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Pancreatic duct epithelium secretes a HC[O.sub.3.sup.-]-rich fluid by a mechanism dependent on cystic fibrosis transmembrane conductance regulator (CFTR) in the apical membrane. However, the exact role of CFTR remains unclear. One possibility is that the HC[O.sub.3.sup.-] permeability of CFTR provides a pathway for apical HC[O.sub.3.sup.-] efflux during maximal secretion. We have therefore attempted to measure electrodiffusive fluxes of HC[O.sub.3.sup.-] induced by changes in membrane potential across the apical membrane of interlobular ducts isolated from the guinea pig pancreas. This was done by recording the changes in intracellular pH ([pH.sub.i]) that occurred in luminally perfused ducts when membrane potential was altered by manipulation of bath [K.sup.+] concentration. Apical HC[O.sub.3.sup.-] fluxes activated by cyclic AMP were independent of [Cl.sup.-] and luminal [Na.sup.+], and substantially inhibited by the CFTR blocker, [CFTR.sub.inh]-172. Furthermore, comparable HC[O.sub.3.sup.-] fluxes observed in ducts isolated from wild-type mice were absent in ducts from cystic fibrosis ([DELTA]F) mice. To estimate the HC[O.sub.3.sup.-] permeability of the apical membrane under physiological conditions, guinea pig ducts were luminally perfused with a solution containing 195 mM HC[O.sub.3.sup.-] and 24 mM [Cl.sup.-] in the presence of 5% C[O.sub.2]. From the changes in [pH.sub.i], membrane potential, and buffering capacity, the flux and electrochemical gradient of HC[O.sub.3.sup.-] across the apical membrane were determined and used to calculate the HC[O.sub.3.sup.-] permeability. Our estimate of ~0.1 [micro]m [sec.sup.-1] for the apical HC[O.sub.3.sup.-] permeability of guinea pig duct cells under these conditions is close to the value required to account for observed rates of HC[O.sub.3.sup.-] secretion. This suggests that CFTR functions as a HC[O.sub.3.sup.-] channel in pancreatic duct cells, and that it provides a significant pathway for HC[O.sub.3.sup.-] transport across the apical membrane.

Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) have opposite actions on the gallbladder; PACAP induces contraction, whereas VIP induces relaxation. Here, we have attempted to identify key residues responsible for their interactions with PACAP ([PAC.sub.1]) and VIP (VPAC) receptors in the guinea pig gallbladder. We synthesized PACAP-27/VIP hybrid peptides and compared their actions on isolated guinea pig gallbladder smooth muscle strips using isotonic transducers. [[Ala.sup.4]]- and [[Val.sup.5]]PACAP-27 were more potent than PACAP-27 in stimulating the gallbladder. In contrast, [[Ala.sup.4], [Val.sup.5]]- and [[Ala.sup.4], [Val.sup.5], [Asn.sup.9]]PACAP-27 induced relaxation similarly to VIP. [[Asn.sup.9]]-, [[Thr.sup.11]]-, or [[Leu.sup.13]]PACAP-27 had 20-70% contractile activity of PACAP-27, whereas [[Asn.sup.24], [Ser.sup.25], [Ile.sup.26]]PACAP-27 showed no change in the activity. All VIP analogs, including [[Gly.sup.4], [Ile.sup.5], [Ser.sup.9]] VIP, induced relaxation. In the presence of a [PAC.sub.1] receptor antagonist, PACAP(6-38), the contractile response to PACAP-27 was inhibited and relaxation became evident. RT-PCR analysis revealed abundant expressions of [PAC.sub.1] receptor, 'hop' splice variant, and [VPAC.sub.1] and [VPAC.sub.2] receptor mRNAs in the guinea pig gallbladder. In conclusion, PACAP-27 induces contraction of the gallbladder via [PAC.sub.1]/hop receptors. [Gly.sup.4] and [Ile.sup.5] are the key N[H.sub.2]-terminal residues of PACAP-27 that distinguish [PAC.sub.1]/hop receptors from [VPAC.sub.1]/[VPAC.sub.2] receptors. However, both the N[H.sub.2]-terminal and [alpha]-helical regions of PACAP-27 are required for initiating gallbladder contraction. [PAC.sub.1] receptor; [VPAC.sub.1] receptor; [VPAC.sub.2] receptor; splice variant

The SLC26 transporters are a family of mostly luminal [Cl.sup.-] and HC[O.sub.3] transporters. The transport mechanism and the [Cl.sup.-]/HC[O.sub.3.sup.-] stoichiometry are not known for any member of the family. To address these questions, we simultaneously measured the HC[Osub.3] and [Cl.sup.-] fluxes and the current or membrane potential of slc26a3 and slc26a6 expressed in Xenopus laevis oocytes and the current of the transporters expressed in human embryonic kidney 293 cells, slc26a3 mediates a coupled 2[Cl.sup-]/1HC[O.sub.3.sup.-] exchanger. The membrane potential modulated the apparent affinity for extracellular [Cl.sup.-] of [Cl.sup.-]/HC[O.sub.3] exchange by slc26a3. Interestingly, the replacement of [Cl.sup.-] with N[O.sub.3.sup.-] or SC[N.sup.-] uncoupled the transport, with large N[O.sub.3.sup.-] and SC[N.sub.-] currents and low HC[O.sub.3.sup.-] transport. An apparent uncoupled current was also developed during the incubation of slc26a3-expressing oocytes in HC[O.sub.3.sup.-]-buffered [Cl.sup.-]-free media. These findings were used to develop a turnover cycle for [Cl.sup.-] and HC[O.sub.3.sup.-] transport by slc26a3. [Cl.sup.-] and HC[O.sub.3.sup.-] flux measurements revealed that slc26a6 mediates a 1[Cl.sup.-]/2HC[O.sub.3.sup.-] exchange. Accordingly, holding the membrane potential at 40 and -100 mV accelerated and inhibited, respectively, [Cl.sup.-]-mediated HC[O.sub.3.sup.-] influx, and holding the membrane potential at -100 mV increased HC[O.sub.3.sup.-]-mediated [Cl.sup.-] influx. These findings indicate that slc26a6 functions as a coupled 1[Cl.sup.-]/2HC[O.sub.3.sup.-] exchanger. The significance of isoform-specific [Cl.sup.-] and HC[O.sub.3.sup.-] transport stoichiometry by slc26a3 and slc26a6 is discussed in the context of diseases of epithelial [Cl.sup.-] absorption and HC[O.sub.3.sup.-] secretion.

Ethanol strongly augments secretin-stimulated, but not acetylcholine (ACh)-stimulated, fluid secretion from pancreatic duct cells. To understand its mechanism of action, we examined the effect of short-chain n-alcohols on fluid secretion and intracellular [Ca.sup.2+] concentration ([[Ca.sup.2+].sub.i]) in guinea pig pancreatic ducts. Fluid secretion was measured by monitoring the luminal volume of isolated interlobular ducts. [[Ca.sup.2+].sub.i] was estimated using fura-2 microfluorometry. Methanol and ethanol at 0.3-10 mM concentrations significantly augmented fluid secretion and induced a transient elevation of [[Ca.sup.2+].sub.i] in secretin- or dibulyryl adenosine 3',5'-cyclic monophosphate (DBcAMP)-stimulated ducts. However, they failed to affect fluid secretion and [[Ca.sup.2+].sub.i] in unstimulated and ACh-stimulated ducts. In contrast, propanol and butanol at 0.3-10 mM concentrations significantly reduced fluid secretion and decreased [[Ca.sup.2+].sub.i] in unstimulated ducts and in ducts stimulated with secretin, DBcAMP, or ACh. Both stimulatory and inhibitory effects of n-alcohols completely disappeared after their removal from the perfusate. Propanol and butanol inhibited the plateau phase, but not the initial peak, of [[Ca.sup.2+].sub.i], response to ACh as well as the [[Ca.sup.2+].sub.i] elevation induced by thapsigargin, suggesting that they inhibit [Ca.sup.2+] influx. Removal of extracellular [Ca.sup.2+] reduced [[Ca.sup.2+].sub.i] in duct cells and completely abolished secretin-stimulated fluid secretion. In conclusion, there is a distinct cutoff point between ethanol (C2) and propanol (C3) in their effects on fluid secretion and [[Ca.sup.2+].sub.i] in duct cells. Short-chain n-alcohols appear to affect pancreatic ductal fluid secretion by activating or inhibiting the plasma membrane [Ca.sup.2+] channel. intracellular calcium; acetylcholine

Aquaporins in rat pancreatic interlobular ducts. Am J Physiol Gastrointest Liver Physiol 282: G324-G331, 2002; 10.1152/ajpgi.00198. 2001.--The aquaporin (AQP) family of water channels is distributed ubiquitously in many epithelia and plays a fundamental role in transmembrane water transport. The aim of this study is to identify the water transport pathway in pancreatic duct cells where most of the HC[O.sup.-.sub.3]-rich fluid originates. Using digital videomicroscopy, we measured the osmotic water permeability ([P.sub.f]) of pancreatic duct epithelium by exposing isolated rat interlobular ducts to the hypotonic solution (145 mosM). To identify mRNA and protein of AQPs expressed in duct cells, we conducted RT-PCR analysis and immunohistochemistry of the isolated duct and pancreas. The calculated [P.sub.f] (160-230 [micro]m/s) of the isolated ducts was significantly reduced to 16-35 [micro]m/s by 80-90% with either basolateral or luminal applications of Hg[Cl.sub.2]. Fluid secretion evoked by secretin was almost completely abolished by a basolateral or luminal application of Hg[Cl.sub.2]. A large amount of AQP1 and small amounts of AQP5 transcripts were detected in the isolated duct cells by RT-PCR. AQP1, but not AQP5, immunoreactivity was present in both luminal and basolateral membranes of the interlobular duct cells. Mercury-sensitive water channels are present in both luminal and basolateral membranes of rat pancreatic ducts. AQP1 of the known AQPs appears to be the main water pathway in interlobular ducts. isolated pancreatic duct; mercury-sensitive water channel; water permeability; fluid secretion

NMR data show that helodermin, the peptide found in lizard's venom, exhibits a stable secondary structure in pure water. This is in contrast to the secretin/vasoactive intestinal peptide family of hormones to which helodermin is homologous by sequence. NMR data also indicate that the alpha-helix conformation of helodermin in water is necessary to bind receptors. A fitting program that uses summed RMS differences between all structures is useful in defining the stable structure of helodermin in water.

Neuropeptide pituitary adenylate cyclase activating polypeptide amide (PACAP) is a hormone and neurotransmitter with 68% homology to vasoactive intestinal peptide. Nuclear magnetic resonance spectroscopy was used to analyze the solution structure of PACAP. The PACAP38 form initially showed a disordered N terminal domain of eight amino acids. It also revealed an alpha helical structure stretching from serine-9 to valine-26 with gaps between lysine-20 and lysine-21 plus a short alpha helix between glycine-28 and arginine-34. The PACAP27 form closely resembles that of PACAP38.