An intermediate-conductance Ca2+-activated K+ channel is important for secretion in pancreatic duct cells.

Potassium channels play a vital role in maintaining the membrane potential and the driving force for anion secretion in epithelia. In pancreatic ducts, which secrete bicarbonate-rich fluid, the identity of K(+) channels has not been extensively investigated. In this study, we investigated the molecular basis of functional K(+) channels in rodent and human pancreatic ducts (Capan-1, PANC-1, and CFPAC-1) using molecular and electrophysiological techniques. RT-PCR analysis revealed mRNAs for KCNQ1, KCNH2, KCNH5, KCNT1, and KCNT2, as well as KCNN4 coding for the following channels: KVLQT1; HERG; EAG2; Slack; Slick; and an intermediate-conductance Ca(2+)-activated K(+) (IK) channel (K(Ca)3.1). The following functional studies were focused on the IK channel. 5,6-Dichloro-1-ethyl-1,3-dihydro-2H-benzimidazole-2-one (DC-EBIO), an activator of IK channel, increased equivalent short-circuit current (I(sc)) in Capan-1 monolayer, consistent with a secretory response. Clotrimazole, a blocker of IK channel, inhibited I(sc). IK channel blockers depolarized the membrane potential of cells in microperfused ducts dissected from rodent pancreas. Cell-attached patch-clamp single-channel recordings revealed IK channels with an average conductance of 80 pS in freshly isolated rodent duct cells. These results indicated that the IK channels may, at least in part, be involved in setting the resting membrane potential. Furthermore, the IK channels are involved in anion and potassium transport in stimulated pancreatic ducts.

Pancreatic bicarbonate secretion involves two proton pumps.

Pancreas secretes fluid rich in digestive enzymes and bicarbonate. The alkaline secretion is important in buffering of acid chyme entering duodenum and for activation of enzymes. This secretion is formed in pancreatic ducts, and studies to date show that plasma membranes of duct epithelium express H(+)/HCO(3)(-) transporters, which depend on gradients created by the Na(+)/K(+)-ATPase. However, the model cannot fully account for high-bicarbonate concentrations, and other active transporters, i.e. pumps, have not been explored. Here we show that pancreatic ducts express functional gastric and non-gastric H(+)-K(+)-ATPases. We measured intracellular pH and secretion in small ducts isolated from rat pancreas and showed their sensitivity to H(+)-K(+) pump inhibitors and ion substitutions. Gastric and non-gastric H(+)-K(+) pumps were demonstrated on RNA and protein levels, and pumps were localized to the plasma membranes of pancreatic ducts. Quantitative analysis of H(+)/HCO(3)(-) and fluid transport shows that the H(+)-K(+) pumps can contribute to pancreatic secretion in several species. Our results call for revision of the bicarbonate transport physiology in pancreas, and most likely other epithelia. Furthermore, because pancreatic ducts play a central role in several pancreatic diseases, it is of high relevance to understand the role of H(+)-K(+) pumps in pathophysiology.

Adenosine receptors in rat and human pancreatic ducts stimulate chloride transport.

Previously, we have shown that pancreatic acini release adenosine triphosphate (ATP) and ATP-handling enzymes, and pancreatic ducts express various purinergic P2 receptors. The aim of the present study was to establish whether pancreatic ducts also express adenosine receptors and whether these could be involved in secretory processes, which involve cystic fibrosis transmembrane regulator (CFTR) Cl- channels or Ca2+-activated Cl- channels and H(+)/HCO(-)(3) transporters. Reverse transcriptase polymerase chain reaction analysis on rat pancreatic ducts and human duct cell adenocarcinoma lines showed that they express A1, A2A, A2B, and A3 receptors. Real-time PCR revealed relatively low messenger RNA levels of adenosine receptors compared to beta-actin; the rank order for the receptors was A2A>A2B>or=A3>>A1 for rat pancreas and A2B>A2A>>A3>or=A1 for duct cell lines. Whole-cell patch-clamp recordings on rat pancreatic ducts showed that, in about half of the recordings, adenosine depolarized the membrane voltage, and this was because of the opening of Cl- channels. Using a Cl--sensitive fluorophore and single-cell imaging on duct cell lines, it was found that 58% of PANC-1 cells responded to adenosine, whereas only 9% of CFPAC-1 cells responded. Adenosine elicited Ca2+ signals only in a few rat and human duct cells, which did not seem to correlate with Cl- signals. A2A receptors were localized in the luminal membranes of rat pancreatic ducts, plasma membrane of many PANC-1 cells, but only a few CFPAC-1 cells. Taken together, our data indicate that A2A receptors open Cl- channels in pancreatic ducts cells with functional CFTR. We propose that adenosine can stimulate pancreatic secretion and, thereby, is an active player in the acini-to-duct signaling.

Characterization of transport mechanisms and determinants critical for Na+-dependent Pi symport of the PiT family paralogs human PiT1 and PiT2.

The general phosphate need in mammalian cells is accommodated by members of the P(i) transport (PiT) family (SLC20), which use either Na(+) or H(+) to mediate inorganic phosphate (P(i)) symport. The mammalian PiT paralogs PiT1 and PiT2 are Na(+)-dependent P(i) (NaP(i)) transporters and are exploited by a group of retroviruses for cell entry. Human PiT1 and PiT2 were characterized by expression in Xenopus laevis oocytes with (32)P(i) as a traceable P(i) source. For PiT1, the Michaelis-Menten constant for P(i) was determined as 322.5 +/- 124.5 microM. PiT2 was analyzed for the first time and showed positive cooperativity in P(i) uptake with a half-maximal activity constant for P(i) of 163.5 +/- 39.8 microM. PiT1- and PiT2-mediated Na(+)-dependent P(i) uptake functions were not significantly affected by acidic and alkaline pH and displayed similar Na(+) dependency patterns. However, only PiT2 was capable of Na(+)-independent P(i) transport at acidic pH. Study of the impact of divalent cations Ca(2+) and Mg(2+) revealed that Ca(2+) was important, but not critical, for NaP(i) transport function of PiT proteins. To gain insight into the NaP(i) cotransport function, we analyzed PiT2 and a PiT2 P(i) transport knockout mutant using (22)Na(+) as a traceable Na(+) source. Na(+) was transported by PiT2 even without P(i) in the uptake medium and also when P(i) transport function was knocked out. This is the first time decoupling of P(i) from Na(+) transport has been demonstrated for a PiT family member. Moreover, the results imply that putative transmembrane amino acids E(55) and E(575) are responsible for linking P(i) import to Na(+) transport in PiT2.

P2Y2 and P2Y4 receptors regulate pancreatic Ca(2+)-activated K+ channels differently.

Extracellular ATP is an important regulator of transepithelial transport in a number of tissues. In pancreatic ducts, we have shown that ATP modulates epithelial K+ channels via purinergic receptors, most likely the P2Y2 and P2Y4 receptors, but the identity of the involved K+ channels was not clear. In this study, we show by RT-PCR analysis that rat pancreatic ducts express Ca(2+)-activated K+ channels of intermediate conductance (IK) and big conductance (BK), but not small conductance (SK). Possible interactions between P2Y receptors and these Ca(2+)-activated K+ channels were examined in co-expression experiments in Xenopus laevis oocytes. K+ channel activity was measured electrophysiologically in oocytes stimulated with UTP (0.1 mM). UTP stimulation of oocytes expressing P2Y4 receptors and BK channels resulted in a 30% increase in the current through the expressed channels. In contrast, stimulation of P2Y2 receptors led to a 20% inhibition of co-expressed BK channel activity, a response that was sensitive to TEA. Furthermore, co-expression of IK channels with P2Y4 and P2Y2 receptors resulted in a large hyperpolarization and 22-fold and 5-fold activation of currents by UTP, respectively. Taken together, this study shows that there are different interactions between the subtypes of P2Y purinergic receptors and different Ca(2+)-activated K+ channels.