The tachykinin peptide family certainly represents one of the largest peptide families described in the animal organism. So far, more than 40 tachykinins have been isolated from invertebrate (insects, worms, and molluscs), protochordate, and vertebrate (skin, gastrointestinal tract, peripheral and central nervous system) tissues. Substance P (SP), first identified by bioassay as early as 1931 but sequenced only in 1971, several years after the elucidation of the structure of eledoisin from molluscan tissues and of physalaemin from amphibian skin, may be considered as a prototype of the tachykinins. Hitherto, as many as 19 tachykinins have been isolated from amphibian integument, and eight additional peptides have been isolated from amphibian gut and brain. Counterparts of skin tachykinins in mammalian tissues are SP, neurokinin A, and neurokinin B. Three main receptor subtypes for the tachykinins have been identified (NK1, NK2, and NK3), but their number is probably destined to increase. It is obvious that the peripheral and central effects of the tachykinins may substantially vary depending on the activation of different receptor subtypes. Matters are further complicated by the frequent capacity of the single tachykinins to bind, although with different affinity, to more receptors. It has been recognized that tachykinins have a variety of effects in physiological and pathological conditions, and there is evidence suggesting intrinsic neuroprotective and neurodegenerative properties of these neuropeptides. This review provides an update on the current body of knowledge regarding tachykinin occurrence and distribution in the animal kingdom, from the lowest invertebrates to man, and the physiological and pharmacological actions of tachykinins outlining the pregnant importance of this large peptide family.

Opiate-like peptides can regulate many cellular functions. We now map [D-Ala(2)]deltorphin I (DADTI)-like immunoreactivity (DADTI-LI) in developing mouse lung and analyze potential functional roles. Most DADTI-LI-positive cells were alveolar cells negative for prosurfactant protein (proSP)-C immunoreactivity. Peak numbers of DADTI-LI-positive cells occurred on embryonic Day 18, decreasing postnatally. To analyze developmental effects of DADTI, e17-18 lung explants were treated with [D-Ala(2)]deltorphin II (DADTII, soluble DADTI analogue, delta-receptor-specific) versus dermorphin (mu-receptor-specific). Type II pneumocyte differentiation, assessed by [(3)H]choline incorporation into saturated phosphatidylcholine and proSP-C immunostaining, was inhibited by DADTII but stimulated by dermorphin. Cell proliferation, measured as [(3)H]-thymidine incorporation and proliferating cell nuclear antigen immunostaining, was stimulated by DADTII and inhibited by dermorphin. All effects were dose-dependent. DADTII-inhibited choline incorporation was reversed by the delta-blocker, naltrindole. Unexpectedly, DADTII-stimulated thymidine incorporation was augmented by naltrindole and reversed by naloxone (mu-blocker). Although dermorphin-stimulated choline incorporation was appropriately blocked by binaltorphimine, dermorphin-inhibited thymidine incorporation was reversed by delta, kappa-, or mu-blockers. The delta- and mu-receptor messenger RNAs occurred pre- and postnatally, whereas kappa-receptor transcripts occurred mainly prenatally. All three receptor proteins were present in epithelial and mesenchymal cells in e18 lung. Thus, DADTI-LI from proSP-C-immunonegative alveolar cells could regulate development via both direct and indirect effects involving multiple opiate receptors.

Parallel bioassay on smooth muscle preparations demonstrated that: all TKs having a neutral or basic residue at position 7 from the C-terminus show a clear-cut preference for the NK1 TK receptor, reinforced by the presence of the aromatic doublet Phe-Phe or Phe-Tyr (aromatic TKs); all aliphatic TKs (Phe-Ile/Val) having an acidic residue at position 7 show a clear-cut preference for NK2/NK3 receptors, generally without selectivity for a single receptor. However, in aromatic TKs having the same acidic residue, the preference for NK2/NK3 receptors is weakened, with a more or less pronounced co-preference for the NK1 receptor. Amino acid substitutions in the C-terminal tripeptide may influence receptor affinity.

Morphologic and immunohistochemical studies were conducted to ascertain whether pumiliotoxin-B (PTX-B), an indolizine alkaloid from the skin of the Neotropical dendrobatid frog, Dendrobates pumilio, affects the anatomic and immunohistochemical features of the electrically stimulated mouse vas deferens preparations. PTX-B, at a concentration of 1 microM, consistently decreased the density pattern of neuropeptide Y (NPY)-immunoreactive nerve fibers contained within the circular muscular layer. The alkaloid also induced striking morphologic changes. It enlarged the lumen of the vasa and relaxed the muscular wall. Pretreatment with prazosin or haloperidol affected neither the release of NPY nor the morphologic changes; pretreatment with tetrodotoxin and guanethidine abolished NPY release and prevented the PTX-B-induced morphologic changes. PTX-B had no appreciable effect on the density and distribution pattern of nerve fibers immunostained for vasoactive intestinal polypeptide, substance P, calcitonin gene-related peptide, enkephalin, pancreatic polypeptide, 5-hydroxy-tryptamine and tyrosine hydroxylase.

Summary Upon electrical stimulation three transmitters are known to be released from the adrenergic nerve terminals of the isolated MVD preparation: two motor transmitters (noradrenaline (NA) and ATP) acting synergistically to provoke twitch contraction, and an inhibitory transmitter, the peptide NPY. The frog alkaloid pumiliotoxin‐B (PTX‐B) displayed two opposite effects on the electrically stimulated MVD: at low concentrations (0.1–0.3 μM) it caused twitch depression, at higher concentrations (0.5–2 μM) there was a potent twitch stimulation. Transmitters and/or receptors involved in the depressive effect could not be clearly identified, although interference with NPY is possible. On the other hand, the potent twitch stimulation caused by PTX‐B may be due to exaggerated release of the same transmitters (NA and ATP) involved in twitch stimulation produced by electrical stimulation. Opening by PTX‐B of the Na+ channels on the membrane of the adrenergic nerve terminals causes activation of the amine pump facilitating re‐uptake of not only endogenous NA but also of exogenous catecholamines.

In vivo antinociception studies demonstrate that deltorphins are opioid peptides with an unusually high blood-brain barrier penetration rate. In vitro, isolated bovine brain microvessels can take up deltorphins through a saturable nonconcentrative permeation system, which is apparently distinct from previously described systems involved in the transport of neutral amino acids or of enkephalins. Removing Na+ ions from the incubation medium decreases the carrier affinity for deltorphins (-25%), but does not affect the Vmax value of the transport. The nonselective opiate antagonist naloxone inhibits deltorphin uptake by brain microvessels, but neither the selective delta-opioid antagonist naltrindole nor a number of opioid peptides with different affinities for delta- or mu-opioid receptors compete with deltorphins for the transport. Binding studies demonstrate that mu-, delta-, and kappa-opioid receptors are undetectable in the microvessel preparation. Preloading of the microvessels with L-glutamine results in a transient stimulation of deltorphin uptake. Glutamine-accelerated deltorphin uptake correlates to the rate of glutamine efflux from the microvessels and is abolished by naloxone.

An antiserum raised against [D-Ala(2)]deltorphin-I (DADTI: Sr-D-Ala Phe-Asp-Val-Val-Gly . NH2), a highly selective ligand for delta-opioid receptors, was applied for the immunohistochemical study of positive brain structures during postnatal development and in. the adult rat. On. postnatal day 0, neuronal cell bodies containing DADTI-like immunoreactivity were widely distributed in various brain regions. These included the olfactory tubercle, ventral pallidum, hippocampal CA2 and CA3 subfields, ventral tegmental area, pars compacta of the substantia nigra, supramammillary nucleus and dorsal raphe nucleus. Immunoreactive nerve fibres were observed in the main and accessory olfactory bulbs, olfactory tubercle, prefrontal cortex, cingulate cortex, neostriatum, nucleus accumbens, Lateral septal nucleus, Lateral habenular nucleus, optic tract, lateral geniculate nucleus and superior colliculus. As pups grew, positive cell bodies decreased gradually in both density and intensity, and those in the olfactory tubercle, ventral pallidum and hippocampus had disappeared by postnatal day 14. A distribution pattern resembling that in the adult was reached on postnatal day 21. Positive cell bodies were found only in the ventral midbrain including the pars compacta of the substantia nigra, ventral tegmental area, A8 region and supramammillary nucleus. Intensely stained positive nerve fibres could be traced along the medial forebrain bundle. Dense positive terminals occupied the neostriatum, nucleus accumbens shell, olfactory tubercle, septal areas, cingulate and medial prefrontal cortex. Double immunostaining revealed that, in the substantia nigra, almost all (97.8%) the DADTI-positive neurons co-localized tyrosine hydroxylase (TH) representing about one third (29.1%) of the total population of TH-positive neurons. These DADTI/TH-positive cells showed poor staining for calbindin D-28KThe results indicate that [D-Ala(2)]deltorphin-I-like molecule(s) may play an important role in early postnatal development of certain neuronal systems. Furthermore, in the adult, they intervene solely in. certain dopaminergic system functions.

We compared the in vitro and in vivo biological activities of PG-KII (pGlu-Pro-Asn-Pro-Asp-Glu-Phe-Val-Gly-Leu-Met-NH2), a new peptide belonging to the tachykinin family, related to kassinin, isolated and sequenced from extracts of the skin of the Australian myobatrachid frog Pseudophryne güntheri, with those of the well-known tachykinins [substance P (SP), neurokinin A (NKA), neurokinin B (NKB), and kassinin (KASS)] to study its pharmacological and receptor profile. PG-KII always proved inactive in the in vitro and in vivo (gastric emptying) NK2 bioassays. It resulted equipotent to SP and more potent than KASS, NKA, and NKB in all in vitro smooth muscle preparations preferentially activated by the NK1-selective agonists. On an in vivo NK3 receptor-mediated function, gastric acid secretion, PG-KII had a potency similar to that of NKB. In contracting guinea pig ileum, which contains NK1, NK2, NK3, and also new tachykinin receptor subtypes, PG-KII was more potent than SP, NKB, and NKA. The cholinergic antagonist, atropine, significantly reduced the guinea pig contractile activity of both PG-KII and NKB but not that of SP or NKA. Pretreatment with the NK1-selective antagonist, CP 96,345, and with the NK2-selective antagonist, MEN 10,376, modified neither the in vivo nor the in vitro effects of PG-KII. These findings indicate that PG-KII is neither an NK1 nor an NK2 receptor agonist but has a spectrum of biological actions close to that of the NK3 receptor agonists. PG-KII elicits strong contractile activity in guinea pig ileum. Administered centrally in the rat it regulates inhibition of gastric acid secretion.

As many as 55 neogastropod molluscs, all belonging to the Muricoidea superfamily, have been investigated for occurrence and contents, in their hypobranchial gland (HG), of choline esters and, subordinately, biogenic amines. Very high amounts of esters, strictly localized in the median area of the HG, were found in all dye-secreting molluscs. The choline esters were represented by murexine, dihydromurexine and senecioylcholine. A fourth ester, acryloylcholine, occurred in the HG of a single, non dye-secreting mollusc. All the compounds displayed potent neuromuscular blocking actions in all examined vertebrate and invertebrate species, as well as potent nicotinic actions. Muscarinic effects were either lacking or unimportant. In addition to choline esters the HG occasionally contained known and hitherto unknown biogenic amines: tyramine, octopamine, 5-hydroxytryptamine. histamine, urocanylhistamine and imidazole-propionylhistamine. The interest of extending the search of bioactive compounds to carnivorous, predatory molluscs other than those described in this paper and, more, extensively, to any molluscan species provided with 'venomous' glands or apparatuses, is emphasized.

In an immunohistochemical study, a specific antiserum raised against [D-Ala2]deltorphin-I (DADTI), a highly selective ligand for delta opioid receptors, was used to demonstrate immunoreactive structures in the rat retina. [D-Ala2]Deltorphin-I immunoreactivity occurred in a subpopulation of the retinal amacrine cells situated in the inner nuclear layer. Their stained processes were mainly distributed in the sublaminae 1 and 3 of the inner plexiform layer. A few positive cells, probably displaced amacrine cells, were also seen in the ganglion cell layer. Double immunostaining revealed that 12.8% of DADTI-immunoreactive cells costored GABA and 27.7% costored neuropeptide Y, whereas only few DADTI-positive cells colocalized tyrosine hydroxylase (0.3%) and almost no other peptides. These findings suggest that some retinal amacrine cells possess DADTI-like molecule(s), possibly acting as neurotransmitter(s) or neuromodulator(s).

[D‐Ala2]deltorphin‐I, a highly selective ligand for δ opioid receptors, is a heptapeptide originally purified from frog skin. Previous immunohistochemical studies indicate that [D‐Ala2]deltorphin‐I‐like molecule(s) may be present in adult rat brain, including specific neuronal cells and fibers partially overlapping with the mesocortical and nigrostriatal dopaminergic systems. Here, we examined the developmental aspect of such immunoreactive brain structures in early postnatal rats. In newborn to 21‐day‐old rats, positive staining in the brain occurred mainly in subpopulations of neurons and occasionally in tanycytes. On postnatal day 0, neuronal cell bodies containing [D‐Ala2]deltorphin‐I‐like immunoreactivity were found in various brain regions, including the olfactory tubercle, ventral pallidum, hippocampus, ventral tegmental area, pars compacta of the substantia nigra, supramammillary nucleus, and dorsal raphe nucleus. Immunoreactive nerve fibers were observed in the main and accessory olfactory bulbs, olfactory tubercle, prelimbic area, anterior cingulate cortex, neostriatum, accumbens, lateral septal nucleus, lateral habenular nucleus, and superior colliculus. As pups grew, positive staining of cell bodies decreased gradually in both density and intesity, and those in the olfactory tubercle and ventral pallidum were no longer visible on postnatal day 14. On postnatal day 21, positive cells were found only in the ventral midbrain, including the pars compacta of the substantia nigra, ventral tegmental area, A8 region, and supramammillary nucleus. Positive fibers also decreased in density with age except in the accessory olfactory bulb, olfactory tubercle, prelimbic area, and anterior cingulate cortex. © 1994 Wiley‐Liss, Inc.

A novel 17 amino acid peptide, having a d-leucine in position 2 of its sequence, has been isolated from methanol extracts of the skin of the Brazilian frog, Phyllomedusa burmeisteri. The sequence of the peptide is: Tyr-d-Leu-Phe-Ala-Asp-Val-Ala-Ser-Thr-Ile-Gly-Asp-Phe-Phe-His-Ser-Ile-NH2. It displays a poor affinity for δ-opioid binding sites, both in the periphery and in the central nervous system. However, the shorter synthetic amidated analogue (1–10) possesses both on the central and peripheral δ binding sites an agonistic potency equalling in affinity and exceeding in selectivity that of the enkephalins. The shorter amidated analogue (1–7) is virtually inactive on opioid binding sites in the periphery, but displays a clear-cut affinity for both δ and μ binding sites on rat brain membranes. To date six different d-amino acid residues have been found, always in position 2 of the sequence, in as many as 11 natural peptide molecules of animal origin.

The dried skin secretion from Phyllomedusa bicolor, 'sapo', is used by the Matses Indians of the Northern Peru, in shamanic rites mainly designed to improve luck in hunting. When rubbed into burned, exposed areas of the skin, the drug causes the prompt appearance of violent peripheral gastrointestinal and cardiovascular effects soon followed by remarkable central effects (increase in physical strength, heightening of senses, resistance to hunger and thirst, exalted capacity to face stress situations). All the peripheral and most of the central effects of 'sapo' can be ascribed to the exceptionally high content of the drug (up to 7% of its weight) in potently active peptides, easily absorbed through the burned, inflamed areas of the skin. The concentration in 'sapo' of the single peptides (phyllocaerulein, phyllomedusin, phyllokinin, demorphins and deltorphins) has been determined by bioassay, and peptide contents were correlated with the different symptoms of the 'sapo' intoxication.

Using a specific antiserum recently raised against [D-Ala2]deltorphin I (DADTI: Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH2), a highly selective ligand for delta-opioid receptors, we have previously demonstrated the occurrence of positive immunostaining in several structures of mouse brain. We describe here the neuroanatomical distribution patterns of DADTI-immunoreactive neuronal bodies, axons, and tanycytes in rat brain. Positive neuronal somata were localized mainly in the ventral mesencephalon, including the ventral tegmental area and the pars compacta of the substantia nigra. A minor population of positive somata was found in the pars reticulata and pars lateralis of the substantia nigra, raphe nuclei, supramammillary nucleus, and retrorubral reticular nucleus. All these regions, except for the supramammillary nucleus, contain dopamine cell bodies. Intensely stained positive nerve fibers could be traced along the medial forebrain bundle. Dense positive terminals were seen in the neostriatum, nucleus accumbens shell, olfactory tubercle, septal areas, cingulate, and medial prefrontal cortex. Double-immunostaining study revealed that, in the substantia nigra, almost all (97.8%) DADTI-positive neurons colocalized with tyrosine hydroxylase (TH), and the doubly stained cells occupied about one-third (29.1%) of the total population of TH-positive neurons. Only a few DADTI/TH-positive cells also stained for 28-kDa calbindin D, although many neurons double-stained for 28-kDa calbindin D and TH. In contrast, the supramammillary nucleus contained a number of DADTI-positive cells, which nearly always stained positively for 28-kDa calbindin D but did not stain for TH. The association of DADTI-like immunoreactivity with certain dopaminergic pathways seems of particular interest. A small population of DADTI-immunostained tanycytes was present in the ventral part of the third ventricle wall.

A series of imidazo[2,1-b]thiazoles bearing halogens or a sulfonylurea group or an imidazolidone group, were synthesized and subjected to pre- and post-emergence herbicidal tests. 5-Bromo-6-(3-pyridyl)-2,3-dihydroimidazo[2,1-b]thiazole (4e) and 6-(2,3,4-trichlorophenyl)-2,3-dihydroimidazo[2,1-b]thiazole-5-carboxylic acid (8b) showed moderate activity in the post-emergence herbicidal tests only.

Three naturally occurring dermorphin-like peptides from the skin of the frog Phyllomedusa bicolor, the related carboxyl-terminal amides, and some substituted analogs were synthesized, their binding profiles to opioid receptors were determined, and their biological activities were studied in isolated organ preparations and intact animals. The opioid binding profile revealed a very high selectivity of these peptides for mu sites and suggested the existence of two receptor subtypes, of high and low affinity. The peptides tested acted as potent mu opioid agonists on isolated organ preparations. They were several times more active in inhibiting electrically evoked contractions in guinea pig ileum than in mouse vas deferens. When injected into the lateral brain ventricle or peritoneum of rats, the high-affinity-site-preferring ligand, [Lys7-NH2]dermorphin, behaved as a potent analgesic agent. By contrast, the low-affinity-site-preferring ligand, [Trp4,Asn7-NH2]dermorphin, produced a weak antinociception but an intense catalepsy.