Locustatachykinin III and IV: two additional insect neuropeptides with homology to peptides of the vertebrate tachykinin family

Two myotropic peptides termed locustatachykinin III and IV were isolated from 9000 brain-corpora cardiaca-corpora allata-suboesophageal ganglion extracts of the locust, Locusta migratoria. The primary structures of Lom-TK III and IV were established as amidated decapeptides: Ala-Pro-Gln-Ala-Gly-Phe-Tyr-Gly-Val-Arg-NH2 (Lom-TK III) and Ala-Pro-Ser-Leu-Gly-Phe-His-Gly-Val-Arg-NH2 (Lom-TK IV). The locustatachykinins were synthesized and shown to have chromatographic and biological properties identical with those of the native materials. They stimulate visceral muscle contractions of the oviduct and the foregut of Locusta migratoria and of the hindgut of Leucophaea maderae. Both peptides exhibit sequence homologies with the vertebrate tachykinins. Sequence similarity is greater with the fish and amphibian tachykinins (up to 40%) than with the mammalian tachykinins. In addition, the intestinal and oviducal myotropic activity of the locustatachykinins is analogous to that of vertebrate tachykinins. Both chemical and biological similarities of vertebrate and insect tachykinins substantiates the evidence for a long evolutionary history of the tachykinin peptide family.     

Locustakinin, a novel myotropic peptide from Locusta migratoria, isolation, primary structure and synthesis.

The isolated hindgut of the cockroach, Leucophaea maderae is a very efficient bioassay tool for the monitoring of certain structural types of insect myotropic peptides during HPLC purification. Using this detection system, a six residue peptide has been isolated from an extract of 9000 brain corpora cardiaca-corpora allata suboesophageal ganglion complexes of Locusta migratoria. Amino acid composition and sequence analysis combined with enzymatic digestion data established the structure of the novel peptide as Ala-Phe-Ser-Ser-Trp-Gly-amide. The chromatographic and biological properties of the synthetic peptide were the same as those of the native peptide, thus confirming structural analysis. The carboxy-terminal pentamer sequence is the active core of leucokinins II, V and VII and of achetakinin III (myotropic neuropeptides isolated from Leucophaea m. and from Acheta domesticus; Holman et al., 1990). Furthermore, the octapeptide leucokinin VII contains the novel sequence as its carboxy-terminal hexamer and Achetakinin V (AFHSWGamide) differs from it by one residue. This new peptide designated as locustakinin I (locusts) may therefore represent an evolutionary molecular link between leucokinin VII (cockroaches) and achetakinin V (crickets). Using synthetic locustakinin, physiological studies will be performed in the locust. In view of the known effects of leucokinins, locustakinin may be important in the stimulation of ion transport and inhibition of diuretic activity in Malpighian tubules. This study indicates that the AFXSWGamide sequence appears to have been well conserved and that members of this peptide family may be widely distributed among insects and posses a number of functions.     

Comparative pharmacological actions of leucokinins V-VIII on the visceral muscles of Leucophaea maderae

1. Leucokinins V-VIII (Lem-K-V to VIII) did not activate visceral muscles of the cockroach Leucophaea maderae uniformly as a group but rather showed a selective action on the muscles of the hindgut. This organ showed a contractile response to all of the leucokinins at 3 x 10(-10) M that was 2-20% above the mean level of spontaneous activity. The maximum response for each peptide was recorded at 2.1 x 10(-7) M. 2. Both the foregut and the oviduct were 100- to 1000-fold less sensitive than the hindgut, and each of the former required more than 10(-8) M to elicit a detectable excitation. The heart, by comparison, did not respond to any of these peptides. 3. The leucokinins caused a protracted excitation of contractile events in the hindgut that lasted for more than 60 min. Moreover, all four peptides evoked contractions from hindguts after membrane depolarization with 158 mM potassium. These results suggest that nonsynaptic receptors for the peptides exist in visceral muscle.     

Sites of action of a peptide neurohormone that controls hindgut muscle activity in the cockroach Leucophaea maderae

A peptide neurohormone from the brain and nervous system of the Madeira cockroach Leucophaea maderae has stimulating effects on both the mechanical and electrical events of hindgut visceral muscle. The peptide initiated action potentials at silent recording sites in the circular muscles of the rectum after prior treatment with tetrodotoxin (10⁻⁶ g/ml). The neurohormone also caused an increase in the amplitude and frequency of spontaneous postsynaptic potentials. However, the isolated hindgut failed to respond to the neurohormone after depolarization in high potassium saline solutions. Both the potassium contracture and the action of the neurohormone were calcium dependent.Although some hindguts were responsive to the neurohormone in a Ca free medium, such preparations failed to respond in 0·5 mM EGTA. Moreover, 1 mM Mn blocked the action of the peptide. The sodium ion was also essential for effective hormone action. These results suggest the presence of a loosely bound source of Ca at the surface of muscle membranes that in some way interacts with the neurohormone to change muscle excitability.     

EFFECTS OF 5,6-DIHYDROXYTRYPTAMINE ON MONOAMINERGIC NEURONES IN THE CENTRAL NERVOUS SYSTEM OF THE RAT

—The injection of 50 μg of 5,6-dihydroxytryptamine (5,6-HT) into a lateral ventricle of the rat depleted the spinal cord and various regions of the brain of indoleamines (presumably 5-HT) and 5-hydroxyindole acetic acid. The concentrations of 5-HT were measured by two different methods: the formation of a fluorescent derivative with o-phthalaldehyde, and the native fluorescence in hydrochloric acid. When the results of both methods were compared on the pons and medulla 4 days after injecting 5,6-HT, the loss in indoleamine appeared to be greater when o-phthalaldehyde was used. This suggests that the two methods may be measuring different compounds. According to both methods, the loss of 5-HT persisted for several days after the injection of 5,6-HT, but by 2 months 5-HT concentrations (measured only by the native fluorescence procedure), had recovered to near-normal values. The depletion of 5-HT was most pronounced in regions adjacent to the ventricular system and in the spinal cord. Initially, caudate and septum were more affected on the side of the injection, and later showed some permanent atrophy. The injection of up to 50 μg of 5,6-HT did not lead to any significant loss of noradrenaline or dopamine from the brain, or to any reduction in the activity of the enzyme tyrosine hydroxylase. The drug was a potent inhibitor of the uptake of [³H]5-HT by brain slices, but was less effective in inhibiting catecholamine uptake systems. These observations suggest a preferential action on tryptaminergic neurones. Larger doses of 5,6-HT caused a loss of catecholamines and tyrosine hydroxylase from the brain, and were severely toxic.