1 The aim of the present study was to validate the Cytosensor microphysiometer, a novel system that measures the extracellular acidi®cation rate as a reliable index of the integrated functional response to receptor activation, as a method for studying NK 3 receptor pharmacology, and then to use this system to assess the functional activity of novel compounds at this receptor. 2 The selective NK 3 agonist senktide caused reproducible, concentration-related increases in acidi®cation ratein CHO-NK 3 cells, with a pEC 50 value of 8.72+0.11 (n=15). [b-Ala 8 ]NKA(4 ± 10), the selective NK 2 agonist, elicited a much weaker response (pEC 50 =6.68+0.08, n=4), while the NK 1 -selective agonist substance P methylester only caused a very weak response at concentrations 53 mM (n=2). The rank order of potency for the endogenous tachykinins NKB4NKA4substance P (n=3) con®rmed the response was mediated by the NK 3 receptor. Moreover, the actual potencies obtained were consistent with a nities measured in radioligand binding studies. 3 The novel compounds PD156319-121 (0.3 ± 1 mM), PD161182 (10 ± 300 nM), PD168001 (10 ± 100 nM) and PD168073 (10 ± 100 nM) all acted as surmountable antagonists of the senktide-induced acidi®cation response, with pA 2 values of 7.49, 8.67, 9.17 and 9.25 respectively (n=3 ± 5). In comparison the known NK 3 antagonist SR142801 (10 ± 100 nM) had a pA 2 value of 8.83 (n=8) for the interaction with senktide. Again, these values are consistent with the radioligand binding data. 4 Amiloride (1 mM) inhibited the senktide-induced acidi®cation 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 acidi®cation responses. 5 Inhibition of protein kinase C with staurosporine (0.1 mM), or depletion of the intracellular Ca 2+ stores with thapsigargin (1 mM), 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 NK 3 receptor was coupling via phospholipase C (PLC), as would be expected, although this could not be con®rmed by the use of the putative PLC/PLA 2 inhibitor U73122. 6 In conclusion, we have demonstrated the utility of the Cytosensor in the characterization of functional responses to agonists, and assessment of the a nities of antagonists in CHO cells expressing the human NK 3 , and have shown that our series of novel compounds are non-peptide NK 3 antagonists of high a nity, as exempli®ed by PD168073.
