Search

In the present study, we have assessed the effect of opioids (endorphins, enkephalins and neoendorphins) on production of IL-1 activity by bone-marrow-derived macrophages. None of the neuropeptides induced IL-1 production by itself. However, some of the opioids potentiated IL-1 production and release in macrophages concomitantly stimulated by lipopolysaccharide (LPS) or silica. LPS induced predominantly intracellular IL-1 activity, whereas most of the silica-induced IL-1 was released extracellularly. beta-Endorphin, leucine-enkephalin (leu-enkephalin) and beta-neoendorphin all potentiated both intracellular and extracellular release of IL 1 induced by either LPS or silica. In contrast, alpha-endorphin, methionine-enkephalin (metenkephalin) and alpha-neoendorphin did not influence IL-1 production or release. The potentiating effects of beta-endorphin on LPS-induced IL-1 production/secretion were inhibited by naloxone, pointing to an involvement of opioid receptors.

Experiments were conducted (i) to determine the hemodynamic (blood pressure and heart rate) responses of conscious rats following intrathecal (IT) administration of endogenous prodynorphin-derived opioids into the lower thoracic space, (ii) to identify the receptors involved in mediating their cardiovascular responses, and (iii) to reveal any possible hemodynamic interactions with the neuropeptide arginine vasopressin. Male Sprague-Dawley rats were surgically prepared with femoral arterial and venous catheters as well as a spinal catheter (into lower thoracic region, T9-T12). After recovery, hemodynamic responses were observed in conscious rats for 5-10 min after IT injections of artificial cerebrospinal fluid (CSF) solution, prodynorphin-derived opioids (dynorphin A, dynorphin B, dynorphin A (1-13), dynorphin A (1-10), alpha- and beta-neoendorphin, leucine enkephalin (LE), methionine enkephalin (ME), arginine vasopressin (AVP), or norepinephrine (NE)). IT injections of AVP (10 or 20 pmol), dynorphin A (1-13), or dynorphin A (10-20 nmol) caused pressor effects associated with a prolonged and significant bradycardia. Equimolar (20 nmol) concentrations of LE, ME, alpha- and beta-neoendorphin, and dynorphin A (1-10) caused no significant blood pressure or heart rate changes. Combined IT injections of dynorphin A (1-13) and AVP caused apparent additive pressor effects when compared with the same dose of either peptide given alone. IT infusion of the specific AVP-V1 antagonist d(CH2)5Tyr(Me)AVP before subsequent IT AVP, dynorphin A (1-13), or NE administration inhibited only the subsequent pressor responses to AVP. The kappa-opioid antagonist (Mr2266) infused IT blocked the pressor actions of subsequent dynorphin A administration and not AVP or NE.(ABSTRACT TRUNCATED AT 250 WORDS)

The distribution of five major products of proenkephalin B [dynorphin1-17, dynorphin B, dynorphin1-8, alpha-neo-endorphin and beta-neo-endorphin] was studied in regions of rat brain and pituitary. The distribution pattern of immunoreactive (ir) dynorphin B (= rimorphin) was found to be similar to that of ir-dynorphin1-17, with the highest concentrations being present in the posterior pituitary and the hypothalamus. HPLC and gel filtration showed the tridecapeptide dynorphin B to be the predominant immunoreactive species recognized by dynorphin B antibodies in all brain areas and in the posterior pituitary. In addition, two putative common precursor forms of dynorphin B and dynorphin1-17 with apparent molecular weights of 3,200 and 6,000 were detected in brain and the posterior pituitary. The 3,200 dalton species coeluted with dynorphin1-32 on HPLC. In contrast with all other tissues, anterior pituitary ir-dynorphin B and ir-dynorphin1-17 consisted exclusively of the 6,000 dalton species. Concentrations of dynorphin1-8 were several times higher than those of dynorphin1-17 in striatum, thalamus, and midbrain while posterior pituitary, hypothalamus, pons/medulla, and cortex contained roughly equal concentrations of these two opioid peptides. No dynorphin1-8 was detected in the anterior pituitary. Concentrations of beta-neo-endorphin were similar to those of alpha-neo-endorphin in the posterior pituitary. In contrast, in all brain tissues alpha-neo-endorphin was found to be the predominant peptide, with tissue levels in striatum and thalamus almost 20 times higher than those of beta-neo-endorphin. These findings indicate that differential proteolytic processing of proenkephalin B occurs within different regions of brain and pituitary. Moreover, evidence is provided that, in addition to the paired basic amino acids -Lys-Arg- as the "typical" cleavage site for peptide hormone precursors, other cleavage signals also seem to exist for the processing of proenkephalin B.

The distribution of peptides derived from the novel opioid peptide precursor proenkephalin B (prodynorphin) was studied in lobes of the pituitary with antibodies against alpha-neoendorphin (alpha-neo-E) beta-neoE, dynorphin (DYN)-(1-17), DYN-(1-8), and DYN B in combination with gel filtration and high pressure liquid chromatography. In the posterior pituitary, all five opioid peptides occurred in high and about equimolar concentrations, whereas putative precursor peptides were found in only minor quantities. In contrast, in the anterior pituitary immunoreactive (ir-) DYN-(1-17) and ir-DYN B consisted exclusively of a common precursor species with a mol wt of about 6000. Six thousand-dalton DYN may be comprised of the C-terminal portion of proenkephalin B, with the sequence of DYN-(1-17) at its N-terminus. Moreover, the major portions of ir-alpha-neo-E and ir-beta-neoE in the anterior pituitary were found to be of an apparent mol wt of 8000. These findings indicate a differential processing of the opioid peptide precursor proenkephalin B in the two lobes of the pituitary. The anterior pituitary seems to process proenkephalin B predominantly into high mol wt forms of neo-E and DYNs, whereas in the posterior pituitary proenkephalin B undergoes further proteolytic processing to the smaller opioid peptides alpha-neo-E, beta-neo-E, DYN-(1-17), DYN-(1-8), and DYN B. Thus, processing differences may enable the selective liberation of different (opioid) peptides with distinct biological properties from one precursor within different tissues.

The effect of leumorphin (LM), one of big leu-enkephalins derived from preproenkephalin B, on PRL secretion was studied in the rat in vivo and in vitro. Intracerebroventricular injection of synthetic porcine LM (0.06-6 nmol/rat) caused a dose-related increase in plasma PRL levels in urethane-anesthetized male rats and in conscious freely moving rats. Intravenous injection of LM (3 nmol/100 g BW) also raised plasma PRL levels in these animals. The plasma PRL response to intracerebroventricular LM (0.6 nmol/rat) was blunted by naloxone (125 micrograms/100 g BW, iv). The stimulating effect of LM on PRL release was the most potent among the peptides derived from preproenkephalin B. In in vitro studies, PRL release from superfused anterior pituitary cells was stimulated in a dose-related manner by LM (10(-9)-10(-6) M), and the effect was blunted by naloxone (10(-5) M). These results suggest that LM has a potent stimulating effect on PRL secretion from the pituitary in the rat by acting, at least in part, directly at the pituitary through an opiate receptor.

The distribution of immunoreactive (ir)-beta-neo-endorphin in 101 microdissected rat brain and spinal cord regions as well as in the neurointermediate lobe of pituitary gland was determined using a highly specific radioimmunoassay. The highest concentration of beta-neo-endorphin in brain was found in the median eminence (341.4 fmol/mg of protein). High concentrations of ir-beta-neo-endorphin (greater than 250 fmol/mg of protein) were found in 11 nuclei, including dorsomedial nucleus, substantia nigra, parabrachial nuclei, periaqueductal gray matter, anterior hypothalamic nucleus, and lateral preoptic areas. Moderate concentrations of the peptide (between 100 and 250 fmol/mg of protein) were found in 66 brain nuclei such as the amygdaloid and septal nuclei, most of the diencephalic structures (not including the hypothalamus), and the majority of the medulla oblongata nuclei and others. Low concentrations of ir-beta-neo-endorphin (less than 100 fmol/mg of protein) were found in 21 nuclei, e.g., cortical structures (frontal., cingulate, piriform, parietal, entorhinal, occipital), olfactory tubercle, and cerebellum (nuclei and cortex). The olfactory bulb has the lowest beta-neo-endorphin concentration (21.3 fmol/mg of protein). Spinal cord segments exhibit low peptide concentrations. The neurointermediate lobe of the pituitary gland is extremely rich in ir-beta-neo-endorphin.

Endogenous opioids have been implicated as modulators of the central nervous system regulation of blood pressure and heart rate. Whether these neuropeptides participate in blood pressure regulation in hypertension is unknown. To begin to study this question, we examined the response to opiate antagonists and agonists in the spontaneously hypertensive rat (SHR) and the normotensive Wistar-Kyoto (WKY) rat. The long-acting opiate antagonist naltrexone, 2.5 micrograms/kg, was injected into the lateral ventricle of the brain in awake, freely-moving SHR and produced a significant 19 mmHg decrease in mean arterial blood pressure compared to basal blood pressure (p less than 0.01); a decrease was not observed at a two logarithm lower dose. In contrast, naltrexone had no effect on the blood pressure of normotensive Wistar-Kyoto (WKY) rats. To evaluate a possible regulatory role for the predominantly kappa receptor active opioids, alpha- and beta-neo-endorphin, 10 micrograms each, was administered to SHR on separate days by intracerebroventricular injection. alpha- and beta-neo-endorphin caused significant decreases in mean arterial blood pressure of 11 and 9 mmHg respectively, effects reversed by pre-treatment with the opiate antagonist, naloxone. Heart rate was unaffected by any of the injected opioids or antagonists. Our naltrexone results support the hypothesis that an endogenous opioid(s) contributes to the hypertensive state of the SHR. Additionally, alpha- and beta-neo-endorphin can lower blood pressure in this model.

Opioid peptides decrease somatic calcium-dependent action potential duration of a subpopulation of mouse dorsal root ganglion (DRG) neurons grown in dissociated cell culture. Based on rank order of potency and naloxone sensitivity, both mu and delta opioid receptors were demonstrated on the somata of DRG neurons and were shown to have a heterogeneous distribution. The purpose of the present investigation was to determine the actions of dynorphin gene products, dynorphin A, dynorphin B, dynorphin A(1-8), dynorphin A(1-9), alpha-neoendorphin and beta-neoendorphin on DRG neuron somatic calcium-dependent action potentials and to compare the actions of dynorphin and neoendorphin peptides to the action of morphiceptin, a mu receptor-selective ligand, and Leu-enkephalin, a delta receptor-preferring ligand. We report that the dynorphin and neoendorphin peptides decreased DRG neuron somatic calcium-dependent action potential duration in a portion of DRG neurons, an action that was dose-dependent and was antagonized by naloxone. DRG neuron responses to the dynorphins and neoendorphins differed from responses to morphiceptin and Leu-enkephalin. First, many DRG neurons responded to dynorphin A but not to morphiceptin or Leu-enkephalin. Second, dynorphin A responses, unlike responses to morphiceptin or Leu-enkephalin, were present after intracellular injection of cesium, a potassium channel blocker. Dynorphin A effectiveness was decreased after deletions at the carboxy-terminus and Leu-enkephalin [dynorphin A(1-5)] was inactive at 10 microM. Thus, on DRG neurons in cell culture, dynorphins and neoendorphins act at opioid receptors distinct from mu and delta receptors, possibly kappa receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

The choice of opiate receptor subtype by alpha- and beta-neo-endorphin was studied in isolated preparations. Neo-endorphins had significant inhibitory actions on the electrically evoked contractions of guinea-pig ileum, mouse vas deferens and rabbit ileum as well as on the rabbit vas deferens which had been shown to contain kappa-receptors exclusively. Mr 2266, a relatively specific kappa-receptor antagonist, was more effective than naloxone, a relatively mu-receptor antagonist, to antagonize the agonist actions of neo-endorphins in either the guinea-pig and rabbit ileum or in the rabbit vas deferens. By contrast, in the mouse vas deferens, the effectiveness of Mr 2266 to antagonize the agonist actions of neo-endorphins was low and similar to that of naloxone. The potencies of neo-endorphins relative to that of ethylketocyclazocine, a representative kappa-receptor agonist, in the guinea-pig ileum were similar to those in the rabbit ileum but were significant different from those in the mouse vas deferens. The data indicate that neo-endorphins act as kappa-receptor agonists in either the guinea-pig and rabbit ileum or in the rabbit vas deferens while in the mouse vas deferens they act on opiate receptor subtypes other than kappa- and mu-receptors.

Fractionation of Triton-solubilized rat brain membranes on diethylaminoethyl-cellulose resolves two peptidases which hydrolyze beta-neo-endorphin. One of these peptidases was identified as Angiotensin Converting Enzyme by (a) its sensitivity to inhibition by the specific inhibitors MK422 and captopril, (b) by the identification of reaction products, and (c) by comparison to authentic angiotensin converting enzyme. In contrast, alpha-neo-endorphin hydrolysis by angiotensin converting enzyme could not be detected. The second enzyme active on beta-neo-endorphin was identified as an aminopeptidase. This aminopeptidase is identical to the previously described enkephalin-degrading aminopeptidase. The possible involvement of these enzymes in the metabolism of opioid peptides is discussed.

Pro-opiomelanocortin (POMC) related peptides have been studied in rat tissue and plasma, but they have not been well characterized in the rhesus monkey. Since monkey pituitary may be more similar to the human pituitary than the rat, we have characterized POMC related peptides by immunocytochemical, multiple radioimmunoassays (RIA's) and molecular sieving chromatography. Immunocytochemical staining demonstrated N-acetylated- beta-endorphin (N-Ac- beta-End) and alpha-MSH in a few corticotrophs. RIA's of crude anterior pituitary extract and molecular sieving chromatography demonstrates that the major portion is beta-End sized with a significant proportion being N-acetylated and an alpha-MSH peak. Molecular sieving chromatography of extracted plasma demonstrated a similar pattern to that seen in the anterior pituitary. These data suggest that rhesus monkey processes POMC differently than rat or man.

Dehydration significantly reduced the concentration of immunoreactive dynorphin A(1-17), dynorphin A(1-8), alpha-neo-endorphin, beta-neo-endorphin, and leu-enkephalin in the rat pituitary posterior-intermediate lobe. A statistically significant increase in immunoreactive dynorphin A(1-8), alpha-neo-endorphin and leu-enkephalin was observed in the hypothalamus. Comparison of the molar ratios of dynorphin A(1-17): dynorphin A(1-8) and alpha-neo-endorphin: beta-neo-endorphin showed an altered profile of stored pro-dynorphin cleavage products in the posterior-intermediate lobe of the pituitary of dehydrated rats.