New galanin(1-15) analogues modified in positions 9, 10 and 11 act as galanin antagonists on glucose-induced insulin secretion.

Galanin (GAL) is a 29-amino-acid residue peptide originally isolated from porcine upper small intestine. GAL exhibits various physiological activities, such as effects on hormones release, smooth muscles contractions, gastric acid secretion, neurons degeneration and feeding. One of the biological actions of GAL is the inhibition of insulin secretion from the pancreatic beta-cells. In our studies we have designed several new 15-amino-acid-residue galanin fragment analogues modified in positions: 6, 8, 9, 10, 11 and tested for their effects on glucose-induced insulin secretion from isolated rat pancreatic islets of Langerhans. In vitro insulin secretion was studied during static incubation. All peptides were tested at two concentrations: 0.1 microM and 1 microM. Among the analogues derived from GAL(1-15)NH(2) peptide: [Phe(9)]GAL(1-15)NH(2) and [Pro(11)]GAL(1-15)NH(2) were found to be the potent antagonists against the inhibitory effect of GAL on glucose-induced insulin secretion from the isolated rat pancreas. These analogues block the GAL-mediated inhibition of insulin secretion. The present studies have shown that analogues: [Phe(9)]GAL(1-15)NH(2) and [Pro(11)]GAL(1-15)NH(2) may be a key compounds for developing a more potent GAL antagonists.

Synthesis and biological properties of new chimeric galanin analogue GAL(1-13)-[Ala10,11]ET-1(6-21)-NH2.

Several chimeric peptides consisting of the N-terminal fragment of galanin (GAL) and C-terminal fragments of other bioactive peptides (e.g. substance P, bradykinin, neuropeptide Y, mastoparan) have been synthesized and reported as high-affinity galanin receptor antagonists. Recently we have synthesized a new chimeric peptide, GAL(1-13)-[Ala(10,11)]ET-1(6-21)-NH(2), consisting of the N-terminal fragment of GAL and the C-terminal fragment of endothelin-1 (ET-1) analogue. This chimera was previously shown to be a moderate-affinity ligand to hypothalamic galanin receptors with a K(D) value of 205 nM. However, its biological action has been unknown so far. In our studies we characterized the biological properties of this new chimeric analogue, investigating its action on rat isolated gastric smooth muscles and influence on insulin secretion from rat isolated islets of Langerhans. Data acquired in the course of our studies suggest that analogue GAL(1-13)-[Ala(10,11)]ET-1(6-21)-NH(2) does not seem to be a potent galanin receptor antagonist in the gastrointestinal tract.

The contractile effects of several substituted short analogues of porcine galanin in isolated rat jejunal and colonic smooth muscle strips.

The activity of short porcine galanin (Gal) analogues was tested in vitro using rat jejunal and colonic smooth muscle strips. Peptides evoked concentration-dependent tissue contractions yielding typical response curves in concentration range from 0.3 nM to 300 microM, with a characteristic fall-down effect at the supramaximal concentrations. Gal(1-15) was less potent than Gal(1-29). Furthermore, [D-Trp(2)]Gal(1-15), [endo-Trp(2),Cle(4)]Gal(1-15), [D-Leu(4)]Gal(1-15), [des-Leu(4)]Gal(1-15), [Hse(6)]Gal(1-15), [Dab(14)]Gal(1-15), [Dpr(14)]Gal(1-15) or [Arg(14)]Gal(1-15) showed a considerable decrease in potency compared to Gal(1-15) in jejunal and/or colonic smooth muscle cells. Functional evidence confirmed that the integrity of both N- and C-terminals must be preserved in order to preserve a full excitatory myogenic potential of the peptide in rat jejunum and colon. Besides, amino acids located in positions 2, 4, 6 and 14 play a crucial role in recognition and activation of molecular domains responsible for Gal action in the intestinal smooth muscle.

The role and interactions of nitric oxide (NO), carbon monoxide (CO), and prostanoids in the pathogenesis of postoperative ileus in rats.

The effects of heme oxygenase (HO) inhibitors, zinc-protoporphyrin-IX (ZnPP-IX), and tin protoporphyrin-IX (SnPP-IX) and their interactions with L-arginine/nitric oxide synthase (NOS) and cyclooxygenase (COX) pathways were investigated in postoperative ileus in rats. Intestinal transit was measured as Evans blue migration after skin incision, laparotomy or laparotomy plus gut evisceration and handling. Laparotomy and small intestinal manipulations increased blood plasma nitrites/nitrates level 1.88-fold. N(omega)-nitro-L-arginine methyl ester, indomethacin, a selective COX-1 blocker (resveratrol) and COX-2 antagonists (nimesulide, DuP-697, NS-398) reversed the additional inhibitory effects of gut manipulation subsequent to laparotomy. In contrast, N-(3-(aminomethyl)benzyl)acetamidine or S-methylisothiourea, highly selective inducible NOS blockers, remained ineffective. ZnPP-IX and SnPP-IX overturned the effects of laparotomy on dye propulsion, but were only partially effective after laparotomy and gut handling attenuating the additional inhibitory influences of gut manipulation, the intestinal transit reaching 89.21%, 92.87%, 53.46%, and 48.56% of respective controls transit. Salutary effects of L-NAME, ZnPP-IX, and SnPP-IX were dose-dependent, L-arginine or hemin (HO substrate) sensitive. Administration of indomethacin and resveratrol subsequent to SnPP-IX reversed the inhibitory effects of laparotomy and manipulation, amounting to 93.91% and 87.43% of controls. On the other hand, L-NAME injected after SnPP-IX abolished the salutary effects of the latter, study dye migration reached 25.18% of control rat. Therefore we demonstrated that nitric oxide, carbon monoxide, and prostanoids play a role in the pathogenesis of postoperative ileus albeit in different mechanisms. Laparotomy stimulated HO activity, whereas gut manipulation led to an excessive constitutive NOS stimulation accompanied by augmented prostanoid synthesis by COX-1. Unaffected synthesis of either NO or CO enables a return of gastrointestinal transit during postoperative period, whereas a pharmacological blockade of two complementary metabolic pathways provides a most effective measure against postoperative ileus development.

Increased potency of some substituted short peptide analogues in comparison to galanin(1-15)-NH(2)in rat fundus strips.

The activity of short porcine galanin (Gal) analogues was tested in vitro using rat gastric fundus strips. The peptides contracted longitudinal smooth muscle in a concentration-dependent manner with the following order of potency: Gal(1-29) >[Cit(14)]Gal(1- 15) >[Asp(14)]Gal(1- 15) >[Dab(14)]Gal(1- 15) >[Nle(14)] Gal(1-15) >[Dpr(14)]Gal(1- 15) >[Arg(14)]Gal(1- 15) >[Orn(14)]Gal(1- 15) >Gal(1-15). Only in the case of two peptides, namely [Cit(14)]Gal(1-15) and [Dab(14)]Gal(1-15) did the values of Hill coefficients, estimated from the appropriate concentration-contraction curves, differ significantly from unity. Our results indicate that both N- and C-terminals of Gal molecule contribute towards the affinity and activity of Gal in rat gastric smooth muscle cell receptors, indicating that their integrity is essential for its full excitatory myogenic action. The substitution of histidine with citruline, aspartic acid, norleucine or diaminobutyric acid in position 14 of the amino acid chain led to a considerable increase in potency, suggesting that amino acids located at this position might play a crucial role where the strength of short analogues is concerned.

Actions of several substituted short analogues of porcine galanin on isolated rat fundus strips: a structure-activity relationship.

The activity of porcine galanin (Gal) fragments and analogues were tested in vitro using rat gastric fundus strips. The peptides contracted longitudinal smooth muscle in a concentration-dependent manner with the following order of potency: [Nle4]Gal(1-15), Gal(-15), [Cle4]Gal(1-15), [Hse6]Gal(1-15), [Va14]Gal(1-15), [Ile4]Gal(1-15), [endoTrip2a, Cle4]Gal(1-15), [desThr3, Cle4]Gal(1-15), [D-Leu4] Gal(1-15), [desLeu4]Gal(1-15). On the contrary [desTrp2, Val4]Gal (1-15) remained inactive up to 10 microM. The values of Hill's coefficients estimated from the appropriate concentration-contraction curves for all analogues except for [Val4]Gal(1-15), [Hse6]Gal(1-15), [endoTrp2a,Cle4]Gal(1-15), [desLeu4]Gal(1-15) and [D-Leu4] Gal(1-15) did not significantly differ from unity. Our results indicate that the integrity of the first four N-terminal amino acids of Gal molecule is essential for the full excitatory myogenic action of the peptide in rat gastric fundus. Similarly, substitution, addition or deletion of amino acid residues in positions two, three, four and six can considerably influence the ability of Gal analogues to interact with Gal receptors. The data acquired in the course of our structure-activity study suggest that both N- and C-terminals of Gal molecule contribute towards the affinity and activity of Gal in rat gastric smooth muscle cell receptors.

The inhibitory effects of terikalant on the contractile activity of galanin in isolated rat fundus strips.

The maximal responses (E(max)s) of isolated rat gastric fundus strips to 300 n m porcine galanin (Gal) were decreased in a concentration-dependent manner by terikalant (RP 62719). EC(50)of the agent equalled 4.39 microm (2.35-8.22). On the contrary the action of 30 n m of carbachol were not affected by the modulator in concentrations up to 30 microm. It is concluded that potassium currents may contribute to the modulation of Gal myotropic activity in the gut.

Sources of activator Ca2+ for galanin-induced contractions of rat gastric fundus, jejunum and colon.

Galanin (Gal) evoked reproducible contractions of isolated rat gastric fundus, colon and jejunum longitudinal strips in concentrations ranging from 1 nM to 3 microM. EC50 of Gal equalled 12.63, 23.27 and 56.02 nM, respectively. Hill's coefficients were not different from unity in any of the tissues examined. Experiments have been performed in the presence of protease and peptidase inhibitors, a variety of specific antagonists and tetrodotoxin (TTX) to exclude the non-specific stimulatory or inhibitory action of Gal. Gal-evoked contractions were attenuated by diminished extracellular Ca2+ concentration and by diltiazem. Gal activity in gastric fundus and colon, but not in jejunum was inhibited by depleting intracellular Ca2+ stores, thapsigargin, dantrolene, ryanodine, TMB-8, neomycin and U-73122. Our data confirmed that Gal contracts rat fundus, jejunum and colon by stimulating specific receptors, which are coupled both to Ca2+ influx through the voltage-dependent calcium channels and intracellular Ca2+ release from ryanodine- and IP3-sensitive stores (stomach and colon) or the extracellular Ca2+ influx only (jejunum). Phosphatidylinositol-specific phospholipase C (PI-PLC) plays a crucial role in the former but not in the latter signal transduction cascade.