Teresa M. Kubiak

Pharmacology of FMRFamide-related Peptides in Helminths

Abstract: Nervous systems of helminths are highly peptidergic. Species in the phylum Nematoda (roundworms) possess at least 50 FMRFamide‐related peptides (FaRPs), with more yet to be identified. To date, few non‐FaRP neuropeptides have been identified in these organisms, though evidence suggests that other families are present. FaRPergic systems have important functions in nematode neuromuscular control. In contrast, species in the phylum Platyhelminthes (flatworms) apparently utilize fewer FaRPs than do nematodes; those species examined possess one or two FaRPs. Other neuropeptides, such as neuropeptide F (NPF), play key roles in flatworm physiology. Although progress has been made in the characterization of FaRP pharmacology in helminths, much remains to be learned. Most studies on nematodes have been done with Ascaris suum because of its large size. However, thanks to the Caenorhabditis elegans genome project, we know most about the FaRP complement of this free‐living animal. That essentially all C. elegans FaRPs are active on at least one A. suum neuromuscular system argues for conservation of ligand‐receptor recognition features among the Nematoda. Structure‐activity studies on nematode FaRPs have revealed that structure‐activity relationship (SAR) “rules” differ considerably among the FaRPs. Second messenger studies, along with experiments on ionic dependence and anatomical requirements for activity, reveal that FaRPs act through many different mechanisms. Platyhelminth FaRPs are myoexcitatory, and no evidence exists of multiple FaRP receptors in flatworms. Interestingly, there are examples of cross‐phylum activity, with some nematode FaRPs being active on flatworm muscle. The extent to which other invertebrate FaRPs show cross‐phylum activity remains to be determined. How FaRPergic nerves contribute to the control of behavior in helminths, and are integrated with non‐neuropeptidergic systems, also remains to be elucidated.

FMRFamide‐like peptides encoded on the flp‐18 precursor gene activate two isoforms of the orphan Caenorhabditis elegans G‐protein‐coupled receptor Y58G8A.4 heterologously expressed in mammalian cells

Two alternatively spliced variants of an orphan Caenorhabditis elegans G‐protein‐coupled receptors (GPCRs; Y58G8A.4a and Y58G8A.4b) were cloned and functionally expressed in Chinese hamster ovary (CHO) cells. The Y58G8A.4a and Y58G8A.4b proteins (397 and 433 amino acid residues, respectively) differ both in amino acid sequence and length of the C‐terminal tail of the receptor. A calcium mobilization assay was used as a read‐out for receptor function. Both receptors were activated, with nanomolar potencies, by putative peptides encoded by the flp‐18 precursor gene, leading to their designation as FLP‐18R1a (Y58G8A.4a) and FLP‐18R1b (Y58G8A.4b). Three Ascaris suum neuropeptides AF3, AF4, and AF20 all sharing the same FLP‐18 C‐terminal signature, ‐PGVLRF‐NH2, were also potent agonists. In contrast to other previously reported C. elegans GPCRs expressed in mammalian cells, both FLP‐18R1 variants were fully functional at 37°C. However, a 37 to 28°C temperature shift improved their activity, an effect that was more pronounced for FLP‐18R1a. Despite differences in the C‐terminus, the region implicated in distinct G‐protein recognition for many other GPCRs, the same signaling pathways were observed for both Y58G8A.4 isoforms expressed in CHO cells. Gq protein coupling seems to be the main but not the exclusive signaling pathway, because pretreatment of cells with U‐73122, a phospholipase inhibitor, attenuated but did not completely abolish the Ca2+ signal. A weak Gs‐mediated receptor activation was also detected as reflected in an agonist‐triggered concentration‐dependent cAMP increase. The matching of the FLP‐18 peptides with their receptor(s) allows for the evaluation of the pharmacology of this system in the worm in vivo.

Importance of the proline residue to the functional activity and metabolic stability of the nematode FMRFamide-related peptide, KPNFIRFamide (PF4)

PF4 has previously been shown to have potent inhibitory effects on myoactivity of somatic muscle strips from the nematode. Ascaris suum. This study examined the bioactivity and metabolic stability of position 2- and position 5-modified analogues of PF4. Although the analogues [Leu5]PF4,[Ala2]PF4, [Gly2]PF4, [Ala2,Leu5]PF4, and [Gly2,Leu5]PF4 all had qualitatively similar inhibitory effects on A. suum somatic muscle strips, their effects were quantitatively distinguishable and had the order of potency: PF4 = [Leu5]PF4 > > [Ala2]PF4 = [Ala2,Leu5]PF4 > > [Gly2]PF4 = [Gly2,Leu5]PF4, Leu5 for Ile5 substitutions in PF4 did not alter the activity of this peptide: however, Gly2/Ala2 for Pro2 substitutions reduced, but did not abolish, peptide activity. Peptide stability studies revealed that [Gly2]PF4(2-7) and -(3-7) and [Ala2]PF4(2-7), -(3-7), and -(4-7) fragments were generated following exposure to A. suum somatic muscle strips. However, the parent peptide (PF4) was not metabolized and appeared to be resistant to the sequential cleavages of native aminopeptidases. Observed analogue metabolism appeared to be due to the activity of released aminopeptidases as identical fragments were generated by incubation in medium that had been exposed to somatic muscle strips and from which the strips had been removed prior to peptide addition. It was found that the muscle stretching and bath mixing characteristics of the tension assay led to more effective release of soluble enzymes from muscle strips and thus greater peptide degradation. These studies reveal that Pro2 in PF4 is not essential for the biological activity of this peptide; however, it does render the peptide resistant to the actions of native nematode aminopeptidases.

Synthetic Analogues of Growth Hormone Releasing Factor (GHRF) with Improved Pharmaceutical Properties

The protein hormone, somatotropin or growth hormone (GH), is essential for normal somatic growth. The biologic activity of GH is species specific. Currently, recombinant human GH is used for increasing growth in children with short stature, while recombinant bovine growth hormone is registered for enhancing lactation in cows. The secretion of GH from the anterior pituitary is controlled by two hypothalamic peptides, somatostatin (inhibitory) and growth hormone releasing factor (GHRF, stimulatory). GHRF offers a potential alternative to GH in applications where increased circulating concentrations of GH are desirable.