Tommaso Costa

Opiate receptor binding sites in human spinal cord

Opiate receptor binding sites were analyzed in various regions of human spinal cord and compared to results obtained in spinal cord and brain of certain animals. mu-, delta- and kappa binding sites were individually monitored by the overall labeling of opiate binding sites with [3H]diprenorphine followed by the sequential elimination of binding to particular sites by the use of selective ligands. kappa-Receptors were the predominant type (approximately 50%), followed by mu-receptors (approximately 40%), and, in rather small amounts, delta-receptors. A similar proportion of receptor types was found in the spinal cord of guinea pigs.

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.

Receptor binding and biological activity of bivalent enkephalins

Two series of dimeric enkephalin analogues were assayed for opioid activity in two isolated smooth muscle preparations: the guinea pig ileum (GPI) and the mouse vas deferens (MVD). Dimers have the general structure: X-(CH2)n-X, where X is H-Tyr-D-Ala-Gly-Phe-Leu-NH-(n = 0, 2, 4, 6, 8, 10, 12), for the first series of dimeric pentapeptide enkephalins (DPEn), and H-Tyr-D-Ala-Gly-Phe-NH-(n = 2, 4, 6, 8, 12), for the series of dimeric tetrapeptide enkephalins (DTEn). Comparison of biological activities with binding affinities revealed that: (1) the DPE series with n = 2-8 showed increased potency in the MVD assay relative to monomeric [D-Ala2, Leu5]enkephalinamide (DALEA); (2) there was an associated increase affinity for the delta receptor of rat brain or neuroblastoma-glioma hybrid cells. (however, the relative potencies were higher in the MVD assay then predicted on the basis of binding affinities); (3) the DTE series also showed an increase in delta receptor affinities and MVD potencies relative to DALEA, for n = 2-12; (4) for the DTE series, the increase in MVD activities was less than that expected on the basis of delta binding affinity; (5) for both the DPE and DTE series, activities in the GPI assay and mu-receptor affinities were highly correlated: as the length of the methylene bridge increased from 2 to 12, there was a progressive loss of activity in both assays, with a similar pattern for DPE and DTE. Two selected dimers and their corresponding monomers were also assayed for antinociceptive activity in vivo: results were consistent with GPI and mu-binding but not with MVD and delta-binding. Two alkylamide analogs of penta- and tetrapeptide monomers, representing the monomer with the attached spacer of the most active dimers, were also assayed in biological and binding assays. Comparison of these compounds with the corresponding dimers suggest that the changes in activities and selectivities induced by dimerization are not a spurious effect of the presence of an akylamide derivative of the carboxy terminal of enkephalin but rather may represent a specific effect due to the bivalent nature of the ligands.

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.

▽EPhe4‐enkephalin analogs Delta receptors in rat brain are different from those in mouse vas deferens

Conformationally restricted enkephalin analogs containing E‐cyclopropylphenylalanine (▽ E Phe), [D‐Ala2, (2R,3S)‐▽ E Phe4,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 δ and a very weak affinity for the μ receptors in rat brain. The extent of δ 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 δ receptors in rat brain, but not with those in the mouse vas deferens, and they suggest that the δ receptors in the central and peripheral nervous systems are different from each other.

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

The distribution of α‐ and β‐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 Giα and Goα but low levels of G40α. The intermediate clone SH‐IN contains high levels of Goα and Giα and moderate levels of G40α. The non‐neuronal clone SH‐EP shows high levels of G40α but lacks Goα. Differentiation of the neuroblast‐like clone SH‐SY5Y by retinoic acid or nerve growth factor does not change the amount of Giα or Goα in the membrane.

Cholera toxin ADP-ribosylates the receptor-coupled form of pertussis toxin-sensitive G-proteins

Cholera toxin catalyzes the ADP-ribosylation of 40 kDa pertussis toxin substrates in membranes from NG108-15 cells, which is increased in the presence of the opioid agonist DADLE. The basal ADP-ribosylation can be abolished by the opioid antagonist ICI 174864, suggesting that unoccupied opioid receptors interact spontaneously with the pertussis toxin substrates Gi/Go in the membrane. Treatment of NG108-15 cells with the opioid agonist DADLE leads to a reduction of agonist-stimulated and basal ADP-ribosylation of 40 kDa substrates catalyzed by cholera toxin. This indicates that the spontaneous interaction between opioid receptors and G-proteins is decreased in membranes of cells in which the receptor was desensitized by prolonged exposure to the agonist.

A highly selective ligand for brain δ opiate receptors, a ▿ EPhe4‐enkephalin analog, suppresses μ receptor‐mediated thermal analgesia by morphine

[D‐Ala2,(2R,3S)‐▿ E Phe4,Leu5]enkephalin (CP‐OH)[ ▿denoting cyclopropyl; superscript E indicating the E‐configuration about the cyclopropane ring], a highly selective opioid ligand for δ 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 μg) showed no analgesic activity in the hot plate (51°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 κ receptors to which dynorphin, an opioid peptide that inhibits morphine analgesia, binds predominantly. These results suggest that, besides the conventional δ receptors which mediate analgesia, the rat brain contains another δ‐like receptor which has a modulatory role to attenuate morphine‐induced analgesia mediated through the μ receptors, and that this modulatory receptor does not exist in the mouse vas deferens.

Sodium regulation of opioid agonist binding is potentiated by pertussis toxin

Pretreatment of intact NG108-15 cells with pertussis toxin suppresses opioid inhibition of cyclic AMP accumulation mediated by the inhibitory guanine nucleotide-binding regulatory protein, Ni, which apparently also mediates the inhibitory nucleotide effects on opioid against binding. The toxin treatment had no effect on opioid agonist binding measured in NG108-15 cell membranes without sodium present. However, the toxin potentiated the inhibitory effect of sodium on agonist binding, leading to an agonist-specific reduction of opioid receptor affinity in the presence of sodium in the binding reaction. The potency of the stable GTP analog, GTP gamma S, to reduce agonist binding in the presence of sodium was little changed in membranes prepared from pertussis toxin-treated cells compared to control membranes, whereas the potency of the stable GDP analog, GDP beta S, was magnified. The data indicate that ADP-ribosylation of Ni by pertussis toxin potentiates sodium regulation of opioid agonist binding and that the communication between Ni and opioid receptors is not lost by the covalent modification of Ni.

Guanine nucleotide-mediated inhibition of opioid agonist binding: modulatory effects of ions and of receptor occupancy

We have analysed the potency of GTP, GDP and their analogues in reducing [3H]DADLE binding to opioid receptors in NG 108-15 cell membranes. Under conditions where non-specific hydrolysis and transphosphorylation is inhibited, the following rank order of potency was found: GDP greater than or equal to GTP gamma S greater than GTP greater than GDP beta S greater than or equal to GDPNH2 greater than GppNHp much greater than GMP. Remarkably, the slopes for the inhibition curves of GTP, GDP and their thiosubstituted analogues, but not of GDPNH2 and GppNHp, were extremely shallow, indicating either negative cooperativity or the existence of two states for the guanine nucleotide binding proteins, that both can mediate the effect of nucleotides on agonist receptor binding. The potencies of the different guanine nucleotide analogues, except that of GppNHp, were increased by the presence of sodium or chloride ions in the assay medium. Magnesium also affected GTP-mediated inhibition of opioid agonist binding since it decreased the IC50 of the nucleotide and steepened the slope of the inhibition curve. The IC50s of nucleotides and the slopes of their inhibition curves were also dependent on the extent of receptor occupancy by the agonist. From these data we conclude that (1) either diphospho- or triphosphonucleotides can regulate agonist binding. (2) Magnesium, sodium and chloride, by acting at different components of the receptor/G protein complex produce similar effects on nucleotide mediated regulation of agonist binding. (3) A mutual influence exists between receptor occupancy by agonists and G protein-mediated guanine nucleotide effect on the receptor.