Opioid effects of short enkephalin fragments containing the Gly-Phe sequence on contractile responses of guinea pig ileum.

1. Effects of the fragments H-Gly-Phe-OH, H-Gly-Phe-NH2 or H-Gly-Phe-OMe on the electrically stimulated cholinergic contractions of the longitudinal layer in isolated guinea pig ileum and on the Morphine-, Met-enkephalin- or Leu-enkephalin-induced inhibition of these contractions were analyzed for opioid activity in respect to Gly-Phe sequence. 2. H-Gly-Phe-OH or H-Gly-Phe-NH2 exerted no effects, while H-Gly-Phe-OMe applied cumulatively (1 pM-1 mM), concentration-dependently reduced the contractions to electrical stimulation, the IC50 value being 1.96 +/- 0.06 microM. Naloxone (1-5 microM) did not reverse the H-Gly-Phe-OMe effects. 3. H-Gly-Phe-OMe at single concentrations (1-10 microM) significantly decreased the maximum inhibition produced by cumulatively added (0.1 nM-100 microM) morphine, Met-enkephalin or Leu-enkephalin. The regression lines for the opioids were shifted to the right but not always in a parallel fashion; the IC50 values were higher as compared to the controls and lower as compared to the IC50 values after naloxone. 4. The pA2 value for H-Gly-Phe-OMe with respect to morphine (6.43 +/- 0.14) did not differ from that to Met-enkephalin (6.68 +/- 0.35) or Leu-enkephalin (9.06 +/- 0.98); the slope of the pA2 plot to morphine was near unity. 5. These data indicated that H-Gly-Phe-OMe exerted predominantly a potent non-competitive opioid antagonistic effect suggesting that short enkephalin fragments containing the Gly-Phe sequence might possess an opioid activity.

Nephrotoxic effect of the specific brain serotonin depletor para-chlorophenylalanine.

Brain serotonin depletion induced by peripheral parachlorophenylalanine (pCPA) is frequently used to evaluate the role of the central serotoninergic system in the regulation of a number of physiological functions, including the secretion of renin by the kidney. We found that due to the treatments applied in the protocol used for the investigation of pCPA effect on renin and vasopressin secretion in rats (300 mg/kg i.p. 64 and 40 h before sacrifice) renal injury was induced as well. Typical changes indicating acute renal failure were observed--an initial polyuria, natriuresis and body mass loss, succeeded by oliguria, decreased glomerular filtration rate, and salt and creatinine retention. Morphological changes in the glomeruli included a thickening of the basal membranes, a confluence and a reduced number of podocyte pedicles. A slight to moderate granular degeneration was observed in epithelial cells of the proximal convoluted tubule, combined with mitochondrial changes--an increase in number, matrix disorganization, and myelin degeneration. In conclusion, the renal function changes after i.p. pCPA may be due not to brain serotonin depletion-alone, but also to nephrotoxic effect.

Biological tests for opiate activity of newly-synthesized compounds--opioid peptides and cyclic beta-diketones.

A group of newly-synthesized (Leu5) enkephalinamides and their derivatives, with incorporated D-amino acids, as well as a group of cyclic beta-diketones from the group of 1, 3-indandiones, were partially pharmacologically characterized in experiments in vivo and in vitro. The substances studied, applied subcutaneously in doses of 50 to 1250 micrograms/kg bogy mass in mice, did not induce symptoms of acute and late toxicity. According to the parameters of the neuropharmacological screening tests performed, no significant differences were observed between the mice treated with the agents tested and the control mice. All five agents investigated significantly shortened the duration of hexobaribital-induced sleep. In the experiments using the conflict test after Vogel et al. (1971), (D-Ala2, D-Leu5) enkephalinamide induced a behavioural effect which can be qualified as anxiolytic. The same analogue also manifested a marked analgesic effect with the two tests used: hot plate test and the peritoneal test with glacial acetic acid. The cyclic beta-diketones-(methindiones) tested also manifested good analgesic effect with both test. (D-Ala2, D-Leu5), enkephalinamide strongly reduced the electrically induced contractile responses of the ileum. This effect was completely prevented by naloxone. The fact that (D-Ala2, D-Leu5) enkephalinamaide, known as a selective agonist of the delta-opiate receptors, proved to be particularly active suggests that both mu- and delta-type opiate receptors exist in the guinea-pig ileum. The pronounced analgesic effect of the leucine-enkephalinamide analogue with two substituted D-amino acids and of the two cyclic beta-diketone methindiones opens up new possibilities for synthesizing efficient analgesic agents.