The regulatory pathways that stimulate pancreatic ductal HCO3– secretion are well described, but less is known about inhibitory pathways. Inhibitory pathways may be important in terms of limiting the hydrostatic pressure within the ducts (so preventing leakage of enzymes into the parenchyma of the gland), and in terms of switching off pancreatic secretion after a meal. Substance P (SP) inhibits secretin-stimulated HCO3– secretion (Ashton et al. 1990), and we have recently reported that SP exerts its effect by inhibiting an apical Cl–/HCO3– exchanger in the duct cell (Hegyi et al. 2001). The purpose of this study was to identify the intracellular signalling pathway utilized by SP.
Guinea-pigs (150-250 g) were humanely killed, the pancreas was removed and small intra/interlobular ducts were isolated and cultured overnight as previously described (Argent et al. 1986). The rate of HCO3– secretion was determined by measuring the initial rate of intracellular acidification (using BCECF) following sudden block of basolateral NaHCO3 cotransporters and Na+/H+ exchangers with DIDS (100 µM) and amiloride (200 µM), respectively (Szalmay et al. 2001). The buffering capacity of duct cells was estimated and the rate of transmembrane H+ flux, JH, was calculated. Since H+ and HCO3– will largely be derived from H2HCO3, we have assumed that JH is equivalent to JHCO3. All the experiments were performed in HCO3–-buffered Ringer solution at 37 °C.
Secretin (10 nM) elevated the basal JH about 3-fold from 2.18 ± 0.2 mM min-1 to 6.3 ± 1.5 mM min-1 (n = 6 ducts, P < 0.05, means ± S.E.M. and Student’s t test). SP (20 nM) had no effect on the basal JH, but totally inhibited the secretin-stimulated elevation of JH (n = 6 ducts). This inhibitory effect of SP could be relieved by 20 nM spantide, a SP receptor antagonist (n = 6 ducts). Phorbol 12,13-dibutyrate (100 nM), an activator of protein kinase C (PKC), reduced the basal JH by 38 % (n = 7 ducts), and totally blocked secretin-stimulated JH (n = 6 ducts). In addition, bisindolylmaleimide (1 µM), an inhibitor of PKC, partially relieved the inhibitory effect of SP on secretin-stimulated JH.
Our data suggest that SP exerts its inhibitory effect on the duct cell by activating PKC, which then inhibits apical Cl–/HCO3– exchange.
This work was funded by The Wellcome Trust (Grant No. 022618) and the Hungarian Scientific Research Fund (Grant No. D42188).