Two novel bombesin-like neuropeptides from the skin secretion of Pelophylax kl. esculentus: Ex vivo pharmacological characterization on rat smooth muscle types

Abstract

Mammalian bombesin-like neuropeptides (BLPs) play an important role in regulation of physiological and pathophysiological processes. Frog skin-derived BLPs, of smaller size and diverse lengths and sequences at their N-terminus, have attracted the attention of many researchers. However, these N-terminal variants and the receptors modulating their pharmacological actions are poorly studied and less understood. In this study, two BLPs, namely, [Asn³, Lys⁶, Thr¹⁰, Phe¹³]3-14-bombesin and [Asn³, Lys⁶, Phe¹³]3-14-bombesin with primary structures NLGKQWATGHFM and NLGKQWAVGHFM were isolated from the skin secretion of hybrid Pelophylax kl. esculentus. Both BLPs share a similar primary structure with only a single amino acid substitution at the eighth position (threonine to valine), while they have quite different myotropic potencies with EC₅₀ values in the range of 22.64 ± 9.7 nM (N = 8) to 83.93 ± 46.9 nM (N = 8). The potency of [Asn³, Lys⁶, Thr¹⁰, Phe¹³]3-14-bombesin was approximately 3-fold higher than that of [Asn³, Lys⁶, Phe¹³]3-14-bombesin. Through the investigation of receptor selectivity using a canonical bombesin receptor antagonist, it was found that [Asn³, Lys⁶, Thr¹⁰, Phe¹³]3-14-bombesin and [Asn³, Lys⁶, Phe¹³]3-14-bombesin had an affinity to both BB1 and BB2 receptors. Their contractile functions are mainly modulated by both BB1 and BB2 receptors on rat urinary bladder and BB2 alone on rat uterus smooth muscle preparations. These data may provide new insights into the design of potent and selective ligands for bombesin receptors. Moreover, [Asn³, Lys⁶, Thr¹⁰, Phe¹³]3-14-bombesin and [Asn³, Lys⁶, Phe¹³]3-14-bombesin did not induce significant hemolysis and toxicity in normal human cells, suggesting that these two natural novel BLPs have great potential for development into new drug candidates.

Keywords: N-terminal variants; bombesin receptor; bombesin-like neuropeptides (BLPs); frog skin; molecule cloning; pharmacological properties; smooth muscle.

FIGURE 1

RP-HPLC chromatogram of Pelophylax kl. esculentus skin secretion (240 min). The retention time of [Asn³, Lys⁶, Thr¹⁰, Phe¹³]3–14-bombesin and [Asn³, Lys⁶, Phe¹³]3–14-bombesin is indicated by arrows.

FIGURE 2

Detection of the cellular toxicities of [Asn³, Lys⁶, Thr¹⁰, Phe¹³]3–14-bombesin and [Asn³, Lys⁶, Phe¹³]3–14-bombesin. (A) Hemolysis of peptides on horse erythrocytes after 2 h of incubation. (B) Percentage of LDH release from HMEC-1 cells treated with peptides for 24 h; Error bar represents standard error mean (SEM) from three individual experiments, nine replicates. Statistical analysis was performed using the Kruskal–Wallis test in one-way ANOVA, (*p < 0.05, **p < 0.01 versus negative control). NS stands for not significant.

FIGURE 3

Dose–response curves of [Asn³, Lys⁶, Thr¹⁰, Phe¹³]3–14-bombesin (red) and [Asn³, Lys⁶, Phe¹³]3–14-bombesin (green) using (A) rat urinary bladder and (B) rat uterus smooth muscle preparations. Each point represents the mean of eight replicates, which are measurements of individual tissue preparations on different days, and bars represent the SEM.

FIGURE 4

Dose–response curves of [Asn³, Lys⁶, Thr¹⁰, Phe¹³]3–14-bombesin in the presence and absence of the BB1 antagonist and BB2 antagonist using (A) rat urinary bladder and (B) rat uterus smooth muscle preparations. Dose–response curves of [Asn³, Lys⁶, Phe¹³]3–14-bombesin in the presence and absence of BB1 antagonist and BB2 antagonist using (C) rat urinary bladder and (D) rat uterus smooth muscle preparations. Each point represents the mean of six replicates, which are measurements of individual tissue preparations on different days, and error bars represent SEM.

FIGURE 5

Contractile responses of [Asn³, Lys⁶, Thr¹⁰, Phe¹³]3–14-bombesin (1 μM) and [Asn³, Lys⁶, Phe¹³]3–14-bombesin (1 μM) on various smooth muscle preparations in the presence and absence of BB1 and BB2 receptor antagonists. (A) Rat urinary bladder smooth muscle preparations; (B) rat uterus smooth muscle preparations. Statistical significance of the difference was analyzed by using one-way ANOVA (*p < 0.05, ***p < 0.001), NS means not significant. Each error bar represents the SEM of six repeats which are measurements of individual tissue preparations on different days.