Jan Amstrup

P2X7 receptor activates extracellular signal-regulated kinases ERK1 and ERK2 independently of Ca2+ influx.

P2X7 nucleotide receptors modulate a spectrum of cellular events in various cells including epithelia, such as exocrine pancreas. Although the pharmacology and channel properties of the P2X7 receptors have been studied intensively, signal transduction pathways are relatively unknown. In this study we applied a heterologous expression system of rat P2X7 receptors in HEK-293 cells. We followed the receptor expression and function using the enhanced green fluorescent protein (EGFP) tag, activation of intracellular proteins and increases in cellular Ca2+. EGFP-P2X7 receptors localized to the plasma membrane, clusters within the membrane and intracellularly. Stimulation of P2X7 receptors in HEK-293 cells led to an activation of extracellular signal-regulated kinases ERK1 and ERK2 and this activation was seen after just 1 min of stimulation with ATP. Using C- and N-terminal P2X7-receptor mutants we show that the N-terminus is important in activation of ERKs, whereas deletion of the last 230 amino acids in the C-terminus did not effect ERK activation. On the other hand, Ca2+ entry was impaired in C-terminal but not in N-terminal mutants. In cell suspensions prepared from rat pancreas we show that P2X7 receptors also activate ERK1 and ERK2, indicating that these signalling pathways are also turned on in native epithelium.

Rat pancreas secretes particulate ecto-nucleotidase CD39

In exocrine pancreas, acini release ATP and the excurrent ducts express several types of purinergic P2 receptors. Thereby, ATP, or its hydrolytic products, might play a role as a paracrine regulator between acini and ducts. The aim of the present study was to elucidate whether this acinar‐ductal signalling is regulated by nucleotidase(s), and to characterize and localize one of the nucleotidases within the rat pancreas. Using RT‐PCR and Western blotting we show that pancreas expresses the full length ecto‐nucleoside triphosphate diphosphohydrolase, CD39. Immunofluorescence shows CD39 localization on basolateral membranes of acini and intracellularly. In small intercalated/ interlobular ducts, CD39 immunofluorescence was localized on the luminal membranes, while in larger ducts it was localized on the basolateral membranes. Upon stimulation with cholecystokinin‐octapeptide‐8 (CCK‐8), acinar CD39 relocalizes in clusters towards the lumen and is secreted. As a result, pancreatic juice collected from intact pancreas stimulated with CCK‐8 contained nucleotidase activity, including that of CD39, and no detectable amounts of ATP. Anti‐CD39 antibodies detected the full length (78 kDa) CD39 in pancreatic juice. This CD39 was confined only to the particulate and not to the soluble fraction of CCK‐8‐stimulated secretion. No CD39 activity was detected in secretion stimulated by secretin. The role of secreted particulate, possibly microsomal, CD39 would be to regulate intraluminal ATP concentrations within the ductal tree. In conclusion, we show a novel inducible release of full length particulate CD39, and propose its role in the physiological context of pancreatic secretion.

Purinergic receptors have different effects in rat exocrine pancreas. Calcium signals monitored by fura-2 using confocal microscopy.

Pancreatic ducts have several types of purinergic P2 receptors, however, nothing is known about P2 receptors in acini. The aim was to establish whether acini express functional P2 receptors coupled to intracellular Ca2+ signals and to measure the signals ratiometrically in a confocal laser scanning microscope. Rat acini and ducts were loaded with various Ca2+ sensing fluorophores (Fluo-4, Fura-Red, Calcium Green-1, Indo-1 and Fura-2). Only Fura-2 loaded evenly into acinar clusters and ducts and UV laser excitation at 351 and 364 nm gave signals showing opposite sensitivity to Ca2+ concentration changes. About 15% of acini or single cells within acini responded to ATP or UTP, yet all responded to carbachol. In contrast, all ducts responded to ATP/UTP. ATP stimulated uptake of a fluorescent anion, luciferin, but again only in a few acinar cells. RT-PCR analysis on acini samples collected from whole pancreas suspensions revealed transcripts for P2Y2, P2Y4 and P2X1, P2X4 receptors. The low number of functional P2 receptors in acini might be related to the fact that they release ATP. Thereby acini would avoid autocrine stimulation and initiation of autodigestive processes, such as occurs in pancreatitis.

Mitochondria-rich cells as experimental model in studies of epithelial chloride channels

The mitochondria-rich (mr) cell of amphibian skin epithelium is differentiated as a highly specialised pathway for passive transepithelial transport of chloride. The apical membrane of mr cells expresses several types of Cl(-) channels, of which the function of only two types has been studied in detail. (i) One type of channel is gated by voltage and external chloride concentration. This intriguing type of regulation leads to opening of channels only if [Cl(-)](o) is in the millimolar range and if the electrical potential is of a polarity that secures an inwardly directed net flux of this ion. Reversible voltage activations of the conductance proceed with long time constants, which depend on V in such a way that the rate of conductance activation increases when V is clamped at more negative values (serosal bath grounded). The gating seems to involve processes that are dependent on F-actin localised in the submembrane domain in the neck region of the flask-shaped mr cell. (ii) The other identified Cl(-) pathway of mr cells is mediated by small-conductance apical CFTR chloride channels as concluded from its activation via beta-adrenergic receptors, ion selectivity, genistein stimulation and inhibition by glibenclamide. bbCFTR has been cloned, and immunostaining has shown that the gene product is selectively expressed in mr cells. There is cross-talk between the two pathways in the sense that activation of the conductance of the mr cell by voltage clamping excludes activation via receptor occupation, and vice versa. The mechanism of this cross-talk is unknown.

Expression of cystic fibrosis transmembrane conductance regulator in the skin of the toad, Bufo bufo and possible role for Cl− transport across the heterocellular epithelium

Evidence is discussed that apical CFTR Cl- channels of mitochondria-rich (MR) cells of Bufo bufo skin conduct beta-adrenergic receptor-activated Cl- currents. Ussing chambers studies revealed the following selectivity sequence of the receptor activated conductance, Cl- > Br- > NO3- > I-. With ion selective microelectrode-techniques, it was shown that receptor-coupled Cl- channels are not located in principal cells. A small conductance (7-10 pS) CFTR-like Cl- channel is located in the apical plasma membrane of MR cells. Short life times of sealed patches prevented detailed study of its selectivity to other halide ions and its molecular regulation. With monoclonal hCFTR-antibodies, selective expression in MR cells of the targeted antigens could be demonstrated. A transcript of CFTR was amplified in the skin, and a bbCFTR cDNA clone was generated from toad skin mRNA that exhibits 89% amino acid identity with the human homologue. The frequency of obtaining channels in patch clamp studies was too low for accounting quantitatively for the macroscopic conductance. Since MR cells were isolated by trypsin, and a putative extracellular loop of the deduced bbCFTR protein contains a target peptide bond for trypsin, enzyme treatment may have destroyed apical CFTR molecules.

Corrigendum to “β-Adrenergic receptors couple to CFTR chloride channels of intercalated mitochondria-rich cells in the heterocellular toad skin epithelium” [Biochim. Biophy. Acta 1618 (2003) 140–152]

The authors regret that there are errors in Figs. 10 and 11 of the article referenced above. In subsequent experimentation, the results of the original study generated by J. Amstrup (GenBank: AY02676) could not be reproduced. In the laboratory of E.H. Larsen, this was discovered by Svend Erik Westh Hansen, who subsequently cloned and verified a significant piece of the gene as listed in GenBank: AY02676[1.2] (submitted by Westh Hansen, S.E. and Hviid Larsen, E.). The wrong data has been rectified in this corrigendum by replacing the original Figs. 10 and 11 by the revised versions given below. To accommodate these changes in the text, revised versions of the abstract and Section 3.5 are also included. The corrections do not affect the conclusions of the article, which are still well supported and unchanged. The authors apologize for any inconvenience the errors in the original version of this article may have caused.