Mismatch of neurokinin A functional and binding data in porcine urinary bladder mucosa and detrusor

Prajni Sadananda, Lu Liu, Elizabeth Burcher. University of New South Wales, Sydney, Australia.

Introduction and Aims: Neurokinin A (NKA) is a tachykinin neuropeptide acting predominantly via NK2 receptors. In the urinary bladder from many species, NKA is potent in contracting the detrusor muscle (e.g. porcine detrusor, pD2, 7.3). The NK1 receptor preferring tachykinin substance P (SP) was only a weak agonist in this preparation. Although most studies have focussed on the detrusor muscle, it is now apparent that the bladder lining (the mucosa) has important functions in its own right, particularly with regard to afferent sensations. We recently found that NKA is also able to contract strips of porcine bladder mucosa via tachykinin NK2 receptors, probably mediated by myofibroblasts under the urothelium [1]. Mucosal responses to NKA (pD2 7.5) were higher than those in intact strips and were inhibited in a non-competitive manner by the NK2 receptor antagonist SR48968 (pKB 10.0) but not the NK1 receptor antagonist SR140333 [1]. The aim of the present study was to characterise the receptors present in the mucosa and detrusor, using radioligand binding in homogenates, with the NK2 receptor preferring radioligand [125I]NKA.

Methods: Pig bladders from an abattoir were transported on ice and placed in cold Krebs-Henseleit solution. Mucosa was initially separated from detrusor and then pinned out to carefully dissect off all smooth muscle remnants, then frozen for later binding studies. Using standard techniques [2], time course studies (15 min–3 h) with 70 pM [125I]NKA were performed separately in detrusor and “dissected mucosal” preparations, using NKA, and later SP, SR48968, SR140333 and CP9994 to define nonspecific binding. Subsequent competition studies were carried out using 10μM SP to define nonspecific binding. Data were analysed by GraphPad Prism 3.0, and competition data compared for binding to a one-site versus a two-site model. Immunostaining was also attempted using an anti-human NK2 receptor antibody, with the peroxidase/DAB method, but this was unsuccessful.

Results: Using 10 μM SP to define nonspecific binding, specific binding of [125I]NKA was optimised at 75% and 72% after 120 min and 60 min incubation, in detrusor (n = 3) and “dissected mucosal” (n = 4) homogenates, respectively. No specific binding was seen when 1μM NKA was used to define nonspecific binding. Competition curves for SP were clearly biphasic and data were resolved into two sites in both tissues (n = 2). For detrusor, the high (H) affinity site (IC50 7.9 pM, 22% of sites) and the low (L) affinity site (IC50 29 nM) were rather different from the values in the “dissected mucosa” (IC50H 6.6 pM, 51% of sites; IC50L 1.1 nM).

Discussion and Conclusions: These preliminary binding data show that although [125I]NKA is NK2 receptor preferring, it appears to bind to sites which are more like NK1 receptors, in both porcine detrusor and “dissected mucosa” homogenates. The low affinity site may correspond to the functional receptor, although its affinity is significantly different in the two preparations. Although NKA clearly contracted the isolated preparations via NK2 receptors, the radioligand was unable to detect these, using standard binding conditions. A similar phenomenon was reported in guinea-pig lung [3]. NK1 receptors may be present in these bladder preparations e.g. on blood vessels, but not coupled to contractile mechanisms. In conclusion, NK2 receptors in porcine detrusor and mucosa appear to have unusual characteristics.

1 Sadananda P et al. (2008) Br J Pharmacol 153:1465-1473.
2 Mussap CJ et al. (1996) J Pharmacol Exp Ther 279:423-434.
3 Geraghty DP et al. (1992) Mol Puharmacol 41:147-153.