Merely touching the pancreas can lead to premature zymogen activation and pancreatitis but the mechanism is not completely understood. Here we demonstrate that pancreatic acinar cells express the mechanoreceptor Piezo1 and application of pressure within the gland produces pancreatitis. To determine if this effect is through Piezo1 activation, we induce pancreatitis by intrapancreatic duct instillation of the Piezo1 agonist Yoda1. Pancreatitis induced by pressure within the gland is prevented by a Piezo1 antagonist. In pancreatic acinar cells, Yoda1 stimulates calcium influx and induces calcium-dependent pancreatic injury. Finally, selective acinar cell-specific genetic deletion of Piezo1 protects mice against pressure-induced pancreatitis. Thus, activation of Piezo1 in pancreatic acinar cells is a mechanism for pancreatitis and may explain why pancreatitis develops following pressure on the gland as in abdominal trauma, pancreatic duct obstruction, pancreatography, or pancreatic surgery. Piezo1 blockade may prevent pancreatitis when manipulation of the gland is anticipated.

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tion of biliary secretion through apical purinergic receptors in cultured rat cholangiocytes. Am. J. Physiol. 273 (Gastrointest. Liver Physiol. 36): G1108-G1117, 1997.-To evaluate whether ATP in bile serves as a signaling factor regulating ductular secretion, voltage-clamp studies were performed using a novel normal rat cholangiocyte (NRC) model. In the presence of amiloride (100 µM) to block Na ϩ channels, exposure of the apical membrane to ATP significantly increased the shortcircuit current (I sc ) from 18.2 Ϯ 5.9 to 52.8 Ϯ 12.7 µA (n ϭ 18). The response to ATP is mediated by basolateral-to-apical Cl Ϫ transport because it is inhibited by 1) the Cl Ϫ channel blockers 4,4Ј-diisothiocyanostilbene-2,2Ј-disulfonic acid (1 mM), diphenylanthranilic acid (1.5 mM), or 5-nitro-2-(3-phenylpropylamino)benzoic acid (50 or 100 µM) in the apical chamber, 2) the K ϩ channel blocker Ba 2ϩ (5 mM), or 3) the Na ϩ -K ϩ -2Cl Ϫ cotransport inhibitor bumetanide (200 µM) in the basolateral chamber. Other nucleotides stimulated an increase in I sc with a rank order potency of UTP ϭ ATP ϭ adenosine 5Ј-O-(3)-thiotriphosphate, consistent with P 2u purinergic receptors. ADP, AMP, 2-methylthioadenosine 5Ј-triphosphate, and adenosine had no effect. A cDNA encoding a rat P 2u receptor (rP 2u R) was isolated from a liver cDNA library, and functional expression of the corresponding mRNA in Xenopus laevis oocytes resulted in the appearance of ATP-stimulated currents with a similar pharmacological profile. Northern analysis identified hybridizing mRNA transcripts in NRC as well as other cell types in rat liver. These findings indicate that exposure of polarized cholangiocytes to ATP results in luminal Cl Ϫ secretion through activation of P 2u receptors in the apical membrane. Release of ATP into bile may serve as an autocrine or paracrine signal regulating cholangiocyte secretory function.

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Background Endoscopic retrograde cholangiopancreatography (ERCP) has a high risk of pancreatitis although the underlying mechanisms are unclear. Transient receptor potential vanilloid 1 (TRPV1) is a cation channel expressed on C and Ad fibres of primary sensory neurons and is activated by low pH. TRPV1 activation causes release of inflammatory mediators that produce oedema and neutrophil infiltration. We previously demonstrated that neurogenic factors contribute to the pathogenesis of pancreatitis. Resiniferatoxin (RTX) is a TRPV1 agonist that, in high doses, defunctionalises C and Ad fibres. When we discovered that the pH of radio-opaque contrast solutions used for ERCP was 6.9, we hypothesised that low pH may contribute to the development of contrast-induced pancreatitis via activation of TRPV1. Methods Rats underwent equal pressure pancreatic ductal injection of contrast solutions at varying pH with or without RTX. Results Contrast solution (pH 6.9) injected into the pancreatic duct caused a significant increase in pancreatic oedema, serum amylase, neutrophil infiltration, and histological damage. Solutions of pH 7.3 injected at equal pressure caused little damage. The severity of the pancreatitis was significantly increased by injection of solutions at pH 6.0. To determine if the effects of low pH were mediated by TRPV1, RTX was added to the contrast solutions. At pH levels of 6.0 and 6.9, RTX significantly reduced the severity of pancreatitis. Conclusions Contrast solutions with low pH contribute to the development of pancreatitis through a TRPV1-dependent mechanism. It is possible that increasing the pH of contrast solution and/or adding an agent that inhibits primary sensory nerve activation may reduce the risk of post-ERCP pancreatitis.

Expression of the recombinant human U1-70K protein in COS cells resulted in its rapid transport to the nucleus, even when binding to Ul RNA was debilitated. Deletion analysis of the U1-70K protein revealed the existence of two segments of the protein which were independently capable of nuclear localization. One nuclear localization signal (NLS) was mapped within the U1 RNA-binding domain and consists of two typically separated but interdependent elements. The major element of this NLS resides in structural loop 5 between the 14 strand and the t2 helix of the folded RNA recognition motif. The C-terminal half of the U1-70K protein which was capable of nuclear entry contains two arginine-rich regions, which suggests the existence of a second NLS. Site-directed mutagenesis of the RNA recognition motif NLS demonstrated that the U1-70K protein can be transported independently of Ul RNA and that its association with the Ul small nuclear ribonucleoprotein particle can occur in the nucleus.U1 small nuclear ribonucleoprotein (snRNP) particles are located in the nuclei of eukaryotic cells and perform a transactivating role in splicing of pre-mRNAs (for reviews, see references 11 and 38). U1 snRNP particles contain U1 RNA and a set of nine proteins termed the Sm complex, which is also present in the other abundant U snRNPs (21, 23). In addition, the U1-70K, U1-A, and U1-C proteins are unique to the U1 snRNP particle (3,29).Assembly of the U1 snRNP particle is thought to occur in the cytoplasm (25). It is believed that cytoplasmic assembly and nuclear transport require both hypermethylation of the 5' cap structure of U1 RNA (12) and a binding site for the Sm proteins on U1 RNA (8,12). However, more recent experiments suggested that the Sm core domain can bind to a transport receptor and can be transported to the nucleus independently of the m3G cap (9). Also, pulse-chase experiments suggest that the Sm proteins associate with U1 RNA in the cytoplasm (7). Consistent with this model of U1 snRNP transport were the findings that the U1-70K and U1-A proteins are dispensable for nuclear import of U1 snRNP particles (12). Furthermore, microinjection experiments in Xenopus oocytes using U1-A protein indicated that this protein contains sequences necessary for nuclear localization within a stretch of 110 residues between amino acids 94 to 204 (17). These amino acids belong to the spacer region separating the two RNA recognition motifs (RRMs). The U1-C protein has also been reported to migrate to the nuclei of oocytes on its own and to associate with preassembled U1 snRNP (15).The U1-70K protein possesses a single defined U1 RNAbinding domain near the N terminus and two Arg-rich domains in the C-terminal portion (30). Similar Arg-rich sequences are also present in known splicing factors including tra, tra-2 (30), su (wa) (4), and in a unique set of proteins termed SR (Ser-Arg-rich) proteins (41 posed of 80 amino acids (1, 18), and a model of the threedimensional structure has been deduced for the first RRM of U1-A by crystallography (...

TRPV4 ion channels represent osmo-mechano-TRP channels with pleiotropic function and wide-spread expression. One of the critical functions of TRPV4 in this spectrum is its involvement in pain and inflammation. However, few small-molecule inhibitors of TRPV4 are available. Here we developed TRPV4-inhibitory molecules based on modifications of a known TRPV4-selective tool-compound, GSK205. We not only increased TRPV4-inhibitory potency, but surprisingly also generated two compounds that potently co-inhibit TRPA1, known to function as chemical sensor of noxious and irritant signaling. We demonstrate TRPV4 inhibition by these compounds in primary cells with known TRPV4 expression - articular chondrocytes and astrocytes. Importantly, our novel compounds attenuate pain behavior in a trigeminal irritant pain model that is known to rely on TRPV4 and TRPA1. Furthermore, our novel dual-channel blocker inhibited inflammation and pain-associated behavior in a model of acute pancreatitis – known to also rely on TRPV4 and TRPA1. Our results illustrate proof of a novel concept inherent in our prototype compounds of a drug that targets two functionally-related TRP channels, and thus can be used to combat isoforms of pain and inflammation in-vivo that involve more than one TRP channel. This approach could provide a novel paradigm for treating other relevant health conditions.