morphinans and Pheochromocytoma

We studied the binding of [3H]D-Ala2-D-Leu5-enkephalin ([3H]DADLE) and [3H] diprenorphine to crude plasma membrane fraction obtained from the bovine adrenal medulla (bovine adrenal medullary membranes) in order to characterize adrenal medullary opioid receptors. The [3H] diprenorphine binding was the highest in crude plasma membrane-mitochondrial fraction among all subcellular fractions studied. The amount of [3H] diprenorphine bound to bovine adrenal medullary membranes was proportional to the protein concentration. Association kinetics of the [3H] diprenorphine binding to bovine adrenal medullary membranes showed that the maximal binding was achieved following 8 min incubation and that the binding conformed the second-order kinetics. [3H] DADLE and [3H] diprenorphine bound to bovine adrenal medullary membranes with high affinities. The Kd and Bmax for the [3H] DADLE binding were found to be 2.9 nM and 57.5 fmole/mg protein, respectively, while those for the [3H] diprenorphine binding were 0.31 nM and 250 fmole/mg protein, respectively. Displacement studies showed that the [3H] diprenorphine binding was inhibited dose-dependently by levorphanol, dynorphin (1-13), beta-endorphin and DADLE. Levorphanol was at least 1000-fold more potent to inhibit the [3H] diprenorphine binding than dextrorphan, indicating stereospecificity of the [3H] diprenorphine binding. Na+, Li+ and K+ (100 mM) diminished the [3H] DADLE binding and enhanced [3H] diprenorphine binding. Na+ (100 mM) increased the Kd value for the [3H] DADLE binding from 2.9 nM to 14.1 nM. Mn++, Ca++ and Mg++ diminished the [3H] diprenorphine binding. Mn++ (1 mM) increased the Bmax value for the [3H] DADLE binding from 95 fmole/mg protein to 450 fmole/mg protein. These effects of Na+ and Mn++ on the [3H] diprenorphine binding were found to be dose-dependent. [3H] Diprenorphine binding to the digitonin-solubilized opioid receptor was also inhibited dose-dependently by Mn++. These results suggest that bovine adrenal medullary membranes contain high affinity and stereospecific opioid receptors and that the binding of opioids to the bovine adrenal medullary opioid receptors is influenced by cations. Binding study also revealed the presence of opioid receptors in human malignant pheochromocytoma. The Kd and Bmax of the [3H] diprenorphine binding to crude membrane fraction obtained from malignant pheochromocytoma were found to be 0.14 nM and 10.4 fmole/mg protein, respectively.

Topics: Adrenal Medulla; Animals; Cattle; Diprenorphine; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Humans; In Vitro Techniques; Metals; Morphinans; Pheochromocytoma; Receptors, Opioid; Tritium