Fred A. Opmeer

Behavioral profile of ψ-MSH: Relationship with acta and β-endorphin action

Abstract In view of the close structural similarity of the pro-opiocortin fragment γ-MSH and of ACTH/MSH type peptides, the behavioral profile of γ-MSH was explored. Attention was first focussed on behavioral procedures in which ACTH/MSH related neuropeptides have been found effective. Using different procedures to test avoidance behavior, it was found that γ-MSH and ACTH-like neuropeptides had opposite effects. In this respect the activity of γ-MSH resembles that of opiate antagonists rather than that of β-endorphin. Accordingly, ACTH 1–24 -induced excessive grooming which is blocked by opiate antagonists, is attenuated by γ-MSH. In addition, γ-MSH injected into the periaqueductal gray matter of the brainstem of opiate naive rats elicited symptoms reminiscent of those seen after opiate withdrawal. γ-MSH attenuated more or less several effects of intracerebroventricularly administered β-endorphin (e.g. antinociception, hypothermia, α-MSH release) and decreased acquisition of heroin self-administration. Although γ-MSH at rather high doses displaced naloxone from its specific binding sites in brain homogenates, it did not interfere with β-endorphin-induced effects on in vitro muscle preparations (guinea pig ileum, rat rectum). Interestingly, γ-MSH induced relaxation of the rat rectum in vitro . It is postulated that γ-MSH may attenuate β-endorphin-induced effects by acting via γ-MSH receptor sites (functional antagonism), although a pharmacological antagonism cannot be excluded as yet.

ACTH-Induced lipolysis in rat adipocytes: Structure-activity relationships

SummaryThe lipolytic action of natural porcine ACTH1–39 and of a number of highly purified synthetic ACTH peptide fragments was studied using rat adipocytes. Of the analogues tested, only ACTH1–24 exhibited full lipolytic activity with respect to intrinsic activity and affinity. Several shorter fragments appeared to be full agonists but had lower affinity. Fragments ACTH5–10 and ACTH7–10 were inactive. No antagonistic effects against the lipolytic action of ACTH could be demonstrated with substimulatory doses of ACTH1–16, ACTH1–10, ACTH7–24 and ACTH11–24. Based on the relative potency derived from dose-response curves, a more refined model with respect to the active centers being encoded in various sequences of the hormone, is proposed.

Altered levels of β-endorphin fragments after chronic morphine treatment of guinea-pig ileum in vivo and in vivo

Abstract The isolated myenteric plexus-longitudinal muscle of the guinea-pig ilem (GPI) was used as testsystem to study the influence of chronic morphine treatment on the levels of enkephalins, β-endorphin and some of its fragments. The peptides were assayed by means of a combination of high pressure liquid chromatography and radioimmunoassays. It was found that the levels of methionine- and leucine-enkephalin and β-endorphin were not altered by chronic morphine treatment of guinea-pigs in vivo nor in GPI exposed to morphine in vitro . However, the levels of some β-endorphin fragments i.c. γ-endorphin and des-tyrosine-γ-endorphin were elevated after morphine treatment in vitro and in vivo respectively. It is suggested that β-endorphin and its fragments are involved in homeostatic processes during development of opiate tolerance.

Quantification of the in vitro induced tolerance to morphine of the isolated guinea pig ileum.

Abstract Exposure of isolated ileum segments to morphine (0.8 – 80 μM) for 18 – 22 h results in development of a dose-related state of tolerance as measured by reduced ability of morphine to suppress electrically evoked contractions of strips prepared from the segments. Subsequent to high frequency stimulation (HSF) contractions of tolerant strips showed increased inhibition as compared to control strips. This enhanced inhibition appeared to be related to the dose of morphine, to which the ileum had been exposed. It was completely antagonized by naloxone, and gradually disappeared as was observed for the diminished response to morphine. It is concluded that the inhibition subsequent to HFS can reliably be used to quantify the degree of in vitro tolerance.

β-endorphin proteolysis by guinea-pig ileum myenteric plexus membranes: Increased γ-endorphin turnover after chronic exposure to morphine

The influence of chronic morphine exposure in vitro on the biotransformation of beta-endorphin (beta E) was investigated using the myenteric plexus-longitudinal muscle of guinea-pig ileum. A membrane preparation was incubated with beta E and the degradation of beta E as well as the accumulation of several beta E fragments in the incubation medium were followed with time. The levels of peptides were determined by specific radioimmunoassays after separation by high-pressure liquid chromatography. It was found that exposure to morphine did not affect the disappearance of beta E, but altered the time course of accumulation of beta E fragments. In fact, the accumulation of gamma-endorphin, alpha-endorphin and des-tyrosine1-alpha-endorphin was enhanced, while that of des-tyrosine1-gamma-endorphin was not change. Additionally, the disappearance of gamma-endorphin appeared to be stimulated by morphine exposure. These data provide evidence that the fragmentation of beta E is changed by chronic morphine exposure in such a way that the turnover of gamma-endorphin is increased.

Interaction of calcium with3H-morphine binding to synaptosomes of the guinea-pig ileum

SummarySpecific binding sites for3H-morphine were assayed in subcellular fractions of a crude mitochondrial P2 pellet from the guinea-pig ileum longitudinal muscle-myenteric plexus, prepared by discontinuous sucrose-gradient fractionation. The highest specific binding (ca. 100 fmol/mg protein) was obtained in the fraction containing synaptosomes, as examined by electron microscopy. A synaptosomal fraction prepared from guinea-pig brain had comparable specific binding (ca. 130 fmol/mg) to that of the ileal synaptosomal fraction. Addition of calcium (10 mM) to the binding assay medium resulted in a marked decrease in particular of the specific3H-morphine binding. Detailed analysis of the specific3H-morphine binding in the synaptosomal fraction of the ileum revealed that 1) a saturable component of specific opiate binding was present between 0.34 and 21.74 nM of3H-morphine; 2) in the presence of 3 and 10 nM of calcium similar decreases of specific3H-morphine binding were obtained, indicating that this binding was maximally inhibited already by 3 mM of calcium; 3) both in the absence and presence of calcium theKD of specific3H-morphine binding was about 38 nM, indicating a non-competitive nature of the calcium inhibition; 4) addition of magnesium exhibited a similar effect as that of calcium, although magnesium appeared to be less potent than calcium in this respect. The data are discussed in the context of previously observed calcium effects on opioid actions in the electrically stimulated guinea-pig ileum bioassay and may contribute to the evidence that the interaction of calcium and opioids on the stimulus-release coupling mechanism at the neuromuscular junction of the guinea-pig ileum is occurring beyond opioid receptor activation as well.