Multiple cleavage sites of cholecystokinin heptapeptide by "enkephalinase"

Degradation of Boc CCK7 (Boc Tyr1 (SO3H)-Met2-Gly3-Trp4-Met5-Asp6-Phe7-NH2), a fully active analog of CCK8, by purified rabbit kidney neutral metalloendopeptidase (enkephalinase) was studied as a basis for the rational design of potent peptidases-resistant analogs of cholecystokinin. Characterization of the metabolites was performed by HPLC using several elution procedures. Three cleavage sites were evidenced: one major at the Asp6-Phe7 bond and two minor at Gly3-Trp4 and Trp4-Met5 bonds. All cleavages were fully inhibited by thiorphan, a potent inhibitor of enkephalinase. The relative importance of the different cleavages was established using several cholecystokinin analogs. At 25 degrees C the half-disappearance time was 18 min for Boc CCK7, Boc[diNle2,5]CCK7 and 70 min for Boc[diNle2,5 D.Asp6]CCK7. Although, half-life of Boc CCK7 and Boc[diNle2,5]CCK7 were identical, the replacement of Met by Nle, a more hydrophobic aminoacid, greatly favoured the cleavage at the Trp4-Nle5 bond which became the major breakdown. This feature was exemplified by the substitution of L.Asp by D.Asp, preventing the Trp4-Nle5 cleavage, which gave rise to the most enkephalinase-resistant analog in this series.     

Competitive antagonists of bradykinin

The first sequence-related competitive inhibitors of the classic kinin in vitro (rat uterus guinea pig ileum) and in vivo (rat blood pressure) assays have been developed. Replacement of the proline residue at position 7 of bradykinin (BK) with a D-phenylalanine residue is the key modification which converts BK agonists into antagonists. [D-Phe7]-BK exhibits moderate (pA2 = 5.0) inhibition of BK activity on the guinea pig ileum but possesses weak BK-like myotropic activity on the isolated rat uterus and 2-4% of BK depressor potency in the rat blood pressure assay. The additional replacement of the phenylalanine residues at positions 5 and 8 of [D-Phe7]-BK with the isosteric beta-(2-thienyl)-alanine residue produces a potent antagonist of BK activity on the uterus (pA2 = 6.4), ileum (pA2 = 6.3), and in the rat blood pressure assay. The antagonism of BK action on smooth muscle is specific for kinins (BK, kallidin, Met-Lys-BK), but neither inhibitor antagonizes the smooth muscle activity of angiotensin or substance P. Inhibition is competitive and fully reversible.     

Effect of DN-1417, a thyrotropin releasing hormone analog, on dopaminergic neurons in rat brain

Acute and chronic effects of γ-butyrolactone-γ-carbonyl-histidyl-prolinamide (DN-1417) were investigated on motor activity, dopamine (DA) metabolites and DA receptors in various brain regions of rats. The motor activity, as measured with Automex recorder, was enhanced after a single injection with DN-1417 (20 mg/kg, IP), and the motor stimulating action persisted during 21 daily injections. Acute DN-1417 elevated both homovanillic acid (HVA) and 3,4-dihydroxyphenylacetic acid (DOPAC) levels in 7 brain regions, prefrontal cortex polar, medial and lateral fields, nucleus accumbens, olfactory tubercles, amygdala and striatum. After chronic treatment for 7 days, the acute effect of DN-1417 on DA metabolites disappeared in all regions except for the striatum in which DN-1417 still increased HVA and DOPAC. The response of striatal DA metabolites was also observed after chronic treatment for 21 days. Chronic DN-1417 produced no significant change in ³H-spiperone binding in the prefrontal cortex, nucleus accumbens, olfactory tubercles and striatum, while striatal ³H-DA binding displaced by 30 nM spiperone was enhanced after chronic treatment. These results indicate that DN-1417 interacts with mesocortical, mesolimbic and nigrostriatal DA systems in the different modes of action. The lack of tolerance to motor hyperactivity, however, raises the question as to whether DN-1417-induced hyperactivity may be mediated by the activation of mesolimbic DA neurons. The involvement of nigrostriatal neurons in DN-1417-induced motor hyperactivity is suggested.     

Effect of oral contraceptives on the rat brain and pituitary opioid peptides

This study was designed to explore the hormonal regulation of CNS opioid peptide levels in female Sprague Dawley rats. Forty-eight animals were divided into 2 equal groups for acute and chronic studies. Each group was further divided into 4 subgroups, each containing 6 animals. Each rat in the control group received an inert pill (in 0.25 ml corn oil daily by gavage); the second group, 15 micrograms norethindrone (NE, a potent progestin present in the oral contraceptive Micronor); the third group, 15 micrograms NE and 1 microgram ethinyl estradiol, EE2 (present in the oral contraceptive Modicon) and the fourth group, 10 times the dose of the third group. Rats were treated either acutely for 5 days or chronically for 7 weeks. Opioid peptides were estimated by radioimmunoassay. Acute administration of 150 micrograms NE + 10 micrograms EE2 decreased the levels of methionine-enkephalin (ME), leucine-enkephalin (LE), dynorphin (DYN) and beta-endorphin like immunoreactivity (beta-EI) by about 50% in the pituitary. The same dose on chronic administration also decreased DYN, but increased the levels of ME and LE in the pituitary by 331 and 69%, respectively. In the hypothalamus, chronic administration of NE + EE2 increased the level of ME (155%) and LE (87%) as well as of DYN (97%). In the striatum, the levels of LE (33%) and DYN (115%) were elevated during chronic administration. It is concluded that the acute administration of NE + EE2, in general, reduces the levels of ME, LE, DYN and beta-EI.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dynorphin-A-(1–13) antagonizes morphine analgesia in the brain and potentiates morphine analgesia in the spinal cord

Intrathecal injection of subanalgesic doses of morphine (7.5 nmol) and dynorphin-A-(1–13) (1.25 nmol) in combination resulted in a marked analgesic effect as assessed by tail flick latency in the rat. The analgesic effect of the composite dynorphin/morphine was dose-dependent in serial dilutions so that a composition of 1/8 of the analgesic dose of dynorphin and 1/3 that of morphine produced an analgesic effect equipotent to full dose of either drug applied separately. The analgesic effect induced by dynorphin/morphine mixture was not accompanied by motor dysfunction and was easily reversed by a small dose (0.5 mg/kg) of naloxone. Contrary to the augmentatory effect of dynorphin on morphine analgesia in the spinal cord, intracerevroventricular (ICV) injection of 20 nmol of dynorphin-A-(1–13) exhibited a marked antagonistic effect on the analgesia produced by morphine (120 nmol, ICV). The theoretical considerations and practical implications of the differential interactions between dynorphin-A-(1–13) and morphine in the brain versus spinal cord are discussed.     

Hypermotility induced by vasoactive intestinal peptide in the rat: its reciprocal action to cholecystokinin octapeptide

Intracerebroventricular administration of vasoactive intestinal peptide (VIP) shortened the duration of pentobarbital-induced sleep and produced significant hypermotility in the rat. Although hypermotility induced by methamphetamine was not potentiated by central administration of VIP, L-DOPA-induced hypermotility in pargyline-pretreated rats was markedly enhanced by VIP and this hypermotility was suppressed by simultaneous administration of cholecystokinin octapeptide (CCK-8) in a dose-related manner. Apomorphine-induced hypermotility was also potentiated by VIP. These results suggest that VIP may stimulate postsynaptic dopaminergic receptor, causing an increase in motility, and that a possible reciprocal interaction exists between VIP and CCK-8.     

Experimental dissociation of food intake and plasma beta-endorphin following 2-deoxy-D-glucose in rats

The present studies were undertaken to further assess the role of plasma beta-endorphin (beta-EP) in the hyperphagia induced by the glucose antimetabolite, 2-deoxy-D-glucose (2-DG). Plasma concentrations of immunoreactive beta-EP (ir-beta-EP) were measured at the end of the first hour of feeding in all animals treated with 400 mg/kg 2-DG. Previous studies had shown a consistent, positive association between 2-DG hyperphagia and plasma ir-beta-EP concentrations, but the present data revealed dissociations between hyperphagia and plasma ir-beta-EP. Dexamethasone administration blocked the 2-DG-induced rise in plasma ir-beta-EP, but had no effect on the 2-DG hyperphagia measured at 1 hour. Forced drinking of a 2% NaCl solution decreased 2-DG hyperphagia, but not the 2-DG induced rise in plasma ir-beta-EP. Thus, elevations in plasma ir-beta-EP are not necessary for the full expression of 2-DG-induced hyperphagia in dexamethasone-treated rats. Furthermore, decreased feeding responses to 2-DG could coexist with increased levels of plasma ir-beta-EP in NaCl-treated normal rats. Elevations in plasma ir-beta-EP do not appear to be the critical opiate link in 2-DG induced hyperphagia.     

In vitro translation of human pheochromocytoma messenger RNAs: characterization of tyrosine-hydroxylase and dopamine-beta-hydroxylase

mRNAs extracted from human pheochromocytoma were translated in vitro in a lysate of a rabbit reticulocytes. Two enzymes of the biosynthetic pathway of the catecholamines, tyrosine-hydroxylase (TH) and dopamine-beta-hydroxylase (DBH), were characterized as translation products after immunoprecipitation by specific antisera and electrophoretic analysis. The precursor of TH is a polypeptide having a molecular mass of 62,000 identical to that found for the mature protein. The molecular mass of the precursor of DBH 73,000 while that of the mature form is 79,000. TH and DBH have been translated from mRNAs having sedimentation coefficients of 22S and 25S, respectively.