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.

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.

Digoxin effects in the guinea pig heart: interaction with rimalkalim.

The aim of this study was to examine the influence of pre-treatment with rimalkalim (formerly HOE 234), an activator of ATP-sensitive K+ channels (KATP), on the chronotropic, inotropic and electrophysiological effects of digoxin in the guinea pig heart. To study these effects we used spontaneously beating right atria and isolated papillary muscles from the guinea pig heart. The following parameters were measured: the heart rate (b.p.m.), 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 time to 10% of total amplitude of force (tt10), action potential (RP, APA, APD, Vmax) and effective refractory period (ERP). Apart from the positive inotropic effect, digoxin induced negative chronotropic action at the concentration of 30 microM. Digoxin also significantly decreased APD50 and APD90 duration and reduced APA and RP. Rimalkalim itself, has similar electrophysiological and chronotropic effects to digoxin, but opposite to digoxin, a negative inotropic action and hyperpolarisation of the isolated tissue. After pre-treatment with rimalkalim (1 microM), the significant potentiation of maximum positive inotropic effect (Emax) of digoxin has been observed, as well as a significant shortening of the duration of ttp, tt10 and ERP when compared with the values obtained with digoxin alone. Furthermore, a significant increase in Vmax occurred, compared to the value obtained with rimalkalim. Glibenclamide (1 microM), a selective inhibitor of ATP-sensitive K+ channels, added to rimalkalim, prevented the changes in digoxin action observed after pre-treatment with rimalkalim. The results demonstrate significant influence of pre-treatment with rimalkalim on digoxin-induced inotropic and electrophysiological effects in the guinea pig heart. An abolishing action of glibenclamide on this effect of rimalkalim suggests that an activation of ATP-sensitive K+ channels might cause these changes.

Negative inotropic action of alpha-1a adrenoceptor blocking agents: role of adenosine and ATP-sensitive K+ channels.

1. The effects of alpha1 adrenoceptor blocking agents doxazosin, indoramin, 5-methylurapidil, niguldipine, WB-4101 and chloroethylclonidine (CEC) on the force of contraction (Fc), velocity of contraction (+dF/dt) and relaxation (-dF/dt) of guinea pig papillary muscles were studied. 2. All examined substances were applied in a wide concentration range (0.01-30.0 microM) for at least 30 min at each concentration. Only alpha1a blockers [i.e., niguldipine (0.01-0.3 microM), 5-methylurapidil (1-30 microM) and WB-4101 (1-30 microM)] showed a concentration-dependent negative inotropic action. 3. This effect was significantly attenuated in the presence of glibenclamide (1 microM) and almost completely abolished by 1,3-dipropyl-8-p-sulfophenylxanthine (1 microM), an antagonist of adenosine receptors with a slight selectivity for the A1 subtype. 4. Pretreatment with dibenamine, an irreversible blocker of alpha1 adrenoceptors (0.6 microM for 40 min), abolished this effect, whereas pretreatment with CEC, an irreversible blocker of alpha1b adrenoceptors (1 microM for 20 min), and pertussis toxin (10 microg/kg IP, 4 to 5 days before experiments) diminished it. 5. The alpha1a adrenoceptor blocking agents in the presence of the unblocked alpha1b adrenoceptor trigger the negative inotropic action, which seems to include adenosine receptor stimulation and activation of ATP-sensitive K+ channels (K[ATP]) through an inhibitory G protein.

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.

Galanin, galantide and galanin (1-14)-[alpha-aminobutyric acid8]-scyliorhinin-I: structure dependent effects on the rat isolated gastric fundus.

The study was undertaken using selected pharmacodynamic parameters to describe the effects of porcine galanin(1-29)-NH2; porcine galanin fragments; galantide; porcine galanin(1-14)-[alpha-aminobutyric acid8]scyliorhinin-I and the analogues of the latter peptides on rat isolated gastric fundus muscle. All tested peptides, apart from galanin(16-29)-NH2 evoked reproducible concentration-dependent contractions with significantly decreased activities in comparison to the potency of the native galanin(1-29)-NH2 molecule. The order of the contractile ability in the group of galanin(1-29)-NH2 short fragments was as follows: [lysine14]galanin(1-15)-NH2 > galanin(1-15)-OH > galanin(1-15)-NH2 > [glycine5] galanin(1-15)-NH2 > galanin(2-15)-NH2 > [glycine5,lysine14]galanin(1-15)-NH2. Aside from [lysine14]galanin(1-15)-NH2 which had a lower efficacy, none of the peptides showed significant changes in this respect in comparison to the intact galanin(1-29)-NH2 molecule. The concentration-response curves of the tested peptides were to the right and their slopes besides from: galanin(1-15)-OH; galanin(2-15)-NH2; [glycine5]galanin(1-15)-NH2 remained not significantly different from galanin(1-29)-NH2. Hill's coefficient for galanin(1-29)-NH2 is 1.03 indicating an interaction of one galanin(1-29)-NH2 molecule with one receptor, fulfilling criteria of classical receptor theory. For galanin fragments Hill's coefficients are < 1 implying that the rules of classical theory may not apply. Galantide and analogues exhibited a subsequent decrease in potency: [cycloleucine4] galantide > galantide > [homoserine6]galantide > [phenylalnine(4fluor)17] galantide. Galanin(1-14)-[alpha-aminobutyric acid8]-scyliorhinin-I and its analogues contracted the gastric fundus with a decline in strength: galanin(1-13)-[norleucine10]-scyliorhinin-I(3-10) > galanin(1-13)-[phenylalanine(4fluor)7]-scyliorhinin-I > galanin(1-14)-[alpha-aminobutyric acid8]-scyliorhinin-I > galanin(1-13)-[alpha-aminobutyric acid8, norleucine10]-scyliorhinin-I(3-10). They all displayed a greater efficacy than galanin(1-29)-NH2, and the concentration-response curves were slightly to the right, almost parallel to that of galanin(1-29)-NH2. Slopes of the curves were not significantly different from galanin(1-29)-NH2. Hill's coefficient for the galantide, [cycloleucine4]galantide; [homoserine6]galantide; [phenylalanine(4fluor)17]galantide and galanin(1-13)-[phenylalanine(4fluor)7]-scyliorhinin-I are < 1. Hill's coefficients for galanin(1-13)-[norleucine10]-scyliorhinin-I(3-10); galanin(1-14)-[alpha-aminobutyric acid8]-scyliorhinin-I; galanin(1-14)-[alpha-aminobutyric acid8, norleucine10]-scyliorhinin-I(3-10) are > 1. A Hill's coefficient markedly different from 1 might indicate that an activation of more than one type of receptors, negative or positive receptor cooperativity or multiple-step agonist-receptor reaction.

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.

Contractile action of galanin analogues on rat isolated gastric fundus strips is modified by tachyphylaxis to substance P.

This study was undertaken to characterize the interaction of porcine galanin (Gal) and some of its analogues with their receptors on rat gastric fundus muscle strips. Gal, galantide (M15) and Gal(1-14)-[Abu8]SCY-I evoked concentration-dependent contractions of gastric smooth muscle strips. Reproducible effects were observed in concentrations of 1-300, 3-1000 and 100-3000 nM, respectively. Specific EC50 for the contractile effect equalled 13.70 and 187 nM. Hill's coefficient for Gal is 1.03 indicating an interaction of one Gal molecule with one receptor, fulfilling the criteria of classical receptor theory. For M15 and Gal(1-14)-[Abu8]SCY-I Hill's coefficients are different from 1, namely 0.73 and 1.56, pointing out that the principle of interaction of one drug molecule with one receptor may not apply. The contraction induced by 300 nM of Gal was not significantly modified by tachyphylaxis to substance P (SP). On the contrary the introduction of tachyphylaxis to SP decreased the contractile effects of M15 and Gal(1-14)-[Abu8]SCY-I by about 57.7 +/- 3% and 39.6 +/- 5%, respectively. The findings suggest that contractile actions of M15 and Gal(1-14)-[Abu8]SCY-I are probably not only due to their agonist activities at Gal receptors but may result from a subsequent stimulation of receptors for SP or release of endogenous SP.

Actions of galanin and some of its analogues on rat isolated gastric fundus.

The study was undertaken to characterize the effects of porcine galanin (pGal) and some of its analogues on rat gastric fundus muscle strips. pGal, galantide (M15) and pGal(1-14)-[Abu8]SCY-I evoked reproducible concentration-dependent contractions in concentrations of 1-300, 3-1,000 and 100-3,000 nM, respectively, with EC50 values of 13, 70 and 187 nM. Hill's coefficient for pGal is 1.03, indicating an interaction of one pGal molecule with one receptor, fulfilling criteria of classical receptor theory. For M15 and pGal(1-14)-[Abu8]SCY-I, Hill's coefficients are significantly different from 1, namely 0.73 and 1.56, so that one drug molecule may not interact with one receptor. The stimulatory effects of pGal were not modified by dibenamine 10 microM or glybenclamide 1 or 10 microM. Diltiazem 0.1, 1 and 10 microM, papaverine 0.1, 10 microM or dibutyryl cAMP (dib cAMP) 100 and 300 microM, blocked the contraction to pGal in a concentration-dependent manner, indicating an important role for the influx of extracellular calcium ions and regulation by cAMP the pGal-evoked contraction. Diltiazem, dibutyryl cAMP and papaverine were not competitive antagonists of pGal in the stomach smooth muscle.

Do sodium nitroprusside and L-NAME affect pyrogen fever in rabbits?

Thermoregulatory responses after treatment with nitric oxide (NO) donor, sodium nitroprusside (SNP-3 mg/kg/h), or NO synthase inhibitor, NG-nitro-L-arginine methylester (L-NAME-100 mg/kg) were investigated in febrile rabbits (lipopolysaccharide E. coli-1 meg/kg). Pretreatment with SNP attenuated pyrogen fever as well as metabolic rate. L-NAME also inhibited postpyrogen increases in metabolism; however, this effect did not lead to antipyresis.

Desensitisation of substance P decreases the contractile effect of M15 and Gal (1-14)-(Abu 8) SCY-I on the rat gastric fundus.

Tachyphylaxis to SP decreased the effect of M15 and Gal(1-14)-[Abu8]SCY-I on gastric smooth muscles, without effect on the action of Gal. These findings support our initial hypothesis: the action of M15 and Gal(1-14)-[Abu8]SCY-I on the smooth muscles may not only be due to their agonist activity at Gal receptors, but may result from a subsequent stimulation of receptors for SP and perhaps for other tachykinins as well, however, a possibility that Gal analogues release endogenous SP can not be excluded. Further studies involving a tachykinin antagonist (spantide) are in progress at the moment. [1].

Thermoregulatory activity of sodium nitroprusside and arginine vasopressin.

1. Thermal responses to sodium nitroprusside (SNP, 3 mg/kg/hr) and arginine vasopressin (AVP, 3 micrograms/kg) were investigated in normothermic and febrile rabbits (LPS, 1 microgram/kg) at ambient temperature of 20.0 +/- 1.0 degrees C. Furthermore, blood pressure after these drugs was tested on a separate group of animals. 2. I.v. infusion of SNP produced hypothermia and attenuated pyrogen fever. On the other hand, AVP increased body temperature and intensified the febrile response. 3. Both drugs affected in an opposite way blood pressure, i.e. SNP produced falls and AVP increases in this parameter. 4. The relationship between the activity of the vascular and thermoregulatory systems in normothermic or febrile state is discussed.

Hypotensive drugs, sodium nitroprusside and prazosin inhibit the metabolic rate in rabbits.

1. Thermoregulatory responses (metabolic rate, rectal and ear skin temperatures, respiratory rate) were measured after treatment with two antihypertensive drugs, i.e. when sodium nitroprusside (6 mg/kg/2 hr) or prazosin (0.75 mg/kg/3 hr) were applied to normothermic or febrile rabbits under thermoneutral conditions (19 +/- 1 degree C). 2. Both drugs significantly decreased blood pressure and metabolic rate. 3. These changes were accompanied by antipyresis. 4. In the case of normothermic rabbits only sodium nitroprusside produced prominent falls in body temperature. 5. The relationship between changes in blood pressure and the intensity of metabolic rate is discussed.

The metabolic rate during the time course of salicylate antipyresis in the rabbit.

The processes concerned with the production and loss of body heat in sodium salicylate or acetylsalicylic acid antipyresis were investigated in adult rabbits at an ambient temperature of 21.5 +/- 0.5 degrees C. The experimental fever elicited by i.v. injection of lipopolysaccharide Escherichia coli (1 microgram/kg) was accompanied by increases in O2 consumption and CO2 production as well as decreases in convective heat loss. Pretreatment with 200 mg/kg of sodium salicylate (an hour's i.v. infusion) or with the same dose of acetylsalicylic acid (per os) significantly reduced pyrogen fever but the magnitude of O2 consumption and CO2 production remained at least at the febrile level. In the case of sodium salicylate, the level was even exceeded. At the same time both salicylates activated heat dissipation as manifested by decreases in vasomotor tone and tachypnea. Thus, it is apparent that the antipyretic effect of salicylates may develop without the inhibition of heat production. Heat loss processes initiated by these drugs are responsible for the antipyresis.