the cyclopropane ring], a highly selective opioid ligand for delta receptors in rat brain, but not for those in the mouse vas deferens, was examined for in vivo biological activities by intracerebroventricular administration. CP-OH (5-20 micrograms) showed no analgesic activity in the hot plate (51 degrees C) test using rats. However, it suppressed completely the analgesic effects of intraperitoneally administered morphine (3 mg/kg rat) in a dose-dependent manner. CP-OH showed no binding affinity for brain kappa receptors to which dynorphin, an opioid peptide that inhibits morphine analgesia, binds predominantly. These results suggest that, besides the conventional delta receptors which mediate analgesia, the rat brain contains another delta-like receptor which has a modulatory role to attenuate morphine-induced analgesia mediated through the mu receptors, and that this modulatory receptor does not exist in the mouse vas deferens.

Analgesia↗
PubMed1988

Synthesis and receptor binding characteristics of [D-Ala2, cysteamine 5] enkephalin, a thiol-containing probe for structural elements of opiate receptors.

For the elucidation of structural elements in the opiate receptors, a thiol-containing enkephalin analog [D-Ala2, cysteamine 5]enkephalin, and its dimeric analog were synthesized and evaluated in the radio-ligand receptor binding assays using rat brain membranes. The dimeric analog was very potent in both delta and mu assays. Comparison of receptor affinities of the thiol-containing enkephalin with those of standard mu or delta receptor specific ligands suggested that the mu receptor contains an essential thiol group which may interact with the thiol group at the C-terminus of the enkephalin analog. It also appears that no metal-ion site, postulated for the delta receptors, is present in the delta binding site.

Animals↗
PubMed1988

Opioid receptors are coupled tightly to G proteins but loosely to adenylate cyclase in NG108-15 cell membranes.

Opioid receptors in intact NG 108-15 cells were irreversibly inactivated with increasing concentrations of the alkylating antagonist beta-chlornaltrexamine (CNA). The consequence of the reduction in density of opioid binding sites (quantified by saturation analysis of opioid binding in membranes) was studied at two steps of opioid receptor-mediated responses, (a) stimulation of high affinity GTPase and (b) inhibition of basal adenylate cyclase. Both agonist-mediated stimulation of GTPase and inhibition of adenylate cyclase activities were progressively reduced as the concentration of CNA in the pretreatment was increased. However, the loss of responsiveness for the two enzymes differed in two aspects. First, the diminution of GTPase responsiveness was in agreement with the loss of binding sites and took place at concentrations of CNA that were lower than those necessary to reduce responsiveness of adenylate cyclase. Second, the loss of responsiveness of GTPase occurred simply as reduction of maximal stimulation, whereas that of adenylate cyclase involved an initial reduction of apparent agonist affinity (10-fold) that was followed by a decrease in maximal effect. We next examined the loss of responsiveness of both GTPase and adenylate cyclase in membranes prepared from cells that had been exposed to increasing concentrations of pertussis toxin (PTX) to inactivate PTX-sensitive G proteins in vivo. Also in this case, the extent of reduction in responsiveness was more pronounced for GTPase than for adenylate cyclase, especially in membranes treated with high concentrations of PTX. However, the pattern of loss was identical for the two enzymes and involved a main reduction in maximal effect of the agonist that was followed only after a large degree of inactivation (greater than 60%) by a diminished apparent affinity for the agonist. Opioid receptor-mediated inhibition of cAMP accumulation in intact cells exhibits an IC50 for the agonist that is 30-10 times lower than that measured in membranes for stimulation of GTPase or inhibition of cyclase, respectively. Treatment of cells with either CNA (1 microM) or various concentrations of PTX altered the concentration-response curves for agonist-mediated inhibition of cAMP accumulation in a manner similar to that observed for adenylate cyclase in membranes, inasmuch as both maximal inhibition and apparent affinities for the agonist were decreased. However, this decrease in affinity (5-fold) was not sufficient to eliminate the discrepancy in agonist potency between membranes and intact cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Diphosphate Ribose↗
PubMed1987

Pertussis toxin abolishes the antinociception mediated by opioid receptors in rat spinal cord.

Intrathecal injection of pertussis toxin (1 microgram) in rats produced a marked decrease in the antinociceptive effect of the intrathecally administered opioid agonists [D-Ala2,D-Leu5]enkephalin, [D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin and bremazocine. The effect of the toxin was time-dependent, since it was more pronounced at 6 than at 2 days after its injection. The pertussis toxin-catalyzed ADP ribosylation of a 40 KDa substrate in membranes prepared from the spinal cord of toxin-injected rats was strongly reduced as compared to controls. The data indicate that the antinociceptive effect produced by opioid agonists with different receptor preference is initiated at receptor sites which interact with G-protein substrates of pertussis toxin.

Adenosine Diphosphate↗
PubMed1987

Differential expression of alpha-subunits of G-proteins in human neuroblastoma-derived cell clones.

The distribution of alpha- and beta-subunits of G-proteins was analyzed in membranes of three cell clones which are derived from the human neuroblastoma cell line SK-N-SH. The neuroblast-like clone SH-SY5Y shows a pattern of G-proteins very similar to that of human brain cortex with high levels of Gi alpha and Go alpha but low levels of G40 alpha. The intermediate clone SH-IN contains high levels of Go alpha and Gi alpha and moderate levels of G40 alpha. The non-neuronal clone SH-EP shows high levels of G40 alpha but lacks Go alpha. Differentiation of the neuroblast-like clone SH-SY5Y by retinoic acid or nerve growth factor does not change the amount of Gi alpha or Go alpha in the membrane.

Cell Differentiation↗