The neuropeptide neuromedin U (NmU) has been implicated in feeding, anxiety, pain, regulation of blood flow and smooth-muscle contraction. Two G-protein coupled receptors for NmU (NmU-R1 and NmU-R2) have been cloned from a number of species. We have previously shown that recombinant human receptors expressed in HEK293 cells couple to G_q/11 to mediate Ca²⁺ signalling and Gai to inhibit forskolin-stimulated cAMP accumulation (Brighton et al., 2003a). We have also shown that Ca²⁺ signalling in response to repetitive application of NmU does not occur and that this is a consequence of an essentially irreversible binding of NmU to its receptors rather than receptor desensitization (Brighton et al., 2003b). Here we examine NmU-mediated Ca²⁺ signalling by endogenous receptors in primary cultures of rat colonic smooth-muscle cells. Experiments were at 37^oC unless stated and data represent 3 separate experiments.

The distal colon of male Wistar rats (>300g) was diced and the extracellular-matrix enzymatically digested with papain, hyaluronidase and collagenase. Cells were dissociated by mechanical shear and cultured at 37oC, 5% CO₂ in 231 smooth-muscle cell media with growth supplement (Cascade Biologics, Nottingham, U.K) and antibiotics. Culture conditions were optimised and cells used at 7 days when they were stellated, 50-60% confluent and 90-100% showed -actin staining.

Confocal imaging of fluo-3-loaded (30min, 20^oC) smooth-muscle cells showed that challenge with 10nM human NmU (hNmU-25) resulted in a rapid (5s) rise (1.8-2.4 fold of basal) of intracellular [Ca²⁺] ([Ca²⁺]_i) followed by a smaller sustained signal (1.2-1.5 fold of basal) in approximately 80% of cells. The response was thapsigargin sensitive and the sustained signal absent when Ca²⁺ was excluded from the extracellular buffer. No response was observed to a second NmU challenge even following extensive washing of cells with buffer (12min) suggesting that NmU may still be bound to the receptors. Similar Ca²⁺ signalling was observed in smooth-muscle cells prepared from rat fundus and uterus, and guinea-pig colon and fundus.

In order to confirm our inability to remove receptor-bound NmU by washing cells with buffer we employed porcine NmU-8 N-terminally labelled with fluorescent Cy3b (Cy3b-NmU-8 (Brighton et al., 2003b). NmU-8 and hNmU-25 have similar affinities for recombinant human NmU receptors (Raddatz et al., 2000). Following addition of 10nM Cy3b-NmU-8 at 4oC, confocal imaging of cultured smooth-muscle cells showed membrane fluorescence that was not seen after pre-addition of 1µM unlabelled hNmU-25 and was not diminished by perfusion of buffer (12min, 4oC). Short-term incubation (<4min) at 37oC with Cy3b-NmU-8 resulted in punctate patches of internalized fluorescence.

Thus, activation of NmU receptors endogenously expressed in colonic smooth muscle cells results in mobilization of intracellular Ca²⁺. Furthermore, the binding of NmU is essentially irreversible and followed by ligand internalization.

Brighton P.J. et al., (2003a) Brit. J. Pharmacol. 140, 75P.
Brighton P.J. et al., (2003b) Brit. J. Pharmacol. In press.
Raddatz et al., (2000) J. Biol. Chem. 275, 32452-32459.