Tachykinin family genes and their receptors are differentially expressed in the hypothyroid ovary and pituitary.

Plasma tachykinin levels are known to be altered with sexual acyclicity and loss of reproductive function. Ovulatory dysfunction, as seen in postmenopausal women, is also often encountered in hypothyroid patients. To know the involvement of different tachykinin genes in hypothyroidism-associated reproductive disorders, we performed DD-PCR with the pituitary RNA of control and hypothyroid rats to see the differentially expressed gene profile. Subsequently, we selected a few clones, tachykinin being one of them. Since its expression was up regulated in hypothyroidism as it does in the sexually acyclic females, we wanted to correlate these two phenomena with hypothyroidism associated reproductive disorders. We observed differential expression of tac2 along with other tk genes and their receptors in rat pituitary and ovary, which suggests that hypothyroidism affects the expression of these genes in these tissues. The experiments were repeated in ovarian tissue obtained at surgery from hypothyroid human patients, which showed similar expression pattern of TAC3 (equivalent to rat tac2) and their receptors as in rat ovary. Significant reduction of tac2 expression in reproductively less active rat ovary suggests the association of tac2 with reproductive senescence. Our results suggest that decline in reproductive function in hypothyroidism is associated with altered expression level of tac2 and its receptors. Further investigation in this area could elucidate the possible mechanism of tachykinins' involvement in loss of sexual cyclicity and other reproductive disorders associated with hypothyroidism.

Neuronal expression of tachykinin-related peptides and gene transcript during postembryonic development of Drosophila.

The gene Dtk, encoding the prohormone of tachykinin-related peptides (TRPs), has been identified from Drosophila. This gene encodes five putative tachykinin-related peptides (DTK-1 to 5) that share the C-terminal sequence FXGXRamide (where X represents variable residues) as well as an extended peptide (DTK-6) with the C-terminus FVAVRamide). By mass spectrometry (MALDI-TOF-MS), we identified ion signals with masses identical to those of DTK-1 to 5 in specific brain regions. We have analyzed the distribution of the Dtk transcript and peptides, by in situ hybridization and immunocytochemistry during postembryonic development of the central nervous system (CNS) of Drosophila. Antiserum against a cockroach TRP that cross-reacts with the DTKs was used for immunocytochemistry. Expression of transcript and peptides was detected from first to third instar larvae, through metamorphosis to adult flies. Throughout postembryonic development, we were able to follow the strong expression of TRPs in a pair of large descending neurons with cell bodies in the brain. The number of TRP-expressing neuronal cell bodies in the brain and ventral nerve cord increases during larval development. In the early pupa (stage P8), the number of TRP-expressing cell bodies is lower than in the third instar larvae. The number drastically increases during later pupal development, and in the adult fly about 200 TRP-expressing neurons can be seen in the CNS. The continuous expression of TRPs in neurons throughout postembryonic development suggests specific functional roles in both larval and imaginal flies and possibly also in some neurons during pupal development.

The troubled story of tachykinins and neurokinins.

A family of peptides that shares a common C-terminal sequence (Phe-X-Gly-Leu-MetNH2) exists in mammalian and non-mammalian species. In mammals, three of these peptides (substance P, neurokinin A and neurokinin B) satisfy the criteria to be considered as neurotransmitters either in the central, peripheral or enteric nervous systems. In addition, multiple receptors for these peptides, which belong to the superfamily of G-protein-coupled receptors, exist. These receptors have distinct pharmacological features and selective agonists and antagonists are available for studying their functional roles. The latest update on nomenclature of these peptides and their receptors, which dates back to 1986, agreed to use the terms tachykinins and tachykinin NK1, NK2 and NK3 receptors. This 'nomenclature mismatch' has generated confusion that urges experts in the field of tachykinin research to provide a revised nomenclature.

Neuropeptide families: evolutionary perspectives.

Examination of neuropeptide families can provide information about phyletic relationships and evolutionary processes. In this article the oxytocin/vasopressin family, growth hormone releasing factor (GRF) superfamily and the substance P/tachykinin family have been considered in detail because they have been isolated from an extraordinarily diverse array of species from several vertebrate classes and invertebrate phyla. More important is that the nucleotide sequence of mRNA or cDNA encoding many of these peptides has been determined, which has allowed evolutionary distances to be estimated based on the DNA mutation rate. The origin of a given family lies in a primordial gene that arose many millions of years ago, and through time, exon duplication and insertion, gene duplication, point mutation and exon loss, the family developed into the forms that are now recognised. For example, in birds, GRF and pituitary adenylate cyclase activating peptide (PACAP) are encoded by the same gene, which probably arose as a result of exon duplication and tandem insertion of the ancestral GRF gene. In mammals GRF is the sole product on one gene, and PACAP is the product of a gene that also produces PACAP-related peptide (PRP), which is homologous to GRF. Thus it appears that between birds and mammals the GRF/PACAP gene duplicated: exon loss gave rise to the mammalian GRF gene, while mutation led to the formation of the mammalian PRP/PACAP gene. The neuropeptide Y superfamily is considered briefly, as is cionin, which is an invertebrate peptide that is closely related to the mammalian gastrin/cholecystokinin family.

Identification of multiple tachykinins in bovine adrenal medulla using an improved chromatographic procedure.

Comparison of data based on the reverse-phase HPLC with two ion-pairing reagents, trifluoroacetic acid (TFA) and heptafluorobutyric acid (HFBA), together with the use of two antibodies, has allowed us to identify the various tachykinins in the bovine adrenal medulla. The results show that substance P-like, neurokinin B-like, and neurokinin A-like (including its extended forms, neuropeptide K and neuropeptide gamma) immunoreactivity are present in the bovine adrenal medulla. The concentration of SP-like immunoreactivity in the adrenal medulla was found to be substantially higher than that of NKA-like and NKB-like immunoreactivity. The strategy described here, using radioimmunoassay combined with HPLC employing TFA and HFBA as the ion-pairing reagents, should be useful for the identification of tachykinins and other peptides in the central and peripheral nervous system.

Identification and cDNA sequence of delta-preprotachykinin, a fourth splicing variant of the rat substance P precursor.

The neuropeptides substance P and neurokinin A are synthesised from a family of precursor polypeptides encoded by the preprotachykinin A (PPT) gene. In addition to a mRNA (beta-PPT) containing all 7 exons of the gene, alternatively spliced mRNAs lacking either exon 4 (gamma-PPT) or exon 6 (alpha-PPT) have been identified. We have determined the sequences of cDNA clones encoding four variants of PPT mRNA from rat dorsal root ganglion (DRG), including a novel mRNA species (delta-PPT) in which both exons 4 and 6 are absent. The sequence of delta-PPT predicts the existence of a novel tachykinin precursor polypeptide.