Direct action of opiates on bromocriptine-inhibited prolactin release by human prolactinoma cells in primary culture.

The present study was undertaken in order to examine the existence of opioid binding sites on cell membranes of human PRL-secreting tumors. Determination of opioid binding sites using different opiate ligands revealed one class of high affinity (KD, 1.3 nM) binding sites. Pharmacological characterization revealed kappa-1 selectivity (high affinity ethylketocyclazocine (EKC) binding, insensitive to 5 microM (D-Ala2, D-Leu5]enkephalin). Subsequently EKC was added to hPRL-secreting tumor cells in primary culture, alone or in combination with the dopaminergic agonist bromocriptine, and PRL release was measured. Opiates had no direct effect on PRL release by prolactinoma cells. When cells were preincubated with bromocriptine [6.6 +/- 4.8 (SD) X 10(-11) M], EKC (10(-11) to 10(-9) M) antagonized, in a dose-dependent manner, the dopaminergic inhibition of PRL release. The opiate effect was reversed by the opiate antagonist diprenorphine (10(-7) M). Cross-competition studies indicated that this effect was not due to the interaction of opiates with the dopaminergic receptor. In conclusion, opioid binding sites are found on prolactinoma cells. The binding of kappa-1 type opioid ligands modulates the inhibitory effect of dopamine upon PRL release.

Reassessment of opioid binding sites in the rat brain.

Opioid binding sites have been characterized pharmacologically in membranes from different areas of the rat brain. Delta, mu and sites belonging to the kappa family (K1, K2, K3) have been detected. Delta sites were more abundant in cortex and striatum, mu sites in striatum and hypothalamus, while kappa binding site concentration was higher in deeper enkephalic structures (brainstem, cerebellum, hypothalamus) and the pituitary gland. A distinct distribution of each subtype of the kappa site was found: kappa 1 sites were higher in the spinal cord, kappa 2 sites in the brainstem and kappa 3 sites in cerebellum. The distribution of delta and kappa sites in the central nervous system was correlated with the distribution of proenkephalin-A derived peptides and precursors, suggesting that these peptides could be their endogenous ligands.

Characterization of enkephalins and related peptides in rat hypophysial portal blood.

Rat hypophysial portal blood, collected from the pituitary stalk, was extracted and enkephalins were assayed by different RIA. Met-Enk-IR and Leu-Enk-IR levels were 1635 +/- 470 pg/ml and 125 +/- 50 pg/ml, respectively. Using HPLC characterization, the presence in portal blood of Met-Enk, Leu-Enk, proenkephalins fragments and dynorphin1-17 has been demonstrated. An unidentified Met-Enk-IR peptide has also been found.

Heterotrimeric configuration is essential to the adhesive function of laminin.

Mouse PFHR9 laminin, B1B2-heterodimers, and free B1-chains were separated from one another by gel filtration on Superose 6. The cell attachment promoting activity of these species was measured after immunoprecipitation with monoclonal anti-laminin antibodies coupled to Sepharose 6MB beads. These antibodies, which did not react with the laminin E8 fragment, were directed against epitopes in the NH2-terminus of the laminin B1-chain and in the central region of laminin. After incubation with purified EHS laminin, the immunosorbents revealed efficient adhesion substrates for a rat rhabdomyosarcoma cell line which attached preferentially to the laminin E8 fragment. Although both were immunoprecipitated efficiently, B1B2-heterodimers and B1-chains, unlike PFHR9 laminin, did not support the attachment of RMS cells. On a molar basis B1B2-heterodimers were 24 times less efficient than PFHR9 laminin or EHS laminin in supporting cell attachment. These data suggest that heterotrimeric configuration is essential to the adhesive function of the laminin E8 fragment.

Comparative thermodynamics of opioid receptor ligand interaction in the bovine adrenal medulla membranes--evidence of opioid site heterogeneity.

1. A marked dependence on temperature of agonist binding delta, mu and kappa 1-3 opioid sites in the bovine adrenal medulla was observed, at the range of 0 to 37 degrees C. These changes concern kinetic (k1) and equilibrium constants (Kd), but not binding capacities (Bmax). 2. These dependences are different for each ligand and each opioid receptor, suggesting their molecular heterogeneity. 3. The comparative thermodynamics indicates that the interaction of opioid agonists with their receptor is exergonic (delta G degree < 0) and entropy driven (delta S degree > 0). 4. The comparison of Van't Hoff and Arrhenius plots indicates a discrete mechanism in the binding of each opioid receptor.

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.

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.

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.

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.

[Direct interaction of tricyclic antidepressants with opiate binding sites in the bovine adrenal medulla]. / Les antidépresseurs tricycliques interagissent directement avec les sites de liaison opiacés dans la médullosurrénale bovine.

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.

Crosslinking of mammalian lectin (galectin-1) by complex biantennary saccharides.

Galectins are beta-galactoside-binding proteins that occur intra- and extracellularly in many animal tissues. They have been proposed to form networks of glycoconjugates on the cell surface, where they may modulate various cell response pathways such as growth, activation and adhesion. The high resolution X-ray crystallographic analyses of three crystal forms of bovine galectin-1 in complex with biantennary saccharides of N-acetyllactosamine type reveal infinite chains of lectin dimers cross-linked through N-acetyllactosamine units located at the end of the oligosaccharide antenna. The oligosaccharide adopts a different low energy conformation in each of the three crystal forms.