Origin of the acetyl moiety of acetylcholine synthesized in rat striatal synaptosomes.

The subcellular localization of the AcCoA compartment supplying the cytoplasmic choline acetyltransferase (ChAc, EC 2.3.1.6) was investigated using a purified preparation of rat striatal synaptosomes (B fraction). It was first demonstrated that the SRA of the [14C]ACh synthesized during a 10 min incubation period was equal to the SRA of the [2-14C] and the [3-14C]pyruvate added to the isolated nerve terminal suspension. The experimental results can be summarised as follows: (i) No modification in the amount of [14C]ACh synthesized from [2-14C]pyruvatetion in the amount of [14C]ACh synthesized from [2-13C]pyruvate could be detected after the addition of high concentrations of either carnitine, acetylcarnitine or acetyl phosphate to the synaptosomal suspension. (ii) Under experimental conditions in which the amount of [1,5-14C]citrate taken up by passive diffusion into the cholinergic nerve endings would allow detection of the possible formation of the labelled ester, no [14C]ACh could be recovered. (iii) The SRA's of the individual carbon atoms of the Krebs cycle intermediary compounds when the cycle is fed with [2-14C] and [3-14C]pyruvate were calculated as a function of the STA's of each of these two precursors (a and a' respectively), of the number of 14CO2 dpm produced in the Krebs cycle from each of these two labelled compounds (D2 and D3 respectively), and as the function of the rate y of exchanges of molecules between the tricarboxylic acid cycle and other metabolic compartments. The experimental value obtained from a 10 min incubation, after the nerve endings had reached a steady metabolic activity, indicate that if the acetyl moiety of ACh was derived from some Krebs cycle intermediary compounds, its SRA could never exceed 55 per cent that of the [2-14C]pyruvate from which it is produced, (iv) No correlation could be found between the rate of [14C]ACh formation and changes in the Krebs cycle activity induced by sodium cyanide, 2-4 dinitrophenol and Ca2+ free medium. (v) The lack of significant [14C]ACh synthesis from [1-14C]acetate in striatal synaptosomes is consistent with the failure of fluoroacetate to modify the amounts of 14CO2 as well as of [14C]ACh formed from [2-14C]pyruvate. These results were interpreted as a confirmation of the presence of a low AcCoA synthetase activity in the nerve terminals. To reconcile all these data, it is proposed that pyruvate is transformed into AcCoA outside the mitochondria by the action of some cytoplasmic pyruvate dehydrogenase-like enzyme.

Design and synthesis of side-chain conformationally restricted phenylalanines and their use for structure-activity studies on tachykinin NK-1 receptor.

Constrained analogues of phenylalanine have been conceptually designed for analyzing the binding pockets of Phe7 (S7) and Phe8 (S8), two aromatic residues important for the pharmacological properties of SP, i.e., L-tetrahydroisoquinoleic acid, L-diphenylalanine, L-9-fluorenylglycine (Flg), 2-indanylglycine, the diastereomers of L-1-indanylglycine (Ing) and L-1-benz[f]indanylglycine (Bfi), and the Z and E isomers of dehydrophenylalanine (delta ZPhe, delta EPhe). Binding studies were performed with appropriate ligands and tissue preparations allowing the discrimination of the three tachykinin binding sites, NK-1, NK-2, and NK-3. The potencies of these agonists were evaluated in the guinea pig ileum bioassay. According to the binding data, we can conclude that the S7 subsite is small, only the gauche (-) probe [(2S,3S)-Ing7]SP presents a high affinity for specific NK-1 binding sites. Surprisingly, the [delta EPhe7]SP analogue, which projects the aromatic ring toward the trans orientation, is over 40-fold more potent than the Z isomer, [delta ZPhe7]SP. A plausible explanation of these conflictual results is that either the binding protein quenches the minor trans rotamer of [(2S,3S)-Ing7]SP in solution or this constrained amino acid side chain rotates when inserted in the protein. In position 8, the high binding affinities of [Flg8]SP and [(2S,3S)-Bfi8]SP suggest that the S8 subsite is large enough to accept two aromatic rings in the gauche (-) and one aromatic ring in the trans direction. Peptides bearing two conformational probes in positions 7, 8, or 9 led to postulate that S7, S8, and S9 subsites are independent from each other. The volumes available for side chains 7 and 8 can be estimated to be close to 110 and 240 A3, respectively. The large volume of the S8 subsite raises question on the localization of the SP-binding site in the NK-1 receptor. If SP were to bind in the transmembrane domains, the cleft defined by the seven transmembrane segments must rearrange during the binding process in order to bind a peptide in an alpha-helical structure and at least one large binding subsite in position 8. Thus, indirect topographical analysis with constrained amino acids might contribute to the analysis of the receptor/ligand dynamics. Finally, this study demonstrates that a good knowledge of the peptidic backbone structure and a combination of constrained amino acids are prerequisites to confidently attribute the preferred orientation(s) of an amino acid side chain.

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.

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 selectiveNK2 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.

Involvement of septide-sensitive tachykinin receptors in inositol phospholipid hydrolysis in the rat urinary bladder.

The selective NK2 agonist [Lys5-MeLeu9,Nle10]NKA(4-10) markedly stimulated [3H]inositol monophosphate (PI1) formation in prisms from the rat urinary bladder. This response was blocked by the NK2 antagonist SR 48968. Senktide (NK3 agonist) was inactive. Septide, a short SP analogue, and the NK1 agonists [Pro9]SP and [Sar9,Met(O2)11]SP also stimulated [3H]IP1 formation and several NK1 tachykinin antagonists (RP 67580, CP 96345, GR 82334, and [D-Pro9,t beta-BPr10,Trp11]SP) were more potent in blocking the septide than the [Pro9]SP response. GR 82334 was the most discriminative. SR 48968 (10(-6) M shifted the [Pro9]SP dose-response curve but did not modify the septide dose-response curve. Septide had a low affinity for [3H][Pro9]SP binding sites, suggesting further that septide and NK1 agonists act on different receptors. Finally, both [Pro9]SP and [Sar9,Met(O2)11]SP blocked the septide-evoked response, acting as partial agonists at the septide-sensitive tachykinin receptors.

Pharmacological characterization of tachykinin septide-sensitive binding sites in the rat submaxillary gland.

Binding studies have shown that [125I]NKA is a selective ligand of tachykinin septide-sensitive binding sites from membranes of the rat submaxillary gland. Indeed, this ligand bound with high affinity to a single population of sites. In addition, competition studies indicated that natural tachykinins and tachykinin-related compounds had a similar affinity for these sites than for those labeled with [3H]ALIE-124, a selective ligand of septide-sensitive binding sites. Moreover, selective tachykinin NK2, or NK3 agonists or antagonists exhibited weak or no affinity for [125I]NKA binding sites. As indicated by Ki values of several compounds, the pharmacological characteristics of the septide-sensitive binding sites (labeled with [125I]NKA) largely differ from those of classic NK1 binding sites, as determined on crude synaptosomes from the rat brain using [125I]Bolton-Hunter substance P (SP) as ligand. Indeed, several tachykinins including neurokinin A (NKA), neuropeptide K (NPK), neuropeptide gamma (NKgamma), and neurokinin B, as well as some SP and NKA analogues or C-terminal fragments such as septide, ALIE-124, SP(6-11), NKA(4-10), which have a weak affinity for classic tachykinin NK1 binding sites exhibited a high affinity for the septide-sensitive binding sites. In contrast, SP, classic selective NK1 agonists, and antagonists had a high affinity for both types of binding sites. The presence of a large population of tachykinin septide-sensitive binding sites in the rat submaxillary gland may thus explain why NPK and NPgamma induce salivary secretion and may potentiate the SP-evoked response in spite of the absence of tachykinin NK2 receptors in this tissue.

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.

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.

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.

Pharmacological characterisation of two tachykinin binding sites in the rat cerebral cortex.

The pharmacological properties of two types of tachykinin receptor were characterised on rat cortical synaptosomes using 125I-Bolton Hunter substance P (125I-BHSP) or with 125I-Bolton Hunter eledoisin (125I-BHE). Shorter SP C-terminal fragments, such as SP (6-11) or (pGlu)-SP (6-11), were more potent than SP itself or longer SP C-terminal fragments in competing for 125I-BHE binding; their efficacy was comparable to that of eledoisin. In contrast, longer SP C-terminal fragments exhibited a higher affinity than shorter ones for the 125I-BHSP binding sites as previously reported. SP N-terminal fragments were devoid of activity on either type of binding sites. SP methyl ester inhibited 125I-BHSP binding but was without effect on 125I-BHE binding whilst, DiMe-C7, a metabolically stable tachykinin analog, had the opposite selectivity. Eledoisin related peptide (ERP) was less effective than either SP or eledoisin on 125I-BHSP and 125I-BHE binding sites respectively. Finally, the undecapeptide or octapeptide SP antagonists, which are weak inhibitors of 125I-BHSP binding, had negligable activity on 125I-BHE binding sites.

Sulfhydryl reagents have different effects on substance P and neurokinin B binding sites on cortical synaptosomes in the rat.

The effects of several protein modifying reagents, including phenoxybenzamine, N-ethylmaleimide (NEM), 5,5'-dithiobis (2-nitro)--benzoic acid (DTNB), p-chloromercuryphenyl sulfonic acid (PCMP) and p-bromophenacylbromide (PBPB), on the binding of 125I-Bolton Hunter substance P (125I-BHSP), 125I-Bolton Hunter eledoisin (125I-BHELE) and 3H-neurokinin B (3H-NKB)4 to cortical synaptosomes were examined. PCMP (10(-4) M), DTNB (10(-4) M) and NEM (10(-3) M) were without effect on the 125I-BHSP binding but reduced markedly the specific binding of 125I-BHELE or 3H-NKB. Although phenoxybenzamine and PBPB inhibited the 125I-BHSP binding when used in high concentrations (10(-4) M and 10(-3) M), they were more potent in inhibiting the 125I-BHELE or 3H-NKB binding. These results indicate that the NKB binding site is more sensitive to alkylating reagents than the SP binding sites and that these reagents can be used to distinguish SP and NKB receptors in the brain.

Substance P(6-11) and natural tachykinins interact with septide-sensitive tachykinin receptors coupled to a phospholipase C in the rat urinary bladder.

The rat urinary bladder possesses NK1, NK2 (but not NK3) and 'septide-sensitive' tachykinin receptors coupled to a phospholipase C. The present study performed with SR48968 (10(-6) M) to avoid any interaction of the tested peptides with NK2 receptors, indicates that substance P(6-11) (with a high potency), neurokinin A, neurokinin B and to a lesser extent neuropeptide K (with a lower potency) stimulate [3H]-inositol monophosphate ([3H]-IP1) formation in this tissue by acting on the 'septide-sensitive' tachykinin receptors. Substance P(6-11) had little affinity for NK1 binding sites and stimulated [3H]-IP1 formation with an EC50 value and a maximal amplitude similar to those of septide. As previously observed with septide, this maximal response of substance P(6-11) (insensitive to 10(-6) M SR48968) which was about three-fold that of substance P, was blocked by the NK1 receptor antagonist RP67580 and prevented by [Pro9]substance P (NK1 receptor agonist). Similarly, substance P and several substance P C-terminal fragments prevented the substance P(6-11)-evoked response. In addition, neurokinin A, neuropeptide K and neurokinin B induced SR48968-resistant responses which exhibited a maximal amplitude similar to that of substance P (6-11) and were blocked by RP67580 and totally or partially (neuropeptide K) prevented by [Pro9]substance P.

A peptidases-resistant glycosylated analogue of substance P-(5-11). Specificity towards substance P receptors.

Glycosylated analogues of the C-terminal heptapeptide of substance P either free or blocked on the N-terminal glutamine were synthesized in order to develop a metabolically stable peptide that would have an increased specificity for one type of receptor. Of the analogue described, (N-alpha-Boc-beta-D-Glc-p (1----5) Gln) -Gln-Phe-Phe-Gly-Leu-Met-NH2 is highly resistant to degradation on exposure to rat hypothalamic slices. This glycosylated peptide is about one third as potent as substance P in eliciting contractions of the guinea-pig ileum and is almost devoided of affinity for the 125I-Bolton Hunter-SP specific binding sites on rat brain synaptosomes.

Influence of the replacement of amino acid by its D-enantiomer in the sequence of substance P. 1. Binding and pharmacological data.

The D-enantiomer of residues 2, 4, 5, 6, 7, 8, 10 and 11 was introduced in the sequence of Substance P: Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2. The achiral glycine residue was replaced by a D-Ala residue. Regarding NK-1 binding potencies or activities, changing to the D-enantiomer in positions 2, 4 or 5 did not modify the pharmacological patterns of the resulting peptides. Introduction of a D-residue in the 6 to 11 sequence drastically decreased the potency of the D-analogues with the exception of [D-Leu10]SP which was found only three times less potent than SP in contracting the guinea-pig ileum. No clear cut evidence between the binding potencies and activities on NK-1, NK-2 and NK-3 assays, was observed which allows a more rational design of tachykinins antagonists.

[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).

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.

Differential localization of 3H-[Pro9]SP binding sites in the guinea pig and rat brain.

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 ligand 3H-[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 of 125I-BHSP (a non selective ligand) and 3H-[Pro9]SP binding sites, does not suggest the existence of NK-1 binding site subtypes in the guinea pig brain.

Tachykinin binding sites in the interpeduncular nucleus of the rat: normal distribution, postnatal development and the effects of lesions.

Tachykinin binding sites in the basal midbrain were labeled in adult and neonatal rats using 125I-Bolton Hunter (BH) substance P (SP) and 125I-BH eledoisin as ligands. In the adult, binding was very low in the tegmentum and raphe adjacent to the interpeduncular nucleus (IPN). Within the IPN, no binding with either ligand was seen in the target subnuclei of the habenular SP and substance K projections, the lateral subnuclei and the cap of the rostral subnucleus. Labeling with 125I-BH-SP was very light and was restricted primarily to the central subnucleus of the IPN while 125I-BH-eledoisin labeling was very dense over the dorsal, the ventral sector of the rostral, the intermediate and the central subnuclei. Lesions of major afferents to the IPN, the fasciculus retroflexus or the locus coeruleus, had no effect on the distribution or density of the binding of either ligand. In rats 0, 4 or 7 days or age, 125I-BH-SP binding was very dense in the ventral tegmental region, the raphe and in the dorsal, rostral and central subnuclei. 125I-BH-eledoisin binding was extremely dense in the raphe and in the dorsal, rostral, intermediate and central subnuclei but was less dense in the ventral tegmentum. Adult levels of binding in the midbrain were established by 11 days of age. Neonatal lesions restricted to the fasciculus retroflexus had no effect on the density of labeling with either ligand in animals allowed to reach adulthood.