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

Pharmacological Research 52 (2005) 283–289

The contractile effects of several substituted short analogues of porcine galanin in isolated rat jejunal and colonic smooth muscle strips A. Umer a , H. Ługowska a , J. Sein-Anand b , P. Rekowski c , J. Ruczy´nski c , J. Petrusewicz a , R.P. Korolkiewicz a,∗ b

a Department of Pharmacology, Medical University of Gda´ nsk, Do Studzienki 38, 80-227 Gda´nsk, Poland Department of Internal Medicine & Toxicology, Medical University of Gda´nsk, D˛ebinki 7, 80-211 Gda´nsk, Poland c Faculty of Chemistry, University of Gda´ nsk, Sobieskiego 18, 80-952 Gda´nsk, Poland

Accepted 4 May 2005

Abstract 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 ␮M, with a characteristic fall-down effect at the supramaximal concentrations. Gal(1–15) was less potent than Gal(1–29). Furthermore, [d-Trp2 ]Gal(1–15), [endoTrp2 ,Cle4 ]Gal(1–15), [d-Leu4 ]Gal(1–15), [des-Leu4 ]Gal(1–15), [Hse6 ]Gal(1–15), [Dab14 ]Gal(1–15), [Dpr14 ]Gal(1–15) or [Arg14 ]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. © 2005 Elsevier Ltd. All rights reserved. Keywords: Rat; Galanin; Smooth muscle

1. Introduction Galanin (Gal) is a biologically active neuropeptide, discovered by Tatemoto et al. [1], with immunoreactivities widely distributed in the central and peripheral nervous systems, respiratory, gastrointestinal and urogenital tracts of several mammalian species including man [2]. Gal, named after its N-terminal glycine and C-terminal alanine amide residues displays a plethora of physiological and behavioral effects [3–5], whereas Gal structure is conserved among vertebrates and all peptide molecules, except for the tuna fish Gal, share a 14 amino acid N-terminal and a variant COOHterminal region. Gal acts at least at three subtypes of specific G-protein coupled membrane receptors influencing a vari∗

Corresponding author. Fax: +48 58 349 18 11. E-mail address: [email protected] (R.P. Korolkiewicz).

1043-6618/$ – see front matter © 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.phrs.2005.05.001

ety of metabolic pathways including among others adenylyl cyclase and phospholipase C [3]. Gal regulates gastrointestinal motility and secretion in a species and locus-dependent manner [3,6–8]. However, due to the lack of specific antagonist of Gal in the gut, it is difficult to differentiate between its pharmacological and physiological actions and therefore its actual involvement in the regulatory processes in the digestive tract remains largely unknown. In our current experiments we have studied 15 amino acid porcine Gal analogues substituted in positions 2, 3, 4, 6 and 14 in rat isolated jejunal and colonic smooth muscle strips used as an in vitro assay of peptide contractile activity [9,10]. We aimed at describing the putative differences in structure–activity relationship of jejunal and colonic smooth muscle response to Gal and its analogues in comparison to rat gastric fundus [11].

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2. Materials and methods 2.1. Animals and tissue preparation All procedures were designed and conducted in accordance with the generally accepted ethical standards and the guidelines established by the Animal Welfare Committee of the Medical University of Gda´nsk, Poland. Albino-Wistar rats of either gender (180–250 g) were kept in normal laboratory conditions, with standard chow pellets and tap water available ad libitum. Animals were fasted overnight before experiments, sacrificed by cervical dislocation and intact tubular jejunal or proximal colonic tissue segments were prepared similarly to the method described by Vane [12]. All tissues mounted at 2.0 g of resting tension in water-jacketed glass chambers (15 ml), filled with carbogen-gassed Tyrode solution (mM): NaCl 136.9, KCl 3.35, CaCl2 1.46, MgCl2 1.03, NaHCO3 11.9, NaH2 PO4 0.48, glucose 5.0 at 37 ◦ C (pH 7.2–7.4) were allowed to equilibrate for 90 min before the beginning of experiment. The buffer was changed every 5 min, except for the contact time of the test peptides with tissues. The registration of mechanic activity of longitudinal smooth muscle was conducted isotonically with PIT 212 force displacement transducers (COTM, Białystok, Poland) connected to a TZ-4100 line recorder (laboratorni Pristroje, Prague, Czech Republic). 2.2. Concentration–response curves for Gal and Gal-analogues Experiments were started when reproducible contractile response to 10 and 30 nM carbamylocholine was obtained.

Carbachol-induced contractions of the longitudinal smooth muscle were unaffected by 10 ␮M amastatin and leupeptin, 1 mM PMSF and 1 ␮M phosphoramidon. Thus, all subsequently used solutions contained the protease and peptidase inhibitors. Besides, the experimental solutions contained a mixture of 1 ␮M atropine, 10 ␮M hexametonium, guanethidine and TTX, respectively. Conventional, concentration–contraction, non-cumulative curves were constructed. The contact time of the peptide with muscle strips ranged from 1 to 3 min. As soon as the peak contraction had developed, tissues were washed out until the length of the strip returned to the basal level. To avoid tachyphylaxis to peptides, the substances were applied at 20 min intervals, buffer changed every 5 min. Each tissue fragment was used to construct only two, non-cumulative, concentration–response curves, using one analogue on each strip: a control one for Gal(1–29)/Gal(1–15) and another one for the respective peptide analogue, applied alternately. The viability and reproducible contractility of each strip was examined at the end of each experimental session by a submaximal contractile response to carbachol, at the same concentration as at the start. 2.3. Drugs Atropine sulphate, guanethidine, hexamethonium, carbachol, phosphoramidon, phenylmethanesulphenyl fluoride (PMSF), were obtained from Sigma (St. Louis, MO, USA). Crystalline TTX was obtained from Sankyo Co., Ltd. (Tokyo, Japan). Other chemicals were purchased from P.P.H. Polskie Odczynniki Chemiczne (Gliwice, Poland). Gal peptides were synthesized using the method described elsewhere [13].

Table 1 The primary structures of the synthesized peptides Peptide

Amino acid sequence

Gal(1–29)NH2 Gal(1–15)NH2 [d-Trp2 ]Gal(1–15)NH2 [endo-Trp2a ,Cle4 ]Gal(1–15)NH2 [des-Thr3 ,Cle4 ]Gal(1–15)NH2 [Pro4 ]Gal(1–15)NH2 [Cle4 ]Gal(1–15)NH2 [des-Leu4 ]Gal(1–15)NH2 [Ile4 ]Gal(1–15)NH2 [d-Leu4 ]Gal(1–15)NH2 [Nle4 ]Gal(1–15)NH2 [Val4 ]Gal(1–15)NH2 [Nva4 ]Gal(1–15)NH2 [Hse6 ]Gal(1–15)NH2 [Nle14 ]Gal(1–15)NH2 [Asp14 ]Gal(1–15)NH2 [Orn14 ]Gal(1–15)NH2 [Arg14 ]Gal(1–15)NH2 [Dab14 ]Gal(1–15)NH2 [Cit14 ]Gal(1–15)NH2 [Lys14 ]Gal(1–15)NH2 [Dpr14 ]Gal(1–15)NH2

G-W-T-L-N-S-A-G-Y-L-L-G-P-H-A-I-D-N-H-R-S-F-H-D-K-Y-G-L-A-NH2 G-W-T-L-N-S-A-G-Y-L-L-G-P-H-A-NH2 G-d-Trp-T-L-N-S-A-G-Y-L-L-G-P-H-A-NH2 G-Trp-W-T-Cle-N-S-A-G-Y-L-L-G-P-H-A-NH2 G-W- ∼ -Cle-N-S-A-G-Y-L-L-G-P-H-A-NH2 G-W-T-Pro-N-S-A-G-Y-L-L-G-P-H-A-NH2 G-W-T-Cle-N-S-A-G-Y-L-L-G-P-H-A-NH2 G-W-T- ∼ -N-S-A-G-Y-L-L-G-P-H-A-NH2 G-W-T-Ile-N-S-A-G-Y-L-L-G-P-H-A-NH2 G-W-T-d-Leu-N-S-A-G-Y-L-L-G-P-H-A-NH2 G-W-T-Nle-N-S-A-G-Y-L-L-G-P-H-A-NH2 G-W-T-Val-N-S-A-G-Y-L-L-G-P-H-A-NH2 G-W-T-Nva-N-S-A-G-Y-L-L-G-P-H-A-NH2 G-W-T-L-N-Hse-A-G-Y-L-L-G-P-H-A-NH2 G-W-T-L-N-S-A-G-Y-L-L-G-P-Nle-A-NH2 G-W-T-L-N-S-A-G-Y-L-L-G-P-Asp-A-NH2 G-W-T-L-N-S-A-G-Y-L-L-G-P-Orn-A-NH2 G-W-T-L-N-S-A-G-Y-L-L-G-P-Arg-A-NH2 G-W-T-L-N-S-A-G-Y-L-L-G-P-Dab-A-NH2 G-W-T-L-N-S-A-G-Y-L-L-G-P-Cit-A-NH2 G-W-T-L-N-S-A-G-Y-L-L-G-P-Lys-A-NH2 G-W-T-L-N-S-A-G-Y-L-L-G-P-Dpr-A-NH2

∼: removed amino acid residue.

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Phosphoramidon, PMSF, hexamethonium were dissolved in a small volume of absolute ethanol. Amastatin and TTX were dissolved in 100 mM acetic acid. All agents were adjusted to the desired volume with Tyrode solution (Table 1). 2.4. Statistical analysis of the results Results are expressed as a percentage of the maximum response induced by each peptide. The concentration– contraction curves for control peptides: Gal(1–29) and Gal(1–15) have been pooled for all experiments, whereas for the sake of clarity the results concerning Gal analogues have been presented in three groups depending on the place of amino acid substitution in peptide chain and the smooth muscle strips in which experiments have been conducted. EC50 and the slope of the concentration–response curves are expressed as means with 95% confidence limits. EC50 , the slopes of the dose–response curves, relative potencies of Gal analogues, Hill coefficients and their statistical significance were determined [14,15] and compared using a non-parametric Mann–Whitney, Wilcoxon signed-rank test for pairs or one-way analysis of variance (ANOVA) plus Bonferroni post-ANOVA tests, where required. Two-tailed p values of less than 0.05 were taken to indicate a significant difference.

3. Results Gal(1–29) or Gal(1–15) evoked concentration-dependent contractions of rat colonic and jejunal smooth muscle strips yielding typical response curves in the range from 0.3 nM to 10 ␮M, with a characteristic fall-down effect at supramaximal concentrations. Gal(1–15) was remarkably less potent than Gal(1–29) and therefore its concentration–response curves were to the right of Gal(1–29) (Fig. 1). On the other hand, the calculated Hill coefficient did not significantly differ from unity for any of the constructed curves (Table 2). The substitutions of Trp in position 2 with d-Trp or addition of a second l-Trp residue in position 2 in [endo-Trp2a ] combined in the latter case with replace-

Fig. 1. Non-cumulative concentration–response curves of jejunal and colonic smooth muscle strips exposed to Gal(1–29) or Gal(1–15). Data have been normalized as percentages of the maximal response to a particular peptide and plotted against log peptide concentration. Data are presented as means ± S.E.M. for at least 5–10 experiments performed on different tissue strips.

ment of Leu by Cle in position 4 of the peptide chain and the deletion of Leu [des-Leu4 ] or exchanges of Leu in position 4 for d-Leu or Ser in position 6 for Hse, respectively, evoked a considerable decrease in potency of test peptides in both jejunal and colonic smooth muscle cells. On the other hand, [des-Thr3 ,Cle4 ]Gal(1–15) was almost equipotent to Gal(1–15) tissues (Figs. 2–4). The values of Hill coefficient differed from unity in the case of [endo-Trp2 ,Cle4 ]Gal(1–15), [Ile4 ]Gal(1–15), [Nle4 ]Gal(1–15) in jejunum, [d-Leu4 ]Gal(1–15) in colon, [des-Thr3 ,Cle4 ]Gal(1–15) and [Val4 ]Gal(1–15) in both tissues (Tables 3–5). The substitution of His in position 14 with Arg, Dab or Dap has significantly diminished the potency of test peptides in colonic and jejunal/colonic smooth muscle strips. The values of Hill coefficient were significantly different from unity for [Asp14 ]Gal(1–15), [Orn14 ]Gal(1–15), [Dpr14 ]Gal(1–15), [Nle14 ]Gal(1–15), [Arg14 ]Gal(1–15), [Dab14 ]Gal(1–15), [Cit14 ]Gal(1–15) and [Lys14 ]Gal(1–15) in studied tissues (Tables 6 and 7).

Table 2 A comparison of pharmacological properties of Gal(1–29)NH2 and Gal(1–15)NH2 in jejunal and colonic smooth muscle strips Test peptides

Smooth muscle tissue Jejunum

EC50 (nM) Relative potency Slope Hill coefficient

Colon

Gal(1–29)NH2

Gal(1–15)NH2

Gal(1–29)NH2

Gal(1–15)NH2

66.52 ± 15.25 1 46.87 ± 2.14 1.00 ± 0.09

164 ± 0.26 28.42 ± 2.91*** 0.91 ± 0.03

17.67 ± 4.21 1 46.79 ± 4.37 0.9 ± 0.026

89.82 ± 6.33*** 0.20 31.51 ± 0.63* 1 ± 0.05

30***

Data are expressed as mean values ± S.E.M. Potency of each peptide (EC50 ) was calculated from the appropriate concentration–response curve performed on different tissue fragments (n = 5–12). Relative potency was described as the ratio of the equi-effective concentrations of each peptide obtained from their respective concentration–effect relationships. Statistical comparisons performed vs. respective controls using an unpaired, non-parametric Wilcoxon test. * p < 0.05 vs. Gal(1–29)NH . 2 *** p < 0.001 vs. Gal(1–29)NH . 2

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Table 3 A comparison of pharmacological properties of Gal(1–15) analogues in jejunal and colonic smooth muscle strips Peptide

EC50

Relative potency

Slope

Hill coefficient

Jejunum Gal(1–15) [d-Trp2 ]Gal(1–15) [endo-Trp2a ,Cle4 ]Gal(1–15) [des-Thr3 ,Cle4 ]Gal(1–15)

164 3.80 8.06 110

± ± ± ±

30.0 nM 1.25 ␮M* 1.48 ␮M*** 20.0 nM

1 0.06 0.02 1.49

28.42 43.67 33.08 35.85

± ± ± ±

2.91 0.74* 0.8 1.65

0.91 0.96 0.72 0.72

± ± ± ±

0.03 0.06 0.05† 0.01†

Colon Gal(1–15) [d-Trp2 ]Gal(1–15) [endo-Trp2a ,Cle4 ]Gal(1–15) [des-Thr3 ,Cle4 ]Gal(1–15)

89.82 610 361 101

± ± ± ±

6.33 nM 80.0 nM*** 57.12 nM** 9.20 nM

1 0.20 0.24 0.97

31.51 45.08 47.38 29.89

± ± ± ±

0.63 4.92* 1.52* 0.94

1.0 1.12 0.89 0.69

± ± ± ±

0.05 0.04 0.01 0.05†

Data are expressed as mean values ± S.E.M. Potency of each peptide (EC50 ) was calculated from the appropriate concentration–response curve performed on different tissue fragments (n = 5–12). Relative potency was described as the ratio of the equi-effective concentrations of each peptide obtained from their respective concentration–effect relationships. Statistical comparisons performed vs. respective controls using ANOVA plus Bonferroni post-ANOVA test. † Significantly different from 1.0 (p value < 0.001). * p < 0.05 vs. Gal(1–15). ** p < 0.01 vs. Gal(1–15). *** p < 0.001 vs. Gal(1–15).

Table 4 A comparison of pharmacological properties of Gal(1–15) analogues in jejunal smooth muscle strips Peptide

EC50

Gal(1–15) [Pro4 ]Gal(1–15) [Cle4 ]Gal(1–15) [des-Leu4 ]Gal(1–15) [Ile4 ]Gal(1–15) [d-Leu4 ]Gal(1–15) [Nle4 ]Gal(1–15) [Val4 ]Gal(1–15) [Nva4 ]Gal(1–15) [Hse6 ]Gal(1–15)

164 219 526 5.09 270 1.55 160 170 204 1.79

± ± ± ± ± ± ± ± ± ±

30.0 nM 52.62 nM 57.38 nM 0.73 ␮M*** 60.0 nM 0.15 ␮M* 90.0 nM 30.0 nM 10.98 nM 0.22 ␮M***

Relative potency

Slope

1 0.78 0.84 0.04 0.62 0.13 0.36 0.96 0.78 0.08

28.42 44.42 34.05 45.29 35.95 58.92 38.39 34.89 39.34 43.23

Hill coefficient ± ± ± ± ± ± ± ± ± ±

2.91 3.14*** 1.07 2.87*** 1.46 3.38*** 1.38* 0.72 1.59* 2.29***

0.91 0.89 0.83 0.90 0.68 1.04 0.71 0.64 0.76 0.76

± ± ± ± ± ± ± ± ± ±

0.03 0.06 0.03 0.05 ±0.02‡ 0.12 0.05‡ 0.03† 0.01 ±0.01

Data are expressed as mean values ± S.E.M. Potency of each peptide (EC50 ) was calculated from the appropriate concentration–response curve performed on different tissue fragments (n = 6–10). Relative potency was described as the ratio of the equi-effective concentrations of each peptide obtained from their respective concentration–effect relationships. Statistical comparisons performed vs. respective controls using ANOVA plus Bonferroni post-ANOVA test. † Significantly different from 1.0 (p value < 0.005). ‡ Significantly different from 1.0 (p value < 0.001). * p < 0.05 vs. Gal(1–15). *** p < 0.001 vs. Gal(1–15).

Table 5 A comparison of pharmacological properties of Gal(1–15) analogues in colonic smooth muscle strips Peptide

EC50

Gal(1–15) [Pro4 ]Gal(1–15) [Cle4 ]Gal(1–15) [des-Leu4 ]Gal(1–15) [Ile4 ]Gal(1–15) [d-Leu4 ]Gal(1–15) [Nle4 ]Gal(1–15) [Val4 ]Gal(1–15) [Nva4 ]Gal(1–15) [Hse6 ]Gal(1–15)

89.82 131 224 1.07 144 0.72 185 179 146 1.39

± ± ± ± ± ± ± ± ± ±

6.33 nM 7.24 nM 27.99 nM 0.08 ␮M*** 21.63 nM 0.14 ␮M*** 33.05 nM 18.10 nM 12.97 nM 0.19 ␮M***

Relative potency

Slope

1 0.75 0.45 0.08 0.67 0.11 0.68 0.52 0.68 0.06

31.51 34.79 32.08 41.17 39.27 60.89 28.71 31.48 30.90 45.23

Hill coefficient ± ± ± ± ± ± ± ± ± ±

0.63 0.67 0.47 1.17*** 0.60*** 2.27*** 0.73 1.13 0.40 2.20***

1 0.77 0.78 0.95 0.92 1.46 0.85 0.68 0.79 1.17

± ± ± ± ± ± ± ± ± ±

0.05 0.01 0.03 0.03 0.03 0.15‡ 0.03 0.05† 0.01 0.07

Data are expressed as mean values ± S.E.M. Potency of each peptide (EC50 ) was calculated from the appropriate concentration–response curve performed on different tissue fragments (n = 6–10). Relative potency was described as the ratio of the equi-effective concentrations of each peptide obtained from their respective concentration–effect relationships. Statistical comparisons performed vs. respective controls using ANOVA plus Bonferroni post-ANOVA test. † Significantly different from 1.0 (p value < 0.005). ‡ Significantly different from 1.0 (p value < 0.001). *** p < 0.001 vs. Gal(1–15).

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Table 6 A comparison of pharmacological properties of Gal(1–15) analogues in jejunal smooth muscle strips Peptide

EC50 (nm)

Gal(1–15) [Nle14 ]Gal(1–15) [Asp14 ]Gal(1–15) [Orn14 ]Gal(1–15) [Arg14 ]Gal(1–15) [Dab14 ]Gal(1–15) [Cit14 ]Gal(1–15) [Lys14 ]Gal(1–15) [Dpr14 ]Gal(1–15)

164 269 259 213 239 141 82.61 180 408

± ± ± ± ± ± ± ± ±

30 29.71 25.92 24.35 18.40 9.74 12.35 3.90 48.01***

Relative potency

Slope

1 0.61 0.63 0.77 0.68 1.17 1.98 0.91 0.44

28.42 36.09 36.15 39.57 36.31 39.43 29.04 44.56 33.31

Hill coefficient ± ± ± ± ± ± ± ± ±

2.91 1.43 1.70 3.2** 1.03 0.80* 1.12 0.73*** 1.06

0.91 0.85 0.69 0.72 1.01 0.89 0.79 0.92 0.79

± ± ± ± ± ± ± ± ±

0.03 0.03 0.05*** 0.08** 0.06 0.02 0.01 0.09 0.02*

Data are expressed as mean values ± S.E.M. Potency of each peptide (EC50 ) was calculated from the appropriate concentration–response curve performed on different tissue fragments (n = 9–10). Relative potency was described as the ratio of the equi-effective concentrations of each peptide obtained from their respective concentration–effect relationships. Statistical comparisons performed vs. respective controls using ANOVA plus Bonferroni post-ANOVA test. * p < 0.05 vs. Gal(1–15). ** p < 0.01 vs. Gal(1–15). *** p < 0.001 vs. Gal(1–15).

Table 7 A comparison of pharmacological properties of Gal(1–15) analogues in colonic smooth muscle strips Peptide

EC50

Gal(1–15) [Nle14 ]Gal(1–15) [Asp14 ]Gal(1–15) [Orn14 ]Gal(1–15) [Arg14 ]Gal(1–15) [Dab14 ]Gal(1–15) [Cit14 ]Gal(1–15) [Lys14 ]Gal(1–15) [Dpr14 ]Gal(1–15)

89.82 81.0 109 94.02 218 164 48.87 79.63 171

± ± ± ± ± ± ± ± ±

6.33 nM 4.98 nM 18.48 nM 5.59 nM 9.29*** nM 22.82 nM 18.02 nM 23.19 nM 16.08 nM

Relative potency

Slope

1 1.24 0.87 0.96 0.41 0.53 2.04 1.84 0.60

31.51 27.90 36.85 31.96 36.33 33.68 28.26 33.60 32.76

Hill coefficient ± ± ± ± ± ± ± ± ±

0.63 0.51 1.84* 0.81 1.42 1.46* 0.54 3.05 0.87

1 0.77 1.1 0.76 0.80 0.73 0.64 0.67 0.89

± ± ± ± ± ± ± ± ±

0.05 0.02 0.04 0.03 0.08 0.04 0.01 0.01 0.07

Data are expressed as mean values ± S.E.M. Potency of each peptide (EC50 ) was calculated from the appropriate concentration–response curve performed on different tissue fragments (n = 9–10). Relative potency was described as the ratio of the equi-effective concentrations of each peptide obtained from their respective concentration–effect relationships. Statistical comparisons performed vs. respective controls using ANOVA plus Bonferroni post-ANOVA test. * p < 0.05 vs. Gal(1–15). *** p < 0.001 vs. Gal(1–15).

Fig. 2. Non-cumulative concentration–response curves of jejunal smooth muscle strips exposed to test agents modified in positions 2, 3 and 4 of peptide chain. Data have been normalized as percentages of the maximal response to a particular peptide and plotted against log peptide concentration. Data are presented as means ± S.E.M. for at least 7–12 experiments performed on different tissue strips.

Fig. 3. Non-cumulative concentration–response curves of colonic smooth muscle strips exposed to test agents modified in positions 2, 3 and 4 of peptide chain. Data have been normalized as percentages of the maximal response to a particular peptide and plotted against log peptide concentration. Data are presented as means ± S.E.M. for at least 7–12 experiments performed on different tissue strips.

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Fig. 4. Non-cumulative concentration–response curves of jejunal smooth muscle strips exposed to test agents modified in position 4 of peptide chain. Data have been normalized as percentages of the maximal response to a particular peptide and plotted against log peptide concentration. Data are presented as means ± S.E.M. for at least 5–9 experiments performed on different tissue strips.

4. Discussion Throughout all of our group’s work synthetic Gal analogues were used, although we realize that porcine rather than rat galanin may have affected the range of the biological actions observed in the course of experiments. On the other hand, Rossowski et al. have shown that the potency of rat Gal is comparable to porcine-Gal in rat jejunum [16]. Consistent with the other observations, it is confirmed that partial Gal sequences do not fully satisfy the structural requirements for the full potency and efficacy of the peptide in rat jejunum or colon, similarly to rat stomach, pancreas, guinea-pig taenia coli, canine small intestine and rabbit iris sphincter [7,9,17–19]. Although Gal(1–15) possessed significant myogenic activity in jejunum and colon, it remained about 2.5 and 5.1 times weaker than native Gal(1–29) in respective tissues, an outcome differing from the results of Katsoulis et al. [9], but matching those of Lagny-Pourmir et al. [20] and Ekblad et al. [18]. In the light of the published data, it seems reasonable to state that the biological activity of Gal depends mainly on the N-terminal half of the peptide moiety [9,19–23]. However, the C-terminal seems important for preserving the full potency and efficacy of Gal molecule in dog jejunum, guinea pig taenia coli, rat jejunum and colon [7,18]. The values of Hill coefficients calculated from their concentration–contraction experiments performed in jejunum and colon, were not different from unity for both Gal(1–29) and Gal(1–15), indicating an interaction of one ligand molecule with one receptor according to the classical receptor theory [24]. Similarly to rat stomach, the substitution of amino acids in position 2 and/or 4 of peptide chain have considerably weakened the myogenic activity of test pep-

tides in jejunal and colonic smooth muscle strips, reaching statistical significance in the case of [d-Trp2 ]Gal(1–15), [endo-Trp2a ,Cle4 ]Gal(1–15), [d-Leu4 ]Gal(1–15) and [desLeu4 ]Gal(1–15). On the other hand, the strength of [desThr3 ]Gal(1–15) compared to Gal(1–15) was not different in smooth muscle cells of the small and large bowel strips. The exchange of Ser to Hse in position 6 diminished the effect of test peptide in jejunum and colon, whereas it remained ineffective in rat gastric fundus [19,25]. The values of Hill coefficients calculated from the respective concentration–contraction curves for peptides modified in positions 2, 3, 4 and 6 were either not markedly different from or below unity, implying in the latter case a heterogenity of binding sites or a negative ligand–receptor cooperativity [24]. The only exemption was a concentration–contraction curve constructed in colonic strips using [d-Leu4 ]Gal(1–15). Hill coefficient was above unity, which might indicate a positive ligand–receptor cooperativity [24,26]. We have previously shown that the substitution of histidine in position 14 with Asp, Nle, Cit and Dab rendered such analogues of Gal(1–15) more active than the parent molecule in gastric fundus [25]. In contrast, this phenomenon has not been observed in jejunum and colon, moreover [Dpr14 ]Gal(1–15), [Dab14 ]Gal(1–15), [Arg14 ]Gal(1–15) contracted smooth muscle strips of jejunum or colon in a markedly less potent way than Gal(1–15). Moreover, [Lys14 ]Gal(1–15) has not displayed any features of a partial agonist at Gal receptors of the small or large bowel, which were noted in the stomach [26,27]. In summary, our previous and current work provides functional evidence showing that the integrity of both N- and Cterminals of Gal molecule are necessary for peptide to exert a full excitatory potential in gastric, jejunal and colonic smooth muscle. Amino acids located in positions 2, 4, 6 and 14 of the peptide play a crucial role in determining the molecule potency and affinity to Gal receptors in studied tissues. However, the similarities and differences between gastric fundus, jejunal or colonic smooth muscle cells cannot be explained based on the results of our experiments alone.

Acknowledgments This work was partly supported by a grant no. BW-80005-0189-4 from the University of Gda´nsk and a grant from the Medical University of Gda´nsk, Poland.

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