Adrenorphin

MDR1 P-glycoprotein is generally regarded as an efflux pump for amphipathic toxic compounds. The question remains, however, whether certain endogenous compounds are also substrates for this transporter. Certain peptides have been shown to interact with MDR1 Pgp as well and we have therefore investigated whether endogenous bioactive peptides are substrates. We demonstrate here that the synthetic mu-opioid peptide DAMGO is a good substrate for MDR1 Pgp. In view of its low interaction with the membrane it is an attractive ligand for measurement of MDR1 Pgp-mediated transport activity in membrane vesicles. Various linear peptides with amidated C-termini were found to inhibit MDR1 Pgp-mediated DAMGO transport. This group includes endogenous opioid peptides such as adrenorphin and endomorphin 1 and 2, as well as the neurokinin, Substance P. The latter bioactive peptides have a relatively high affinity for the transporter. Transport of endomorphin 1 and 2 could be directly demonstrated by the uptake of the radiolabeled opioid peptides in membrane vesicles from MDR1-transfected cells with a K(m) of 15 and 12 microM, respectively. This opens the possibility that MDR1 Pgp is involved in the elimination and/or tissue distribution of these bioactive peptides.

Spatial structure of the peptide hormone adrenorphin was investigated by the theoretical conformational method. A solution of the "reverse conformational problem" for adrenorphin made it possible to predict a series of the modified synthetic analogues, which may assume one of the low-energy conformations of the native hormone.

Background:Alpha (α)-amidation of peptides is a mechanism required for the conversion of prohormones into functional peptide sequences that display biological activities, receptor recognition and signal transduction on target cells. Alpha (α)-amidation occurs in almost all species and amino acids identified in nature. C-terminal valine amide neuropeptides constitute the smallest group of functional peptide compounds identified in neurosecretory structures in vertebrate and invertebrate species.Methods:The α-amidated isoform of valine residue (Val-CONH2) was conjugated to KLH-protein carrier and used to immunize mice. Hyperimmune animals displaying high titers of valine amide antisera were used to generate stable hybridoma-secreting mAbs. Three productive hybridoma (P15A4, P17C11, and P18C5) were tested against peptides antigens containing both the C-terminal α-amidated (-CONH2) and free α-carboxylic acid (-COO(-)) isovariant of the valine residue.Results:P18C5 mAb displayed the highest specificity and selectivity against C-terminal valine amidated peptide antigens in different immunoassays. P18C5 mAb-immunoreactivity exhibited a wide distribution along the neuroaxis of the rat brain, particularly in brain areas that did not cross-match with the neuronal distribution of known valine amide neuropeptides (α-MSH, adrenorphin, secretin, UCN1-2). These brain regions varied in the relative amount of putative novel valine amide peptide immunoreactive material (nmol/μg protein) estimated through a fmol-sensitive solid-phase radioimmunoassay (RIA) raised for P18C5 mAb.Conclusions:Our results demonstrate the versatility of a single mAb able to differentiate between two structural subdomains of a single amino acid. This mAb offers a wide spectrum of potential applications in research and medicine, whose uses may extend from a biological reagent (used to detect valine amidated peptide substances in fluids and tissues) to a detoxifying reagent (used to neutralize exogenous toxic amide peptide compounds) or as a specific immunoreagent in immunotherapy settings (used to reduce tumor growth and tumorigenesis) among many others.

Biologically active peptide hormones and neurotransmitters have been shown to be enzymatically liberated from larger, inactive precursor molecules by tissue-specific post-translational processing, particularly at the typical cleavage signals of paired basic residues. Subsequent N-terminal or C-terminal modifications may be of importance in regulating the biological activities of these peptides. C-terminal alpha-amidation is considered to be essential for the biological function of several non-opioid peptides. Here we present the isolation and structure of a novel C-terminally amidated opioid peptide, amidorphin, from bovine adrenal medulla. Amidorphin and the recently isolated octapeptide metorphamide (adrenorphin) are the only endogenous opioid peptides in mammals known to possess a C-terminal amide group. The amino acid sequence of amidorphin corresponds to the sequence 104-129 of bovine proenkephalin A. Very high concentrations of amidorphin were detected in bovine adrenal medulla and in a further endocrinological system, the hypothalamic-neurohypophyseal axis. Amidorphin may therefore be considered to be a major gene product of the opioid peptide precursor proenkephalin A in these endocrine tissues.

A metalloprotease has been identified in ovine chromaffin granules which cleaves the proenkephalin fragment BAM12P to produce adrenorphin-Gly. This cleavage occurs at a single arginine residue and is an intermediate step in the production of the opiate adrenorphin in vivo. The identity of the product was confirmed by reverse-phase and ion-exchange chromatography. The adrenorphin-Gly-generating enzyme (AGE) was determined by chromatofocusing to have a pI value of 5.2 and bound strongly to a metal-chelate affinity column. After purification by gel-filtration and ion-exchange chromatography AGE was free of contaminating activities, as cleavage of radiolabelled BAM12P generated a single product as judged by reverse-phase and ion-exchange chromatography. The enzyme has a molecular mass of approx. 45 kDa and a pH optimum of 8.6 in Mops, Taps and Hepes buffers, but was inhibited by phosphate buffers. It was inhibited by micromolar concentrations of copper and zinc ions, but not by millimolar concentrations of calcium or manganese ions. The addition of BAM22P, dynorphin 1-13 or dynorphin 1-8 to the incubation mixture inhibited the cleavage of radiolabelled BAM12P. The cleavage was also inhibited by the presence of catecholamines at concentrations similar to those found within the chromaffin granule. This may explain the known effect of reserpine on chromaffin cells of reducing catecholamine levels and simultaneously increasing adrenorphin levels. It may also indicate a function for AGE and adrenorphin as reporters of intragranular conditions.

The distribution of immunoreactive (ir)-metorphamide (adrenorphin) in 101 microdissected rat brain and spinal cord regions was determined using a highly specific radioimmunoassay. The highest concentration of metorphamide in brain was found in globus pallidus (280.1 fmol/mg protein). High concentrations of ir-metorphamide (greater than 120 fmol/mg protein) were found in 9 nuclei, including central amygdaloid nucleus, lateral preoptic area, anterior hypothalamic nucleus, hypothalamic paraventricular nucleus, interpeduncular nucleus, periaqueductal grey matter and nucleus of the solitary tract. Moderate concentrations of the peptide (between 60 and 120 fmol/mg protein) were found in 47 brain nuclei such as nucleus accumbens, bed nucleus of stria terminalis, several septal and amygdaloid nuclei, most of the hypothalamic nuclei, ventral tegmental area, red nucleus, raphe nuclei, lateral reticular nucleus, area postrema and others. Low concentrations or ir-metorphamide (less than 60 fmol/mg protein) were measured in 41 nuclei, e.g., cortical structures, hippocampus, caudate nucleus, thalamic nuclei, supraoptic nucleus, substantia nigra, vestibular nuclei, cerebellum (nuclei and cortex). The olfactory bulb has the lowest metorphamide concentration (5.8 fmol/mg protein). Spinal cord segments exhibit very low peptide concentrations.

We studied the secretion and tissue contents of adrenorphin in human pheochromocytomas. In 17 human pheochromocytomas from 11 patients, we found a remarkably wide distribution in immunoreactive adrenorphin levels (3-7771 pg/mg tissue). Adrenomedullary pheochromocytomas contained a significantly larger amount of immunoreactive adrenorphin (2295 +/- 1092 pg/mg, mean +/- SE) than did extramedullary ones (17.8 +/- 8.4 pg/mg). Gel chromatographic studies revealed that immunoreactive adrenorphin consisted largely of material emerging at the position of synthetic adrenorphin in both pheochromocytoma and normal adrenal medulla tissue. Nicotine (10(-5) M) significantly stimulated the secretion of immunoreactive adrenorphin as well as catecholamines from cultured human pheochromocytoma cells. Adrenorphin was a more potent inhibitor of catecholamine secretion evoked by 10(-5) M nicotine than was met-enkephalin in cultured human pheochromocytoma cells. The 50% inhibitory concentrations (IC50) were 1.1 X 10(-6) and 6.5 X 10(-5) M for adrenorphin and met-enkephalin, respectively. The effect of adrenorphin was much the same as that of dynorphin-(1-13) (IC50, 1.0 X 10(-6) M) and BAM-12P (IC50, 4.5 X 10(-6) M). These results indicate the presence and secretion of adrenorphin in human pheochromocytomas. Adrenorphin may play an important role in regulating catecholamine secretion in human pheochromocytoma.

Adrenorphin is the first C-terminally amidated form of opioid peptides isolated from human pheochromocytoma tumor and is considered to be generated out of proenkephalin A by unique processing. By the highly specific and sensitive radioimmunoassay (RIA) procedure utilizing the antiserum against adrenorphin, combined with high performance liquid chromatography (HPLC), immunoreactive adrenorphin in rat brain was verified to be identical with its authentic peptide. It has been revealed that adrenorphin immunoreactivity distributes widely in rat brain but in the unique pattern distinct from those of other endogenous opioid peptides. Note that immunoreactive adrenorphin was most concentrated in the olfactory bulb, and appreciably in the hypothalamus and striatum. Furthermore, immunohistochemical study has revealed that adrenorphin-immunoreactive structures in hypothalamic region of rat were localized in the neurones of the arcuate nucleus. In addition, adrenorphin-immunoreactive fibre plexus was found in the various regions of the hypothalamus, such as median eminence, periventricular zone and paraventricular nucleus. These indicate that adrenorphin may have a unique physiological function.

Adrenorphin is the first C-terminally amidated form of opioid peptide isolated from human pheochromocytoma tumor and is considered to be generated out of proenkephalin A by unique processing. We have developed a highly specific and sensitive radioimmunoassay for adrenorphin as well as for PH- 8P , whose structure and processing are similar to adrenorphin . Prior to the measurement of both peptides in rat brain, immunoreactive adrenorphin and PH- 8P were verified to be identical with their individual authentic peptides by high performance liquid chromatography. Here we have determined the distribution of adrenorphin in rat brain by radioimmunoassay, and compared it with that of PH- 8P . The regional distribution of adrenorphin was found to be quite different from that of other endogenous opioid peptides including PH- 8P . The highest concentration of adrenorphin was found in the olfactory bulb. These results suggest that adrenorphin is generated by a specific processing mechanism and may have a unique physiological function distinct from that of known opioid peptides. In addition, we identified adrenorphin also in human and bovine adrenal medullas.