François Petitet

Possible existence of a new tachykinin receptor subtype in the guinea pig ileum.

The guinea pig ileum possesses NK-1 and NK-3 tachykinin receptors. As expected, [Pro9]SP and senktide, which are selective agonists of NK-1 and NK-3 receptors, respectively, were found to be highly potent in contracting the guinea pig ileum. Surprisingly, similar observations were made with septide, SP-O-CH3, [Apa9-10]SP, or [Pro9,10]SP although, in contrast to [Pro9]SP, these four peptides showed a low affinity for 3H-[Pro9]SP-specific NK-1 binding sites on membranes from the guinea pig ileum. They were also devoid of affinity for NK-2 and NK-3 binding sites. GR 71251, a compound which has been described as a NK-1 antagonist, was more potent in inhibiting the septide- than the [Pro9]SP-evoked contracting response. Altogether, these results suggest that septide, [Apa9-10]SP, and [Pro9,10]SP exert their high contracting activity in the guinea pig ileum by acting on a new subtype of tachykinin receptors.

Selective agonists of NK-2 binding sites highly active on rat portal vein (NK-3 bioassay)

All the synthetized NKA and NKA (4-10) agonists have been found active in the rat portal vein bioassay. Even [Lys5, MeLeu9, Nle10] NKA(4-10), a highly potent competitor of NK-2 binding sites with very low binding potencies for NK-1 and NK-3 sites (IC50 greater than microM) is still active in contracting the rat portal vein. These results suggest that this tissue contains not only a fairly large population of NK-3 receptors but also a minor population of NK-2 receptors. Comparison of the activities of NKA C-terminal analogues on the guinea-pig ileum suggests that 1) only a small population of NK-2 receptors are present in this tissue and 2) beside NK-1, NK-2 and NK-3 receptors, another type of receptor sensitive to C-terminal sequences might be present in the guinea-pig tissue.

Higher potency of RP 67580, in the mouse and the rat compared with other nonpeptide and peptide tachykinin NK1 antagonists

1 This study was undertaken to compare the potency and selectivity of the nonpeptide (RP 67580, (±)‐CP‐96,345 and its chloro‐derivative [(±)‐cis‐3‐(2‐chlorobenzylamino)‐2‐benzhydrylquinuclidine] (CP‐C1)) and peptide (GR 71,251 and spantide) neurokinin1 (NK1) antagonists in mouse and rat preparations. 2 Among the NK1 antagonists tested, RP 67580 was the most potent in inhibiting the specific binding of [125I]‐Bolton Hunter substance P ([125I]‐BHSP) to crude synaptosomes from the rat brain (Ki: 2.9 nm). (±)‐CP‐96,345 was about ten fold less potent (Ki: 31 nm) than RP 67580 while other compounds exhibited even less affinity. 3 All NK1 antagonists inhibit competitively the activation of phospholipase C by [Pro9]substance P ([Pro9]SP) in cultured cortical astrocytes from the newborn mouse, a preparation rich in NK1 receptors but devoid of NK2 and NK3 receptors. pA2 values for the most potent compounds, RP 67580 and (±)‐CP‐96,345, were 8.28 and 7.08 respectively. When used alone, all antagonists showed some agonist activity at 10−5 m, except spantide which was already effective at 10−6 m. 4 An excellent correlation was found between the potency of the NK1 antagonists in blocking the stimulation by [Pro9]SP of phosphoinositide breakdown in cortical astrocytes and in inhibiting [125I]‐BHSP specific binding to rat brain synaptosomes. 5 As shown on single cells by use of the Indo‐1 microfluorometric method, RP 67580 (10−7 m) prevented reversibly the elevation of cytosolic calcium concentration induced by [Pro9]SP (10−8 m) in cultured cortical astrocytes. 6 Several experiments indicated that the antagonists were highly selective for NK1 receptors. RP 67580 did not modify the noradrenaline‐evoked activation of phospholipase C in cortical astrocytes; when used at 10−5 m all antagonists had no or only little affinity for NK2 or NK3 binding sites and did not block the NKA (10−8 m)‐induced activation of phospholipase C in the hamster urinary bladder (a selective NK2 test). 7 In conclusion, RP 67580 appears to be a potent NK1 antagonist in the mouse and the rat. Results obtained with (±)‐CP‐96,345 confirm the lower potency of this compound in these two species when compared with reported data obtained in the guinea‐pig or man.

Selective agonists of tachykinin binding sites.

Summary— Three types of binding sites for the mammalian tachykinins, ie Substance P (SP) Neurokinin A (NKA) and Neurokinin B (NKB), have been found in both the central and peripheral nervous systems. Substance P binds to the NK‐1 subclass of binding site while NKA and NKB are less selective endogenous ligands, which preferentially interact with the NK‐2 and NK‐3 subclasses of binding sites, respectively. Complementary strategies, including 3‐dimensional structure analysis by NMR spectroscopy and structure‐activity relationships led to the design of selective agonists of these binding sites. [Pro9] SP, [Pro10] SP and the cyclic analogues [Cys3,6, Tyr8, Pro9] SP and [Cys3,6, Tyr8, Pro10] SP are selective NK‐1 agonists. [Lys5] NKA(4–10) is a water soluble NK‐2 potent agonist. Finally, [Pro7] NKB, which completely discriminates NK‐2 and NK‐3 binding sites, is a water‐soluble NK‐3 selective agonist.

Synergistic Regulation of Cytosolic Ca2+ Concentration in Mouse Astrocytes by NK1 Tachykinin and Adenosine Agonists

The effects on cytosolic Ca2+ concentration of 2‐chloroadenosine and [L‐Pro9]‐substance P, a selective agonist of NK1 receptors, were investigated on astrocytes from embryonic mice in primary culture. Cells responded to [L‐Pro9]‐ substance P with a transitory increase in cytosolic Ca2+ which was of shorter duration when external Ca2+ was removed. A transient response to 2‐chloroadenosine alone occurred. When simultaneously applied, [L‐Pro9]‐substance P and 2‐chloroadenosine evoked a prolonged elevation of cytosolic Ca2+ (up to 30 min). This phenomenon was dependent on the presence of extracellular Ca2+, but insensitive to dihy‐dropyridines, La3+, and Co2+, excluding the implication of voltage‐operated Ca2+ channels. Arachidonic acid also induced a sustained elevation of cytosolic Ca2+, but did not increase further the response evoked by [L‐Pro9]‐substance P and 2‐chloroadenosine. The activation of protein kinase C by a diacylglycerol analogue mimicked the effect of [L‐Pro9]‐substance P in potentiating the 2‐chloroadenosine‐evoked response. Like 2‐chloroadenosine, pinacidil, which hyper‐polarizes the cells by opening K+ channels, prolonged the elevation of cytosolic Ca2+ concentration induced by [L‐Pro9]‐substance P. Conversely, depolarization with 50 mM KC1 canceled the effects of either pinacidil or 2‐chloroadenosine applied with [L‐Pro9]‐substance P. Pertussis toxin pretreatment suppressed all the effects induced by 2‐chloroadenosine.

NK-1 receptors are the only class of tachykinin receptors found on mouse cortical astrocytes

Extending our previous studies, our results indicate that cultured cortical astrocytes from the mouse possess only NK-1 receptors coupled to phospholipase C. An excellent correlation was found in the potency of tachykinins and selective analogs at inhibiting 125I-BHSP binding and at stimulating phospholipase C activity, their rank order being that of NK-1 receptors. No binding sites could be found with ligands of NK-2 or NK-3 receptors. No additive effect could be shown with NK-2 or NK-3 agonists when phospholipase C activity was estimated with high concentrations of NK-1 agonists. C- or N-terminal SP fragments did not modify SP- or [Pro9]SP-evoked responses.

NK-1 tachykinin receptor in rat and guinea pig brains: Pharmacological and autoradiographical evidence for a species difference

The affinities of a large variety of peptide or nonpeptide tachykinin analogues were determined on membranes from rat and guinea pig brains using the selective NK-1 radioligand 3H-[Pro9]SP. Nonpeptide antagonists clearly revealed a species difference; (+/-)CP-96,345 was more potent in the guinea pig, while RP 67580 was found to be a better competitor of 3H-[Pro9]SP binding to rat brain membranes. This was confirmed on brain slices by autoradiography. Numerous brain structures were analyzed by optical densitometry. From these data, a heterogeneity of NK-1 binding sites among different structures can be excluded in both rat and guinea pig brains.

Differential localization of3H-[Pro9]SP binding sites in the guinea pig and rat brain

Abstract Due to the existence of differences in the pharmacological properties of tachykinin NK-1 receptors in the rat and the guinea pig, the autoradiographic distribution of NK-1 binding sites was compared in the brain of the two species using the selective NK-1 ligand3H-[Pro9]SP. If a good similarity in the distribution of NK-1 binding sites could be seen in basal ganglia, a relative absence of correlation was observed between the estimated optical densities in other brain structures of the two species. For instance, the interpeduncular nucleus, the lateral habenular nucleus and the deep layers of the cerebral cortex were labeled in the guinea pig but not in the rat while the reverse was observed for the columns of the vermis lobules 9–10, the dorsal raphe nucleus, the medial habenular nucleus, the superficial cortical layers and the dorsal hippocampus. Furthermore, the high similarity found in the localization of125I-BHSP (a non selective ligand) and3H-[Pro9]SP binding sites, does not suggest the existence of NK-1 binding site subtypes in the guinea pig brain.

17 - Tachykinin Receptors: Binding and Cellular Activity Assays

Publisher Summary This chapter discusses the technical aspects of the methods related to treatment of tachykinin (TK) receptors. TKs represent a family of structurally related peptides that share a common C-terminal amino acid sequence, exhibit different rank order of potency according to the tissue investigated, and show some analogy in their biological effects. Mammalian TK are derived from large precursors encoded by two genes, namely, the preprotachykinins A (PPTA) and preprotachykinins B (PPTB) genes. The PPTA large precursors generate substance P (SP) alone or both SP and neurokinin A (NKA), SP and neuropeptide K (NPK), or SP and NPγ through alternative splicing, whereas the PPTB precursors give rise to neurokinin B (NKB) alone. NK1 receptors are characterized by their preferential affinity for SP. NK2 receptors are characterized by their preferential affinity for NKA. NK3 receptors are characterized by their preferential affinity for NKB. Binding studies have demonstrated that TK analogs recognize TK receptors, but the agonist or antagonist properties of these compounds have been demonstrated using biological assays on peripheral isolated organs. It is reported that dog carotid artery, rabbit pulmonary artery, and rat portal vein distinguish responses are mediated by NK1, NK2, and NK3 receptors. However, there is not always a perfect correlation between the relative potencies of TK or analogs in binding studies and those in assays made on these peripheral organs.

A new selective bioassay for tachykinin NK3 receptors based on inositol monophosphate accumulation in the guinea pig ileum

The selective agonists of tachykinin NK1, NK2 and NK3 receptors, respectively [Pro9]substance P, [Lys5,MeLeu9,Nle10]neurokinin A-(4-10) and senktide, stimulated phosphoinositide breakdown in slices of the guinea pig ileum. This was also the case with septide which has recently been found to act on a new type of tachykinin receptors in this tissue. The NK1, NK2 and septide-evoked responses were completely antagonized in the combined presence of (+/-)-CP-96,345 and MEN 10,376 which are potent and selective antagonists of tachykinin NK1 and NK2 receptors respectively in the guinea pig ileum. Like senktide, other available NK3 receptor agonists, such as [MePhe7]neurokinin B, [MeVal7]neurokinin B, [Pro7]neurokinin B and DiMe-C7, stimulated phosphoinositide hydrolysis in either the absence or combined presence of (+/-)-CP-96,345 and MEN 10,376, although senktide was the most potent. Therefore, following the blockade of tachykinin NK1, NK2 and septide-sensitive receptors, the accumulation of inositol monophosphate appears to be a valuable, rapid and sensitive bioassay for determining the activity of NK3 receptor agonists and putative NK3 receptor antagonists.