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

Abstract

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.

Cytosensor techniques for examining signal transduction of neurohormones

“…The Cytosensor has been used to study the signal transduction pathways activated by a wide range of hormones, including corticotrophin-releasing factor (CRF; Barstye et al 1999;Robinson et al 1999;Smart et al 1999), parathyroid hormone (PTH; Barrett et al 1997), corticosterone (Redish et al 1993), estrogen (Redish et al 1993), interleukin-1 (IL-1; Hammond et al 1999;Smart et al 1998a), progesterone (Redish et al 1993), and testosterone (Redish et al 1993), as well as those signal transduction pathways activated by other neuropeptides, e.g., bombesin ) and the neurokinins (Jordan et al 1998).…”

“…Transient sharp monophasic responses are usually associated with a phospholipase (PLC)-mediated mobilisation of calcium from intracellular stores Jordan et al 1998), whereas broader monophasic responses are more likely to reflect Gscoupled adenylate cyclase activity . Gicoupled cAMP responses are often associated with an initially delayed onset, but the ECAR is then maintained until the agonist is washed off (Barrett et al 1997), whereas ECARs with delayed onset that persist even after the agonist is removed generally involve the activation of protein kinases (Hammond et al 1999;Laping et al 1998).…”

“…Indeed, as the ECAR is measured in real-time (Jordan et al 1998), the Cytosensor is particularly well suited to examine the kinetics of desensitisation. For example, using microphysiometry, we were able to demonstrate that the CRF 2 receptor desensitises more rapidly then the CRF 1 receptor, but both receptor subtypes display a similar recovery time .…”

“…The main approach used to date for studying the Gprotein coupling receptors in the Cytosensor has been the pretreatment of the cells with various toxins (Jordan et al 1998). The cells are seeded into cups and then cultured overnight in medium containing either pertussis-toxin (Merkouris et al 1997;Pei et al 1997) or cholera-toxin (Jordan et al 1998), and then the subsequent agonist induced ECAR is examined.…”

“…The cells are seeded into cups and then cultured overnight in medium containing either pertussis-toxin (Merkouris et al 1997;Pei et al 1997) or cholera-toxin (Jordan et al 1998), and then the subsequent agonist induced ECAR is examined. If the ECAR is inhibited by pertussistoxin treatment, then the response is mediated by the Gi/Go family, as demonstrated for the D 4 dopamine receptor (Chio et al 1994), whereas if the ECAR is inhibited by treatment with cholera-toxin then Gs-coupling is involved (Jordan et al 1998). However, if the ECAR is not affected by either toxin then the receptor is most likely coupling through the Gq family as reported for the NK 3 tachykinin receptor (Jordan et al 1998) and the D 2 dopamine receptor (Neve et al 1992).…”