Monique Saffroy

Localization of tachykinin binding sites (NK1, NK2, NK3 ligands) in the rat brain.

A comparative autoradiographic analysis of the distribution of tachykinin binding sites was made on brain serial sections using several ligands. (1) 3H-SP, 125I-BHSP and 3H-physalaemin labeled identical binding sites (NK1 type). (2) 3H-NKB, 125I-BHE and 3H-eledoisin also labeled identical sites (NK3 type). (3) 125I-BHNKA preferentially labeled NK3 binding sites, the distribution of 125I-BHNKA binding sites being identical to that of 3H-NKB or 125I-BHE binding sites. (4) The distributions of 3H-SP and 3H-NKB binding sites were markedly different. (5) A very low density of labeling was found with 3H-NKA or 125I-NKA, and these binding sites were distributed only in areas rich in either 3H-SP or 3H-NKB binding sites. (6) Particular efforts were made to look for the presence of tachykinin binding sites in the substantia nigra, since this structure is particularly rich in SP and NKA and contains functional tachykinin receptors of the NK1 and NK2 types as suggested by physiological studies. Confirming previous reports, low or very low labeling was observed in the substantia nigra with 3H-SP or 125I-BHSP and 3H-NKB or 125I-BHE. Similar results were found with 3H-NKA, 125I-NKA or 125I-BHNKA. In conclusion, our data do not provide evidence yet for the existence of NK2 binding sites in the rat brain.

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.

Autoradiographic distribution of tachykinin NK2 binding sites in the rat brain: comparison with NK1 and NK3 binding sites.

The autoradiographic distribution of tachykinin NK(2) binding sites was determined in the adult rat brain using [(125)I]neurokinin A in the presence of either senktide (NK(3) agonist) and [Pro(9)]substance P (NK(1) agonist) or senktide and SR 140333 (NK(1) antagonist). Indeed, this radioligand labels two subtypes of NK(1) binding sites (which present a high affinity not only for SP but also for neurokinin A, neuropeptide K and neuropeptide gamma) as well as NK(3) binding sites. The distribution of NK(2) binding sites was also compared with those of NK(1) and NK(3) binding sites, these sites being labeled with [(125)I]Bolton and Hunter substance P and [(125)I]Bolton and Hunter eledoisin, respectively. In agreement with our results obtained with membranes from various brain structures, NK(2)-sensitive [(125)I]neurokinin A labeling was mainly observed in few structures including the dorsal and ventral hippocampus, the septum, the thalamus and the prefrontal cortex. The density of NK(2) binding sites was weak when compared with those of NK(1) and NK(3) binding sites. Marked differences were observed in the distributions of NK(1), NK(2) and NK(3) binding sites. These results are discussed taking into consideration differences or similarities between the distributions of NK(2)-sensitive [(125)I]neurokinin A binding sites and of their endogenous ligands (neurokinin A, neuropeptide K and neuropeptide gamma) but also local NK(2) agonist responses blocked by NK(2) antagonists. Insights on the roles of endogenous tachykinins in several brain functions are also discussed on the basis of the respective distributions of different neurokinin binding sites.

Quantitative autoradiographic analysis of the distribution of binding sites for [125I]Bolton Hunter derivatives of eledoisin and substance P in the rat brain.

[125I]Bolton and Hunter eledoisin binds to a single class of non-interacting sites in rat cerebral cortex tissue sections with an apparent Kd of 9.9 nM and a Bmax of 244 fmol/mg protein. When concentrations of up to 23 nM [125I]Bolton and Hunter eledoisin were used, [125I]Bolton and Hunter eledoisin binding was specific, saturable and reversible. Kassinin, eledoisin and neurokinin B were more potent than substance P and neurokinin A in inhibiting the specific binding of [125I]Bolton and Hunter eledoisin to cerebral cortex tissue sections. These kinetic and pharmacological characteristics are consistent with results obtained from binding studies on cortical synaptosomes. When the localization of [125I]Bolton and Hunter substance P and [125I]Bolton and Hunter eledoisin binding sites were compared, differences in many areas of the brain were noted. Large differences were seen in the paraventricular and supraoptic hypothalamic nuclei, and in layers IV and V of the cerebral cortex, which were densely labeled by [125I]Bolton and Hunter eledoisin, but not by [125I]Bolton and Hunter substance P. In contrast, nuclei of the septum (diagonal band of Broca, septohippocampal nucleus, dorsal part of the lateral septal nucleus), the rostrodorsal part of the hippocampus and other discrete nuclei [endopyriform nucleus, anterior cortical amygdaloid nucleus, the vermis columns (9-10), the dorsal tegmental nucleus, the hypoglossal and ambiguus nucleus] had high levels of [125I]Bolton and Hunter substance P binding but were only labeled weakly by [125I]Bolton and Hunter eledoisin. Thus, the two ligands seem to label different sites, since these binding sites have different biochemical and pharmacological properties, and are localized in different anatomical structures.

[3H]neurokinin B and 125I-Bolton Hunter eledoisin label identical tachykinin binding sites in the rat brain.

Abstract: [3H]Neurokinin B ([3H]NKB) of high specific activity (75 Ci/mmol) was synthesized for study of its binding to crude synaptosomes from the rat cerebral cortex. The specific binding of [3H]NKB (75% of total binding) was temperature dependent, saturable, and reversible. Scatchard analyses and Hill plots showed the existence of a single population of noninteracting binding sites (KD= 4.3 nM; Bmax= 123 fmol/mg of protein). Competition studies indicated the following rank order of potencies among tachykinins: NKB > eledoisin (E) > kassinin > physalaemin > neurokinin A (NKA) > substance P (SP), a result suggesting that NKB might be the endogenous ligand for [3H]NKB binding sites. It is of interest that 127I‐Bolton Hunter (BH) NKA (127I‐BHNKA) was much more potent than NKA in inhibiting the specific binding of [3H]NKB, which raises certain questions concerning the use of 125I‐BHNKA as a Iigand for NKA binding sites in the brain. These results, as well as those obtained with different SP analogues, show a close similarity to those obtained previously with 125I‐BHE binding to cortical synaptosomes. This suggested that the two ligands labeled identical binding sites. In addition, using either [3H]NKB or 125I‐BHE as ligands, similar displacement curves were obtained with increasing concentrations of NKB and 127I‐BHE. The similarity of the [3H]NKB and 125I‐BHE binding sites was further confirmed by comparison of their localization on rat brain sections by autoradiography. The distribution of binding sites for [3H]NKB and 125I‐BHE was identical throughout the brain, and the highest density of binding sites for the two ligands was found in layers IV and V of the cerebral cortex, the paraventricular nucleus of the hypothalamus (magnocellular part), and the ventral tegmental area.

A 25 year adventure in the field of tachykinins

Several aspects of our 25 year adventure in the field of tachykinins will be successively described. They concern: substance P (SP) synthesis and release in the basal ganglia, the identification and pharmacological characterization of central tachykinin NK(1), NK(2) and NK(3) binding sites and their topographical distribution, the description of some new biological tests for corresponding receptors, the identification of tachykinin NK(1) receptor subtypes or conformers sensitive to all endogenous tachykinins (substance P, neurokinin A (NKA), neurokinin B (NKB), neuropeptide gamma (NP gamma) and neuropeptide K (NPK)) and finally, the functional involvement of these receptors and their subtypes in tachykinin-induced regulations of dopamine and acetylcholine release in the striatum.

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.

[Pro9]SP and [pGlu6, Pro9]SP(6-11) interact with two different receptors in the guinea-pig ileum as demonstrated with new SP antagonists.

Structural considerations led us to postulate that the introduction of the dipeptides DPro9-Pro10 and DPro9-MeLeu10 should lock the C-terminal tetrapeptide of SP in a type II beta-turn structure, a prerequisite for antagonist activity. Indeed, as the GR 71251, [DPro9, Pro10, Trp11]SP was more potent in inhibiting the septide, (pA2 = 6.5), than the [Pro9]SP, (pA2 < or = 5), spasmogenic activity in the guinea-pig ileum bioassay. This result confirms that septide, [pGlu6, Pro9]SP(6-11), a peptide active in the guinea-pig ileum bioassay and practically devoid of binding potencies for the three specific NK-1, NK-2 and NK-3 tachykinin binding sites interacts with a tachykinin receptor different from the NK-1 receptor sensitive to [Pro9]SP. Interestingly enough, the reintroduction of the leucine side-chain in position 10 yielded [DPro9, MeLeu10, Trp11]SP, an antagonist, equipotent in inhibiting both the septide- and the [Pro9]SP-evoked contractile response in the guinea-pig ileum bioassay, (pA2 = 6.6).