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.

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.

Experimental hyperlipidemia prevents the protective effect of ischemic preconditioning on the contractility and responsiveness to phenylephrine of rat-isolated stunned papillary muscle.

This study was designed to establish a hyperlipidemic diet (significant increase in the cholesterol and triglycerides blood levels, but without atherogenic changes in heart muscle and coronary vessels) and to investigate the influence of experimental hyperlipidemia on the effects of ischemic preconditioning (PC) of rat-isolated papillary muscle on the time course of contractility during simulated ischemia and reperfusion and responsiveness to phenylephrine under such a condition. The animals were divided in four experimental groups: standard diet-fed control group (SD), SD underwent ischemic preconditioning (SD-PC), hyperlipidemic diet-fed group (HLD) and HLD underOFFt PC (HLD-PC). Force of contraction (Fc), velocity of contraction (+dF/dt), and velocity of relaxation (-dF/dt) were measured. HLD preparations were more sensitive to ischemia then SD ones. PC, performed by 5-min perfusion with no-substrate solution gassing with 95% N2/5% CO2 in the presence of fast electrical stimulation, and 10 min of reperfusion with normal solution and rate of stimulation, significantly increased the resistance of isolated cardiac tissues to simulated ischemia in SD-PC group, but not in HLD-PC group. Negative inotropic action of phenylephrine occured in SD group of preparations after simulated-ischemia/reperfusion period was also prevented by PC. Therefore, we conclude that experimental hyperlipidemia significantly influenced the function of rat heart muscle including the higher sensitivity to ischemia and different reaction to the same PC procedure.

The effects of potassium channel modulators on the simulated ischaemia-induced changes in contractility and responsiveness to phenylephrine of rat-isolated papillary muscle.

The aim of the present study was to compare the influence of terikalant, a blocker of inwardly rectifying K+ channels, galanin, a neuropeptide of 29 aminoacids with a complex mechanism of action including an activation of inwardly rectifying K+ channels and glibenclamide, a blocker of ATP-sensitive K+ channels, on simulated ischaemia-induced changes in contractility and response to phenylephrine of rat-isolated heart muscle. Experiments were performed on isolated rat heart papillary muscles. The following parameters were measured: force of contraction (Fc), velocity of contraction (+dF/dt) and velocity of relaxation (-dF/dt), time to peak contraction (ttp) and relaxation time at 10% of total amplitude of contraction (tt10). In the presence of 1 microM of galanin, as well as terikalant, simulated ischaemia caused a decrease in Fc, +dF/dt and -dF/dt, however, it significantly increased a drop in Fc and -dF/dt. After 60 min of reperfusion, all the measured parameters recovered completely except Fc in the galanin group. Terikalant, but not galanin, prevents the negative inotropic action of phenylephrine observed in the control group. On the other hand, addition of 1 microm of glibenclamide to the no-substrate solution prevented the simulated ischaemia-induced decrease in Fc, +dF/dt and -dF/dt. In this group phenylephrine did not cause the negative inotropic action. The above mentioned data reveal that pretreatment with the inhibitors of ATP-sensitive and inwardly rectifying K+ channels protect rat-isolated papillary muscle against ischaemia-induced disturbances in contractility.

The influence of experimental hyperlipidemia on the time course of contractility during simulated ischaemia and reperfusion and responsiveness to phenylephrine of rat heart papillary muscle.

The aim of this study was to examine the influence of simulated ischaemia on the contractility and responsiveness to phenylephrine of rat isolated papillary muscle in standard diet fed (SD) and hyperlipidemic diet fed (HLD) rats. The following parameters were measured: force of contraction (Fc), rate of rise (+dF/dt) and rate of fall (-dF/dt) of force of contraction, time to peak contraction (ttp) and relaxation time at 10% of total amplitude of contraction (tt10). The baseline values of Fc and +dF/dt, but, not -dF/dt, were significantly lower in HLD group than in SD group. Tissues from HLD rats were more sensitive to ischaemia regarding Fc, +dF/dt and -dF/dt. Moreover, reprefusion completely reversed the effects of ischaemia only in SD rats, but not in HLD rats, regarding Fc and +dF/dt. In contrast, a recovery of -dF/dt during reperfusion occurred only in the HLD group. In SD rats, phenylephrine (10 and 30 microM) had no effect on the contractility or induced megative inotropic effects (100 and 300 microM). Propranolol (1 microM), a non-selective blocker of beta-adrenoceptors, had no effects on this action. Chloroethylclonidine (CEC) (1 microM), a selectivw blocker of alpha 1b-adrenoceptor subtype, but not WB-4101(2-((2,6-dimethoxyphenoxyethyl)amino-methyl-1,4-benzodioxane), a selective blocker of alpha 1a adrenoceptor subtype, abolishes the negative inotropic action of phenylephrine. In HLD rats, phenylephrine had positive inotropic action (10 and 30 microM). The results indicate that hyperlipidemic diet in rats leads to the suppression of force of contraction and velocity of contraction, but not velocity of relaxation of isolated heart muscle. Under such a condition, heart muscle is more sensitive to ischaemia, but has better responsiveness to phenylephrine after ischeamia-reperfusion period.

Lysine14galanin(1-15)-NH2: a partial agonist at galanin receptors in rat isolated gastric fundus.

The study was undertaken to characterize the effects of the porcine galanin [pGal(1-29)-NH2] analogue [Lys14]pGal(1-15)-NH2 on rat gastric fundus. [Lys14]pGal(1-15)-NH2 is a less potent contractile agent than pGal(1-29)-NH2 (EC50 74.1 vs. 43.7 nmol/l, respectively) and shows a significantly lower maximal response than pGal(1-29)-NH2. Concentration-contraction curves were constructed for pGal(1-29)-NH2 alone (control) and pGal(1-29)-NH2 in the presence of 10, 100, and 1,000 nmol/l of [Lys14]pGal(1-15)-NH2. [Lys14]pGal(1-15)-NH2 shifted the concentration-contraction curves of pGal(1-29)-NH2 significantly to the right, whereas their linear portions remained parallel to that for the pGal(1-29)-NH2 control. [Lys14]pGal(1-15)-NH2 markedly increased the EC50 of the respective pGal(1-29)-NH2 concentration-contraction curves. It did not substantially change the maximal response of the muscles to pGal(1-29)-NH2 and the form of the respective concentration-contraction curves. Schild's plot gave a straight line with a slope of 0.84. The pA2 value for [Lys14]pGal(1-15)-NH2 was 8.23. [Lys14]pGal(1-15)-NH2 seems to be a partial Gal receptor agonist. Since the lack of specific Gal receptor antagonists in the gastrointestinal tract makes a precise characterization of its role as a motility modulator difficult, the position 14 in the pGal(1-29)-NH2 molecule looks as an attractive target in the search of a pure Gal receptor antagonist in the smooth muscles of the gut.

Contractile effects of porcine galanin(1-29)-NH2 on the rat isolated gastric fundus: mediation by potassium ions.

Galanin (3-300 nM) evoked reproducible concentration-dependent contractions of rat isolated gastric fundus strips, EC50 of the peptide equalled 16.7 nM (6.2-39.2) and the slope of the concentration-response curve was 34.8 (24.0-45.7). The maximal response (Emax) to carbachol (30 nM) was not affected by the absence of the potassium ions in the bathing solution. On the contrary the Emax to galanin (300 nM) was decreased by almost 95% by the use of the potassium-free buffer. Re-exposure of the muscle strips to potassium containing bathing medium reversed the inhibition by about 35%, yet the value remained significantly lower than that of the control. Apamin (1 and 2 microM), glybenclamide (10 microM), clofilium tosylate (10 microM) did not significantly influence the Emax to carbachol. Apamin or glybenclamide did not affect the contractile action of galanin, while clofilium attenuated the Emax to the peptide in a concentration-dependent manner, the EC50 of the agent being 9.44 microM (164 nM-541 microM). It was concluded that the potassium ions play a modulatory role in gastric smooth muscle contraction following galanin receptor stimulation, probably by interacting with the extracellular calcium influx.

Pharmacological characterization of the contractile effects of galanin (1-29)-NH2, galantide and galanin (1-14)-(alpha-aminobutyric acid8)scyliorhinin-I in the rat gastric fundus.

Porcine galanin (1-29)-NH2, galantide (M15) and galanin (1-14)-(alpha-aminobutyric acid8)-scyliorhinin-I used in concentrations of 300, 1,000 and 3,000 nM respectively caused contractions of rat fundus strips. The contractile responses to galanin(1-29)-NH2 were not modified by atropine (10 microM), guanethidine (10 microM), naloxone (1 microM), a mixture of propranolol (10 microM) and phentolamine (10 microM), indomethacin (10 microM), a mixture of mepyramine (10 microM) and cimetidine (10 microM), saralasin (10 microM), and spantide (100 microM). The effects of M15 and galanin(1-14)-(alpha-aminobutyric acid8)-scyliorhinin-I were significantly decreased by atropine for 36 and 18% and by spantide for 37 and 26% respectively. Indomethacin inhibited the muscle response to M15 without influence on the galanin (1-14)-(alpha-aminobutyric acid8)-scyliorhinin-I-induced action. These results support findings that galanin (1-29)-NH2 contracts rat gastric fundus strips by stimulating specific receptors localized on the surface of smooth muscle cells. M15 and galanin(1-14)-(alpha-aminobutyric acid8)-scyliorhinin-I seem to contract smooth muscles not only by acting at galanin receptors, but by interacting with muscarinic or tachykinin receptors or modulating the release of acetylcholine and substance P. Diltiazem (EC50 825 nM), dantrolene (EC50 30.2 microM) and the phospholipase C inhibitors U-73122 (EC50 549 microM) and U-73343 (EC50 751 microM) lowered the contraction to galanin(1-29)-NH2 in a concentration-dependent manner. These observations imply that though the extracellular Ca2+ influx plays a major role in the action of galanin(1-29)-NH2, the release of Ca2+ ions from the intracellular stores contributes to the response of smooth muscles of galanin(1-29) NH2. Norepinephrine (30, 60, 100 and 300 nM) concentration-dependently reduced the Emax to galanin (1-29)-NH2 and reduced the slopes of the concentration-contraction curves, without a notable change in EC50. Pertussis toxin pre-treatment (10 and 30 mg/kg intravenous [i.v.]), 120 h before the experiment, notably increased the maximal response of the rat gastric fundus to galanin(1-29)-NH2, without a significant change in the properties of the concentration-contraction curves (EC50, slopes). The observations may suggest that pertussis toxin-sensitive GTP-binding proteins are involved in the modulation of the excitatory effects of galanin(1-29)-NH2 in the rat gastric fundus.