Activation of the cloned human NK₃ receptor in Chinese Hamster Ovary cells characterized by the cellular acidification response using the Cytosensor microphysiometer

Q2 · MEDICINE

Article

Author: R E Jordan ; D Smart ; P Grimson ; A T McKnight ; N Suman-Chauhan

The aim of the present study was to validate the Cytosensor microphysiometer, a novel system that measures the extracellular acidification rate as a reliable index of the integrated functional response to receptor activation, as a method for studying NK3 receptor pharmacology, and then to use this system to assess the functional activity of novel compounds at this receptor.

The selective NK3 agonist senktide caused reproducible, concentration‐related increases in acidification ratein CHO‐NK3 cells, with a pEC50 value of 8.72±0.11 (n=15). [β‐Ala8]NKA(4–10), the selective NK2 agonist, elicited a much weaker response (pEC50=6.68±0.08, n=4), while the NK1‐selective agonist substance P methylester only caused a very weak response at concentrations 3 μM (n=2). The rank order of potency for the endogenous tachykinins NKB>NKA>substance P (n=3) confirmed the response was mediated by the NK3 receptor. Moreover, the actual potencies obtained were consistent with affinities measured in radioligand binding studies.

The novel compounds PD156319‐121 (0.3–1 μM), PD161182 (10–300 nM), PD168001 (10–100 nM) and PD168073 (10–100 nM) all acted as surmountable antagonists of the senktide‐induced acidification response, with pA2 values of 7.49, 8.67, 9.17 and 9.25 respectively (n=3–5). In comparison the known NK3 antagonist SR142801 (10–100 nM) had a pA2 value of 8.83 (n=8) for the interaction with senktide. Again, these values are consistent with the radioligand binding data.

Amiloride (1 mM) inhibited the senktide‐induced acidification response by 68.3±3.3 (n=4), indicating that the Na+/H+ antiporter plays an important role in this response, and this is consistent with the importance of this antiporter in other acidification responses.

Inhibition of protein kinase C with staurosporine (0.1 μM), or depletion of the intracellular Ca2+ stores with thapsigargin (1 μM), both resulted in a reduction in the maximum response to senktide (63.3±1.7 and 68.9±3.2% respectively, n=3–5), and co‐application of these inhibitors abolished the response (n=3). This strongly suggested that the NK3 receptor was coupling via phospholipase C (PLC), as would be expected, although this could not be confirmed by the use of the putative PLC/PLA2 inhibitor U73122.

In conclusion, we have demonstrated the utility of the Cytosensor in the characterization of functional responses to agonists, and assessment of the affinities of antagonists in CHO cells expressing the human NK3, and have shown that our series of novel compounds are non‐peptide NK3 antagonists of high affinity, as exemplified by PD168073.

British Journal of Pharmacology (1998) 125, 761–766; doi:10.1038/sj.bjp.0702156

01 Jun 1997·The Journal of pharmacology and experimental therapeutics

Neurokinin A-induced vasoconstriction and muscular contraction in the rat isolated stomach: mediation by distinct and unusual neurokinin2 receptors.

Article

Author: Lippe, I ; Wachter, C H ; Holzer, P

This study examined the pharmacological identity of the tachykinin receptors which in the rat stomach mediate vasoconstriction and muscular contraction. The vasculature of the rat isolated stomach was perfused with oxygenated Krebs buffer containing 3% dextran. Vasoconstrictor responses were recorded as increases in the vascular perfusion pressure and gastric contractions were measured as increases in the intraluminal pressure. By examining the effects of selective agonists and antagonists for tachykinin neurokinin (NK)1, NK2 and NK3 receptors it was found that the vasculature contained only NK2 receptors that were activated by the NK2 receptor agonist [betaAla8]-NKA-(4-10) and inhibited by the NK2 receptor antagonists MEN-10,627 and GR-94,800. However, the vasoconstrictor action of NKA was blocked only when the preparations were exposed to a combination of NK1, NK2 and NK3 receptor antagonists (SR-140,333, MEN-10,627, PD-161,182). In contrast, the NKA-evoked contraction of the gastric musculature was suppressed by NK2 receptor antagonists but little affected by NK1 or NK3 receptor antagonists. This observation was consistent with the predominance of NK2 receptors on the muscle as revealed by the effects of receptor-selective NK1, NK2 and NK3 agonists and antagonists. These results demonstrate that the major tachykinin receptor type present on the gastric vasculature and musculature is a NK2 receptor that is sensitive to receptor-selective agonists and antagonists. The NKA-evoked gastric contraction is also primarily due to NK2 receptor activation, whereas the NKA-induced vasoconstriction is mediated by a distinct and unusual type of NK2-like receptor that is blocked by a combination of NK1, NK2 and NK3 receptor antagonists only.

01 Jun 1996·Immunopharmacology

‘Targeted’ molecular diversity: design and development of non-peptide antagonists for cholecystokinin and tachykinin receptors

Review

Author: David Horwell ; Giles Ratcliffe ; Martyn Pritchard ; Jennifer Raphy

A drug design strategy to non-peptide small molecule antagonists of neuropeptides is described that targets the molecular diversity which exists in the 'privileged' data set of the physico-chemical properties represented by the side-chains of the 20 genetically encoded amino acids. The strategy is exemplified by the design of a selective and high affinity cholecystokinin CCK-A antagonist PD 140548, CCK-B antagonist CI-988 (formerly PD 134308) tachykinin NK-1 antagonist PD 154075 and NK-2 antagonist Cam-2291. The NK-3 antagonists, PD 157672 and the non-peptide PD 161182, were developed from an information-rich dipeptide library constructed from 256 N-protected dipeptides and 64 hydrophobic biased dipeptides.

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Indication	Highest Phase	Country/Location	Organization	Date
Anxiety Disorders	Discovery	US	Parke-Davis & Co. Ltd.	-

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