Involvement of delta and mu opioid receptors in the acute and sensitized locomotor action of cocaine in mice.

Analogs of deltorphins, such as cyclo(Nδ, Nδ-carbonyl-d-Orn2, Orn4)deltorphin (DEL-6) and deltorphin II N-(ureidoethyl)amide (DK-4) are functional agonists predominantly for the delta opioid receptors (DOR) in the guinea-pig ileum and mouse vas deferens bioassays. The purpose of this study was to examine an influence of these peptides (5, 10 or 20 nmol, i.c.v.) on the acute cocaine-induced (10mg/kg, i.p.) locomotor activity and the expression of sensitization to cocaine locomotor effect. Sensitization to locomotor effect of cocaine was developed by five injections of cocaine at the dose of 10mg/kg, i.p. every 3 days. Our results indicated that DK-4 and DEL-6 differently affected the acute and sensitized cocaine locomotion. Co-administration of DEL-6 with cocaine enhanced acute cocaine locomotion only at the dose of 10 nmol, with minimal effects at the doses 5 and 20 nmol, whereas co-administration of DK-4 with cocaine enhanced acute cocaine-induced locomotion in a dose-dependent manner. Similarly to the acute effects, DEL-6 only at the dose of 10 nmol but DK-4 dose-dependently enhanced the expression of cocaine sensitization. Pre-treatment with DOR antagonist - naltrindole (5 nmol, i.c.v.) and mu opioid receptor (MOR) antagonist, ß-funaltrexamine abolished the ability of both peptides to potentiate the effects of cocaine. Our study suggests that MOR and DOR are involved in the interactions between cocaine and both deltorphins analogs. A distinct dose-response effects of these peptides on cocaine locomotion probably arise from differential functional activation (targeting) of the DOR and MOR by both deltorphins analogs.

Antinociceptive effects of two deltorphins analogs in the tail-immersion test in rats.

The antinociceptive effects of analogs of deltorphins: cyclo(Nδ,Nδ-carbonyl-D-Orn2, Orn4)deltorphin (DEL-6) and deltorphin II N-(ureidoethyl)amide (DK-4) after intracerebroventricular (i.c.v.) administration were investigated in the tail-immersion test in rats. Morphine, the most commonly used µ-opioid receptors (MOR) agonist, was employed as a reference compound. The contribution of the MOR, δ-(DOR) and κ-opioid receptors (KOR) in antinociceptive effects of the deltorphins analogs was studies using selective antagonists of these receptors. The results indicated that DK-4 (5, 10 and 20 nmol) and DEL-6 (5, 10 and 20 nmol) were the most effective in alleviating thermal pain at the dose of 20 nmol. The antinociceptive potency of DEL-6 at the dose of 20 nmol was approximately equal but DK-4 at the dose of 20 nmol was less effective than morphine at the dose of 13 nmol. DOR antagonist - naltrindole (NTI, 5 nmol) very strongly and, to the lower extent MOR antagonist - ß-funaltrexamine (ß-FNA, 5 nmol), inhibited antinociceptive effect of DK-4 (20 nmol). In turn, ß-FNA was more potent than NTI in inhibition of the antinociceptive effects of DEL-6. Co-administration of DEL-6 and morphine at doses of 5 nmol, which do not produce measurable antinociception, generated additive antinociceptive effect. Chronic intraperitoneal (i.p.) injection of morphine (9 days) displayed a marked analgesic tolerance to the challenge dose of morphine and a slight cross-tolerance to challenge doses of DEL-6 and DK-4, given i.c.v. These findings indicate that the new deltorphin analogs recruit DOR and MOR to attenuate the nociceptive response to acute thermal stimuli.

Moderate exercise training reduces arterial chemoreceptor reflex drive in mild hypertension.

The aim of our study was to check the responsiveness the chemoreceptor reflex in 28 young mildly hypertensive men (HTS), aged 18-32 years and 25 normotensive male subjects (NTS) aged 19-32 years, before and after 3-months dynamic exercise training. We tested the hypothesis that dynamic training reduces arterial chemoreceptor drive in mild hypertension. Circulatory response to 3-min hyperoxic inactivation of arterial chemoreceptors induced by 70% oxygen breathing was measured before and after training. Arterial blood pressure (BP) was recorded continuously by Finapres method, stroke volume and arm blood flow were registered by impedance reography, heart rate by ECG. Both groups were submitted to moderate 3-months dynamic exercise training. Before training the hyperoxic breathing caused in HTS a significant decrease in systolic BP by 6+/-1 mmHg p<0.01, in diastolic BP by 2+/-0.6 mmHg p<0.01, and in total peripheral vascular resistance (TPR) by 0.24+/-0.04 TPRU (p<0.01). After training hyperoxia augmented systolic BP by 2.64+/-1.9 mmHg (NS), diastolic BP by 2+/-1 mmHg p<0.05, and TPR by 0.043+/-0.05 TPRU (ANOVA). In NTS before training brief hyperoxia produced insignificant change in BP and TPR. In NTS after training hyperoxia increased systolic BP by 4.2 mm Hg+/-1.23 p<0.01 and diastolic BP by 3.1+/-0.6 mmHg p<0.01 respectively and TPR by 0.053+/-0.02 TPRU. Our results confirm earlier finding on the enhanced arterial chemoreceptor reflex drive in mild human hypertension. We conclude that normalizing arterial blood pressure in subjects with mild hypertension which occurred after 3-months dynamical exercise training is due to attenuation of the sympathoexcitatory chemoreceptor reflex drive by exercise training. The mechanism of this effect requires further study.

Highly potent side-chain to side-chain cyclized enkephalin analogues containing a carbonyl bridge: synthesis, biology and conformation.

Six novel cyclic enkephalin analogues have been synthesized. Cyclization of the linear peptides containing basic amino acid residues in position 2 and 5 was achieved by treatment with bis(4-nitrophenyl)carbonate. It was found that some of the compounds exibit unusually high mu-opioid activity in the guinea pig ileum (GPI) assay. The 18-membered analogue cyclo(N(epsilon),N(beta)-carbonyl-D-Lys2,Dap5)-enkephalinamide turned out to be one of the most potent mu-agonists reported so far. NMR spectra of the peptides were recorded and structural parameters were determined. The conformational space was exhaustively examined for each of them using the electrostatically driven Monte Carlo method. Each peptide was finally described as an ensemble of conformations. A model of the bioactive conformation of this class of opioid peptides was proposed.

Tryptic hydrolysis of hGH-RH(1-29)-NH2 analogues containing Lys or Orn in positions 12 and 21.

Two analogues of the 29 amino acid sequence of human growth hormone-releasing hormone, namely [Nle27]hGH-RH(1-29)-NH2 and [Orn(12,21),Nle27]hGH-RH(1-29)-NH2, have been synthesized and subjected to digestion by trypsin. The course of degradation was followed using RP-HPLC and ESI-MS. Several intermediates and final products of degradation were identified and conclusions regarding the rate of cleavages at different positions occupied by Lys and Arg residues were drawn. The analogue containing ornithine was found to be less susceptible to hydrolysis by trypsin: the 12-13 and 21-22 peptide bonds were completely resistant to the cleavage. The results show that by replacing Lys with Orn, a possibility exists to design new peptides, which could be more stable in biological fluids.

Primary structure of fox (Vulpes vulpes) proinsulin based on sequence studies of pancreatic peptides and cDNA.

Insulin and C-peptide were extracted and purified from fox (Vulpes vulpes) pancreas using gel filtration, ion-exchange chromatography and HPLC. Chromatographic data for the insulin, as well as for its oxidized and carboxymethylated chains proved it to be identical to that of polar fox (Alopex lagopus) and dog. The sequence analysis of a peptide which was assumed to be the corresponding C-peptide revealed that it comprises 23 amino acid residues and is identical to the C-peptide fragment isolated from dog pancreas: it differs from polar fox C-peptide by a single substitution (Asp-->Glu). mRNA was isolated from pancreatic tissue and cDNA was obtained by reverse transcription. A polymerase chain reaction was performed using gene-specific primers to obtain a DNA fragment corresponding to part of fox proinsulin. DNA sequencing revealed 100% identity to dog proinsulin at the protein level, although some amino acids were encoded by different codons. The total sequence of proinsulin was deduced from these results.

Conformational analysis of a novel cyclic enkephalin analogue using NMR and EDMC calculations.

Conformational space of a novel cyclic enkephalin analogue, cyclo(N(epsilon),N(epsilon')-carbonyl-D-Lys2,Lys5)enkephalin amide, was exhaustively examined. A large number of conformations was selected and clustered into families on the basis of their structure and energy. For representative conformations ROESY spectra were generated and their linear combination was fitted to the spectra measured in water and Me2SO-d6. This procedure yielded an ensemble of most populated conformations of the peptide in the two solvents.

Postexercise decrease in arterial blood pressure, total peripheral resistance and in circulatory responses to brief hyperoxia in subjects with mild essential hypertension.

The objective of our study was: (1) to compare the influence of moderate exercise on circulatory after-response in mildly hypertensive (n = 8) and normotensive male subjects (n = 9); (2) to examine the circulatory response to 3-min hyperoxic inactivation of arterial chemoreceptors at rest and during postexercise period in both groups. Hypertensive men (HTS) with a systolic blood pressure (SBP) 148 +/- 5 mm Hg, diastolic blood pressure (DBP) 92.4 +/- 4 mm Hg; and normotensive men (NTS), with a SBP 126 +/- 3 mm Hg, DBP 75.6 +/- 1.3 mm Hg, were submitted to 20-min of moderate exercise on a cycloergometer (up to the level of 55% of each subject's resting heart rate reserve). Finger arterial BP was recorded continuously with Finapres, impedance reography was used for recording stroke volume, cardiac output and arm blood flow. In HTS a significant decrease in SBP by 14.5 +/- 3.4 mm Hg, DBP by 8.9 +/- 1.9 mm Hg, total peripheral resistance (TPR) by 0.45 +/- 0.05 TPR u. (33.7 +/- 2.7%), and in arm vascular resistance (AVR) by 11.0 +/- 2.7 PRU u. (35.6 +/- 7%), was observed over a 60-min postexercise period. NTS exhibited insignificant changes in SBP, DBP, AVR except a significant decrease in TPR limited only to 20-min postexercise period. Hyperoxia decreased SBP, DBP and TPR in HTS. This effect was significantly attenuated during the postexercise period. Long-lasting antihypertensive effect of a single dynamic exercise in HTS suggests that moderate exercise may be applied as an effective physiological procedure to reduce elevated arterial BP in mild hypertension. We suggest also that the attenuation of the sympathoexcitatory arterial chemoreceptor reflex may contribute to a postexercise decrease in arterial BP and in TPR in mildly hypertensive subjects.

Synthesis of a novel side-chain to side-chain cyclized enkephalin analogue containing a carbonyl bridge.

A novel type of cyclic opioid peptide analogue, cyclo[N epsilon,N epsilon'-carbonyl-D-Lys2,Lys5]enkephalinamide, was prepared from a linear precursor peptide. The peptide was synthesized on the Merrified resin and also by a combination of the solid-phase technique and the classical method in solution. In both cases the cyclization was performed by reaction of bis(4-nitrophenyl)carbonate with the free side-chain amino groups of the two lysine residues. The described method permits the convenient preparation of novel peptide analogues cyclized via a ureido group incorporating the side-chain amino groups of two alpha, omega-diamino acid residues. The cyclic enkephalin analogue containing a 21-membered ring structure showed preference for mu over delta opioid receptors in opioid bioassays in vitro.

Evaluation of carbodiimides using a competition method.

A competitive reaction of activated Boc-Ala-OH and Boc-Phe-OH with H-Leu-resin has been developed for assessing the relative efficiencies of different carbodiimides. This allowed a comparison of the efficiency of the carbodiimides N,N'-dicyclohexylcarbodiimide,N,N'-diisopropylcarbodiimide, N-tert-butyl-N'-methylcarbodiimide and N-tert-butyl-N'-ethylcarbodiimide. Comparable results were obtained when these reagents were used for the preformation of symmetrical anhydrides or of 1-hydroxybenzotriazole esters in situ. Differential incorporation was observed when asymmetrical carbodiimides were used for peptide bond formation by the direct carbodiimide procedure.

Chronic administration of a new potent agonist of growth hormone- releasing hormone induces compensatory linear growth in growth hormone-deficient rats: mechanism of action.

To assess the efficacy of a potent agonist analog of GH-releasing hormone (GH-RH), [Dat1,Gln8,Orn12,21,Abu15,Nle27,Asp28,A gm29]hGH-RH(1-29) (JI-38), we investigated the effects of its chronic administration on growth responses in monosodium glutamate (MSG)-lesioned and normal young rats. Body weight (BW), body length (BL), tibia length (TIL), and tail length (TAL) were monitored. Basal serum GH concentrations, GH responses to bolus injections of GH-RH, pituitary GH and serum IGF-I concentrations were measured by RIA. Pituitary GH-RH receptor concentration and binding affinity was also evaluated after the treatment. Neonatal treatment with MSG resulted, as expected, in blunted growth and a decrease in serum and pituitary GH concentration and serum IGF-I levels. A reduction in GH-RH receptor concentration, associated with increased binding affinity of the GH-RH receptor was also found. Chronic administration of GH-RH agonist JI-38 in doses of 2 micrograms at 12-hour intervals for 2 weeks markedly increased the GH responsiveness to GH-RH and stimulated growth, with MSG-treated animals achieving the growth rate of normal controls. Acceleration of growth was associated with stimulated GH synthesis and IGF-I secretion, although basal serum GH levels did not change. Pituitary GH-RH receptor concentration and binding affinity were not significantly modified by the treatment. Treatment of normal young growing rats with agonist JI-38 did not further increase the normal growth acceleration in these rats, but stimulated the GH synthesis and augmented the GH secretory responsiveness. The treatment of MSG-lesioned rats with GH-RH agonist was generally more effective in female than in male animals, and in some cases masked the sex differences in growth rate. Our findings provide the first evidence that the blunted growth rate of the MSG-lesioned rats is associated with a decreased pituitary GH-RH receptor concentration. Our work demonstrates that administration of GH-RH agonist JI-38 is able to restore the normal growth rate of the GH-deficient rats by stimulating GH synthesis and IGF-I secretion.

Effect of chronic administration of a new potent agonist of GH-RH(1-29)NH2 on linear growth and GH responsiveness in rats.

The effects of a repeated or continuous administration of a potent agonistic analog of growth hormone-releasing hormone (GH-RH), [Dat1, Thr8, Orn12,21, Abu15, Nle27, Asp28, Agm29] hGH-RH(1-29)(JI-36), on the linear growth and the GH responses to bolus injections of GH-RH(1-29)NH2 were investigated in male rats about 7 weeks old. Body weight and tail length were monitored. Basal serum GH and IGF-I concentrations and GH responses to GH-RH(1-29)NH2 were measured by RIA. Chronic administration of GH-RH agonist JI-36 by continuous release from osmotic minipumps at the rate of 0.2 microgram/h or twice daily injections of 0.5 and 5 micrograms/rat for 4 weeks significantly speeded up the growth of rats as measured by the tail length. The acceleration of growth was similar in the 3 groups and was associated with stimulation of IGF-I secretion. The GH response to bolus injection of GH-RH(1-29)NH2 was preserved in all groups and no attentuation of the response occurred in rats treated for 4 weeks with agonist JI-36 as compared with the control group. Our results indicate that chronic administration of GH-RH agonist JI-36 significantly increases the growth rate without affecting somatotroph responsiveness in rats. It is therefore likely that this class of GH-RH agonists may be useful clinically.

Hemodynamic responses to brief hyperoxia in healthy and in mild hypertensive human subjects in rest and during dynamic exercise.

Hemodynamic consequences of the withdrawal of arterial chemoreceptor drive (ACD) by brief systemic hyperoxia were studied in 16 mild hypertensive subjects (HT) and in 16 healthy subjects (NT) in horizontal position at resting metabolic rate. In another 9 mild HT and match NT measurements were made in resting sitting position and during steady-state mild physical exercise on cycloergometer, (30% of VO2 max.) Tidal volume, minute ventilation, end tidal CO2 and O2 concentration, K+, Na+, pO2, pCO2 values in blood were recorded. Impedance reography was used for recording stroke volume (SV) and arm blood flow (ABF). Cardiac output (CO), ABF and arterial blood pressure (ABF) values were used for calculation of the total peripheral resistance (TPR) and vascular resistance in the arm (AVR). To assess the neurogenic circulatory response to withdrawal of ACD in HT attenuated by opposite peripheral effects of high oxygen, the values of AVR, ABP, TPR and AVR changes during brief hyperoxia in NT, assumed to be of peripheral origin, were subtracted from respective values in HT, assumed to be of mixed neurogenic and peripheral origin. In HT hyperoxia applied in sitting position produced a brief decrease in systolic and diastolic ABP by 5.4 +/- 0.8% and 3.4 +/- 1.1% respectively, of TPR by 12.4 +/- 3% and of AVR by 4.7 +/- 4.6%. Decrease in AVR during hyperoxia was significantly greater in sitting than in horizontal position. In NT hyperoxia produced opposite effects in ABP, TPR and AVR, as compared to those in HT. In HT subjects during steady-state exercise the TPR decreased by 21 +/- 3.7% reaching a value no different from that in NT. We suggest, that in primary hypertension neurogenic sympathoexcitatory ACD is augmented and interacts with the peripheral mechanisms related to tissue oxygen supply.

Synthesis and biological evaluation of superactive agonists of growth hormone-releasing hormone.

Analogs of the 29 amino acid sequence of human growth hormone-releasing hormone (hGH-RH) with agmatine (Agm) in position 29, desaminotyrosine (Dat) in position 1, norleucine (Nle) in position 27, and L-alpha-aminobutyric acid (Abu) in position 15 have been synthesized, and their biological activity was evaluated. Some peptides contained one or two residues of ornithine (Orn) instead of Lys in positions 12 and 21 and additional replacements in positions 8 and 28. All analogs were found to be more potent than hGH-RH-(1-29)-NH2 in the superfused rat pituitary cell system. In tests in vivo in rats after subcutaneous administration, the analogs JI-22, [Dat1, Orn12,21, Abu15, Nle27, Agm29]hGH-RH-(1-29); JI-34, [Dat1, Orn12,21,Abu15,Nle27, Asp28, Agm29]hGH-RH-(1-29); JI-36, [Dat1, Thr8, Orn12,21, Abu15,Nle27,Asp28,Agm29]hGH-RH-(1-29); and JI-38, [Dat1,Gln8, Orn12,21,Abu15,Nle27,Asp28,Agm29]hGH-RH-(1 -29) displayed a potency 44.6,80.9,95.8, and 71.4 times greater, respectively, than that of hGH-RH-(1-29)-NH2 at 15 min and 217.1, 89.7, 87.9, and 116.8 times greater at 30 min. After intravenous administration, JI-22, JI-36, and JI-38 were 3.2-3.8 times more potent than hGH-RH-(1-29)-NH2 at 5 min and 6.1-8.5 times more active at 15 min. All analogs were found to have higher binding affinities for GH-RH receptors on rat pituitary cells than hGH-RH-(1-29)-NH2. Because of high activity and greater stability, these analogs could be considered for therapy of patients with growth hormone deficiency.

N-cyclohexyl-N'-isopropylcarbodiimide: a hybrid that combines the structural features of DCC and DIC.

N-Cyclohexyl-N'-isopropylcarbodiimide was prepared and used for peptide couplings by various procedures. Reaction between Z-Val and Gly-OEt was used for yield studies and determination of the extent of the side reaction in solution. The value of this reagent for solid peptide synthesis was demonstrated by the synthesis of sequences comprising the 65-74 residues of acyl carrier protein. The efficiency of activation was determined by competition experiments. From all these studies the conclusion can be drawn that N-cyclohexyl-N'-isopropylcarbodiimide is comparable to or even better than the commonly used DCC for mediation of peptide bond formation in solid phase synthesis. By virtue of the solubility of N-cyclohexyl-N'-isopropylurea in dichloromethane, the carbodiimide seems to be a good candidate for practical execution of peptide bond formation by the standard carbodiimide procedure.

Reinvestigation of the reactions of carbodiimides with alkoxycarbonylamino acid symmetrical anhydrides. Isolation of two N-acylureas.

Alkoxycarbonylamino acid symmetrical anhydrides were allowed to react with N,N'-diisopropylcarbodiimide. The determination of the decrease in carbodiimide concentration indicated that the reaction was slow and proceeded indirectly. It is presumed that symmetrical anhydride in the presence of basic carbodiimide is converted into oxazolone and alkoxycarbonylamino acid. The latter reacts with carbodiimide to give O-acylisourea which, in turn, rearranges to N-acylurea. A second minor product was found to be N-(N1,N2-bisalkoxycarbonyldipeptidyl)urea. The main pathway leading to the formation of this product starts with rearrangement of the symmetrical anhydride to N1,N2-bisalkoxycarbonyldipeptide acid. These experiments suggest that the reaction between anhydride and carbodiimide is not the source of N-acylurea in peptide synthesis.

Synthesis and biological evaluation of human preproenkephalin (100-111) and its analogs.

The dodecapeptide sequence, Tyr-Gly-Gly-Phe-Met-Lys-Arg-Tyr-Gly-Gly-Phe-Met (BI), which is totally conserved in the primary structures of human, bovine, rat and toad preproenkephalins, has been synthesized by the solid-phase method. Coupling reactions were achieved by using symmetrical anhydrides of tert.-butyloxycarbonylamino acids performed with N-tert.-butyl,N'-methylcarbodiimide. 6-Arg and 7-Lys analogs have also been obtained. The peptides show opiate activity in both GPI and MVD assay, and possess antinociceptive properties as estimated by the hot-plate test in mice when applied intracisternally.

Primary structure of fox pituitary growth hormone.

Tryptic digests of fox growth hormone (fGH) were separated by reverse phase high performance liquid chromatography (HPLC) and by paper electrophoresis. From the amino acid composition of these tryptic peptides and from their alignment with the expected tryptic peptides from bovine growth hormone (bGH), the primary structure of fGH is proposed. There are only 17 amino acid residues which are different in these two growth hormone molecules.

Isolation and characterization of growth hormone from blue fox pituitary glands.

Growth hormone has been purified to homogeneity from blue fox pituitary glands. It has 191 amino acids with two disulfide bridges and a single tryptophan residue. The somatotropin activity is only 8% when compared with the bovine hormone in the receptor-binding assay. From radioimmunoassay data using baboon antisera to porcine or bovine growth hormone, the fox hormone has 14-17% immunoreactivity of bovine or porcine hormone.