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SR 48968, a non-peptide tachykinin NK2 receptor antagonist, has been shown to possess sub-micromolar affinity for NK3 receptors present in the guinea pig. In the present study, we have compared the binding affinities of SR 48968 to the cloned human and rat NK3 receptors expressed in CHO cells. Using [125I]-[MePhe7]-neurokinin B as the radioligand, SR48968 displayed an IC50 value of 350 nM for the human NK3 receptor as compared with a value of greater than 10 microM for the rat NK3 receptor. Exposure of cells transfected with human NK3 receptor cDNA to [Pro7]-neurokinin B increased inositol phospholipid turnover in a concentration-dependent manner and this response was blocked competitively by SR 48968. Our results demonstrate that SR 48968 is an antagonist at the human NK3 receptor and may be a useful tool for elucidating the species-dependent variations in the non-peptide antagonist binding site(s) on the NK3 receptor.

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The rational discovery of a high affinity NK2 receptor antagonist is described utilizing a general strategy for peptoid design. The contribution to NK2 receptor binding affinity for each amino acid of the hexapeptide 'minimum fragment': Leu-Met-Gln-Trp-Phe-GlyNH2 (8c), was examined by preparing derivatives where each amino acid in turn was replaced with Ala in an 'alanine scan'. The results from this study indicated the primary importance of the Trp and Phe side-chain for binding and led to the observation that Z-Trp-PheNH2 (9a) is a micromolar affinity NK2 receptor dipeptide lead. Further exploration of structure-affinity via conformationally restricted analogues and N- and C-terminus modifications gave a selective, nanomolar affinity NK2 receptor antagonist, (2,3di-CH3OPh)CH2OCO(S)Trp(S)alpha-MePheGlyNH2, PD 147714 (19) with an K(i) = 1.4 nM (hamster urinary bladder membranes and using [125I]-iodohistidyl-NKA (0.1 nM) as the radioligand).

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1. We investigated the effects of the non-peptide NK1 receptor antagonist, CP-96,345, its inactive enantiomer CP-96,344, and the racemic mixture (+/-)-CP-96,345, on the binding of [3H]-nimodipine and [3H]-diltiazem to L-type calcium channels in rat cerebral cortex membranes. In isolated peripheral tissues containing tachykinin receptors, the effects of (+/-)-CP-96,345 have been compared with those of diltiazem. 2. In guinea-pig trachea, (+/-)-CP-96,345 produced antagonism of responses to the selective NK1 agonists [Sar9, Met(O2)11]SP and substance P-methyl ester that was apparently competitive in nature (pKB 7.0-7.5), while in guinea-pig ileum the antagonism was not surmountable. 3. The reduction of maximum responses by (+/-)-CP-96,345 in the guinea-pig ileum was not selective; it was obtained with muscarinic agonists and other agents, and was also observed in the portal vein of the rat where NK1 receptors are not present. 4. The tissue-specific reduction of maximum responses by (+/-)-CP-96,345 in ileum was reproduced by diltiazem. 5. (+/-)-CP-96,345 produced a concentration-dependent enhancement of [3H]-nimodipine binding to rat cerebral cortex membranes with a maximal stimulation of 186 +/- 29% above control (EC50 83.2 nM). Scatchard analysis revealed that (+/-)-CP-96,345 increased the affinity of [3H]-nimodipine for its binding sites without affecting Bmax (control: KD = 0.32 nM; with 100 nM (+/-)-CP-96,345: KD = 0.074 nM). 6. CP-96,345, CP-96,344, and the racemate all inhibited [3H]-diltiazem binding in rat cerebral cortex membranes with Ki values of 22.5 nM, 34.5 nM and 29.9 nM respectively; a similar value was obtained for diltiazem itself (33.6 nM). In comparison, CP-96,345 and ( +/- )-CP-96,345 inhibited the binding of[125I]-Bolton-Hunter-conjugated substance P in this tissue with Ki values of 59.6 nM and 82.0 nM respectively, while CP-96,344 had no measurable affinity (IC50> 10 microM).7. Substance P and a range of ligands selective for NK1, NK2, or NK3 receptors had no significant effect at 10 microM on either [3H]-diltiazem or [3H]-nimodipine binding.8. The results indicate that in addition to possessing affinity for the NK1 receptor, the non-peptide antagonist, CP-96,345, displays high affinity for [3H]-diltiazem binding sites on L-type calcium channels.The functional effect that may be observed in integrated models will be a consequence of either property, or be a composite effect of NK1 receptor antagonism and L-channel blockade.

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1. Daily administration of 1 nmol substance P or 3 pmol recombinant human interleukin-1 alpha (IL-1 alpha) caused intense neovascularization in a rat sponge model of angiogenesis. Lower doses of substance P (10 pmol) or IL-1 alpha (0.3 pmol) were ineffective when given alone. When combined at these low doses, substances P and IL-1 alpha interacted to produce an enhanced neovascular response. 2. By use of selective tachykinin NK1, NK2 and NK3 receptor agonists, ([Sar9,Met(O2)11]substance P, [beta-Ala8]neurokinin A(4-10), Succ-[Asp6,MePhe8]substance P(6-11) (senktide), respectively), it was established that the activation of NK1 receptors is most likely to mediate the angiogenic response to substance P in this model. 3. The angiogenic activity of substance P and IL-1 alpha (10 pmol and 0.3 pmol day-1, respectively) was abolished by co-administration of (i) the selective peptide NK1 receptor antagonist, L-668,169 (1 nmol day-1), (ii) the selective non-peptide NK1 receptor antagonists, RP 67580 and (+/-)-CP-96,345 (both at 1 nmol day-1) or (iii) the IL-1 receptor antagonist, IL-1ra, (50 micrograms day-1). In contrast, the selective NK2 receptor antagonist, L-659,874 (1 nmol day-1) was ineffective. 4. The angiogenic action of substance P and IL-1 alpha was resistant to modification by mepyramine (1 nmol day-1) and/or cimetidine (10 nmol day-1), indomethacin (7 nmol day-1) or the platelet-activating factor (PAF) antagonist, WEB-2086 (22 nmol day-1), indicating that histamine, prostaglandins and PAF are not likely to be involved in this neovascular response. 5. The inhibition of the substance P/IL-1 angiogenic response by selective NK1 receptor antagonists or by an IL-1 receptor antagonist demonstrates that angiosuppression can be achieved by blocking the activity of angiogenic factors at the receptor level.

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The pharmacological profile of [125I][Tyr4]bombesin binding to gastrin-releasing peptide- and neuromedin B-preferring sites has been investigated in rat cerebral cortex and olfactory bulb membranes, respectively. [125I][Tyr4]bombesin specific binding to cerebral cortex membranes was displayed biphasically by gastrin releasing peptide and [D-Phe6]bombesin-(6-13)-ethyl amide. In the presence of 10 mM neuromedin B, displacement curves for bombesin-related peptides were monophasic with gastrin releasing peptide displaying approximately 100-fold higher affinity than neuromedin B. In olfactory bulb membranes, [125I][Tyr4]bombesin binding was also displaced biphasically by gastrin releasing peptide, [D-Phe6]bombesin-(6-13)-ethyl amide and neuromedin B. In the presence of 10 microM [D-Phe6]bombesin-(6-13)-ethyl ester, displacement curves were monophasic with neuromedin B possessing approximately 10-fold higher affinity than gastrin-releasing peptide. Under these conditions, successive deletion of N-terminal amino acids from bombesin-(1-14) was well tolerated at both sites, with little loss in affinity up to bombesin-(5-14). A 5- to 10-fold drop in affinity was observed at both sites with bombesin-(6-14), whilst the octapeptide acetyl-bombesin-(7-14) displayed similar affinities to bombesin-(1-14). Bombesin-(8-14), -(9-14) and -(10-14) were essentially inactive (IC50 > 10 microM). C-terminal deletion of Met24 (bombesin-(1-13)) resulted in 100-fold loss of affinity at the gastrin-releasing peptide site and complete loss of affinity at the neuromedin B site. Fragments smaller than bombesin-(1-13) were virtually inactive at either site.(ABSTRACT TRUNCATED AT 250 WORDS)

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The pharmacological profile of NK2 binding sites has been characterised in homogenates of rabbit urinary bladder and compared with that present in homogenates of hamster bladder. In both species, [125I]neurokinin A-specific binding to urinary bladder membranes was displaced by neurokinin A and the NK2 agonist [beta-Ala8]neurokinin A-(4-10) whilst the NK1 ligands [Sar9,Met(O2)11]substance P and (+/-)-CP-96,345, and the NK3 agonist, senktide, were only weak displacers or ineffective. At rabbit NK2 sites, the rank order of affinity of NK2 receptor-selective antagonists was; MEN 10,376 > MEN 10,207 > L-659,877 >> R 396. In contrast, the rank order of displacement of [125I]neurokinin A-specific binding to hamster bladder membranes was: L-659,877 > R 396 > MEN 10,376 > MEN 10,207. These data demonstrate that [125I]neurokinin A binds to pharmacologically distinct NK2 binding sites in hamster and rabbit urinary bladder.

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1. Tachykinin-stimulated inositol phospholipid hydrolysis was examined in slices of rat parotid gland, hamster urinary bladder and guinea-pig ileum longitudinal muscle. 2. In the presence of lithium, substance P and other naturally-occurring and synthetic tachykinins induced large, dose-dependent increases in [3H]-inositol monophosphate accumulation. 3. In slices of rat parotid gland, [pGlu6,L-Pro9]SP(6-11) was considerably more potent in stimulating inositol phospholipid hydrolysis than [pGlu6,D-Pro9]SP(6-11). 4. In contrast, in slices of hamster urinary bladder, [pGlu6,D-Pro9]SP(6-11) exhibited greater potency in evoking inositol phospholipid breakdown than [pGlu6,L-Pro9]SP(6-11). 5. The differential selectivity of these C-terminal fragments of substance P suggests that they may be useful tools for distinguishing between NK1 and NK2 receptors. 6. L-659,837 and L-659,874 antagonized eledoisin-stimulated inositol phospholipid hydrolysis in slices of hamster urinary bladder. Neither compound significantly reduced substance-P evoked inositol phospholipid breakdown in slices of rat parotid gland, or senktide-induced inositol phospholipid hydrolysis in slices of guinea-pig ileum. 7. L-659,837 and L-659,874 had no effect on the atropine-sensitive, carbachol-stimulated inositol phospholipid hydrolysis in slices of rat parotid gland. 8. These data further support the notion that L-659,837 and L-659,874 are potent and selective NK2 receptor antagonists.

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Clozapine, an atypical neuroleptic drug devoid of extrapyramidal side effects, was a moderately potent, competitive inhibitor of the binding of [3H]quaternised ICS 205-930 to 5-HT3 receptor sites in rat cortical membranes, possessing a pKi value of 7.0. In contrast, several other antipsychotic agents, including fluphenazine, alpha-flupenthixol, haloperidol, spiperone and (-)-sulpiride were essentially inactive. Clozapine also antagonised the 2-methyl 5-HT-induced depolarisation of the rat isolated superior cervical ganglion, a response known to be mediated via 5-HT3 receptors. Clozapine (0.1-1 microM) induced parallel displacements to the right of the dose-response curve to 2-methyl 5-HT in this tissue, possessing a pKb value of 7.3. These data suggest that the atypical antipsychotic profile of clozapine may be related, at least, in part to its ability to interact with central 5-HT3 receptor sites.

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1. The binding properties and pharmacological specificity of the selective NK3 tachykinin receptor agonist [3H))-senktide [( 3H]-succinyl[Asp6,MePhe8] substance P (6-11] have been examined in homogenates of guinea-pig ileum longitudinal muscle-myenteric plexus (LM/MP) and cerebral cortex. 2. Scatchard analysis of saturation binding studies in guinea-pig ileum LM/MP and cerebral cortex membranes indicated that [3H]-senktide bound to a single site with apparent high affinity, KD = 2.21 +/- 0.65 nM; Bmax = 13.49 +/- 0.04 fmol mg-1 protein in ileum and KD = 8.52 +/- 0.45 nM; Bmax = 76.3 +/- 1.6 fmol mg-1 protein in cortex (values are means +/- ranges; n = 2). 3. The pharmacological profile for tachykinins and analogues in displacing [3H]-senktide from ileum membranes was: [MePhe7] neurokinin B greater than neurokinin B (NKB) congruent to senktide greater than eledoisin greater than substance P (SP) greater than neurokinin A(NKA) greater than physalaemin greater than [Sar9,Met(O2)11]SP greater than [Nle10]NKA(4-10) = [Glp6,L-Pro9]-SP(6-11) greater than substance P methyl ester, consistent with [3H]-senktide binding to an NK3 subtype of tachykinin receptor. A similar rank order of affinity was obtained for these peptides in displacing [3H]-senktide from cortex membranes. 4. Several tachykinin receptor agonists were tested for their ability to displace [3H]-senktide from ileal and cortical NK3 binding sites and were found to be either weak displacers (pIC50 less than 5.00) or inactive. 5. The binding of [3H]-senktide to cortex membranes was inhibited by GTP (p1C,0 = 6.49)and GTP-gamma- S (p1C,0 = 6.67) with ATP being at least three orders of magnitude less potent (pIC50 = 3.55). 6. These results indicate that both central and peripheral NK3 receptors share a similar pharmacological specificity and that they may be labelled selectively with the NK3 receptor agonist [3H]-senktide.

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1. Tachykinin-stimulated inositol phospholipid hydrolysis was examined in slices of longitudinal muscle from guinea-pig ileum. 2. Substance P, neurokinin A and neurokinin B induced a concentration-dependent accumulation of total [3H]-inositol phosphates in the presence of 12 mM lithium with similar maximal responses and EC50 values. 3. The selective NK1-receptor agonist, substance P methyl ester, and the selective NK3-receptor agonist succ-[Asp6, MePhe8]-SP(6-11) (senktide) also stimulated [3H]-inositol phosphate formation with maximum responses of 50.69 +/- 0.96 and 45.64 +/- 1.17% relative to 10 microM substance P, respectively. Substance P methyl ester was approximately equipotent with substance P, whereas senktide was approximately 100 times more potent. 4. When added together, maximally effective concentrations of substance P methyl ester and senktide gave responses that were fully additive. In contrast, responses to substance P and neurokinin B were not additive. 5. The stimulation of [3H]-inositol phosphate formation by substance P, neurokinin B and senktide was not affected by atropine (2 microM) or tetrodotoxin (TTX, 0.3 microM). 6. The contractile effect of senktide was inhibited completely by TTX and partially blocked by atropine. Contractions induced by substance P methyl ester were not changed in the presence of TTX or atropine. 7. [D-Pro4, D-Trp7,9,10]-SP(4-11) competitively antagonized the action of substance P methyl ester on inositol phospholipid hydrolysis and contraction, but had no significant effect on senktide-induced inositol phospholipid breakdown or contraction. 8. These results suggest that NK3-receptors in the guinea-pig ileum are coupled to inositol phospholipid hydrolysis.

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1. An inositol monophosphatase was purified to homogeneity from bovine brain. 2. The enzyme is a dimer of subunit Mr 29,000. 3. The enzyme hydrolyses both enantiomers of myo-inositol 1-phosphate and both enantiomers of myo-inositol 4-phosphate, but has no activity towards inositol bisphosphates, inositol trisphosphates or inositol 1,3,4,5-tetrakisphosphate. 4. Several non-inositol-containing monophosphates are also substrates. 5. The enzyme requires Mg2+ for activity, and Zn2+ supports activity to a small extent. 6. Other bivalent cations (including Zn2+) are inhibitors, competitive with Mg2+. 7. Phosphate, but not inositol, is an inhibitor competitive with substrate. 8. Li+ inhibits hydrolysis of inositol 1-phosphate and inositol 4-phosphate uncompetitively with different apparent Ki values (1.0 mM and 0.26 mM respectively).

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