Keith J. Watling

The interaction of the NK1 receptor antagonist CP‐96,345 with L‐type calcium channels and its functional consequences

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 μm). 7 Substance P and a range of ligands selective for NK1, NK2, or NK3 receptors had no significant effect at 10 μm 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.

Characterisation of [125I][MePhe7]neurokinin B binding to tachykinin NK3 receptors : evidence for interspecies variance

Human tachykinin NK3 receptors expressed in Chinese hamster ovary (CHO-K1) cells were characterised using the novel radioligand [125I]iodohistidyl,[MePhe7]neurokinin B ([125I][MePhe7]neurokinin B). [125I][MePhe7]neurokinin B was shown to label human NK3 binding sites with high affinity in a saturable and reversible manner. The rank order of affinity of a range of tachykinin ligands confirmed that the tachykinin receptor expressed was the NK3 receptor type. An interspecies comparison of NK3 binding sites revealed pharmacological differences between human, guinea pig and rat tachykinin NK3 receptors. The NK2 selective antagonist SR 48968, inhibited binding of [125I][MePhe7]neurokinin B to NK3 binding sites with Ki values of 287 nM and 205 nM in human and guinea pig respectively, but was > 30-fold less active in the rat.

Structure-activity requirements of bombesin for gastrin-releasing peptide- and neuromedin B-preferring bombesin receptors in rat brain

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)

Pharmacological characterization of tachykinin-stimulated inositol phospholipid hydrolysis in peripheral tissues.

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.

An SAR study for the non-peptide substance P receptor (NK1) antagonist, CP-96,345.

Abstract Results from an SAR study around the novel, non-peptide substance P receptor NK1 antagonist, (±)CP-96,345 (1) are described. The importance of the 2° nitrogen and the aromatic moieties are clarified.