Search

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.

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.

The interlobular duct segments of guinea-pig pancreas secrete HC[O.sup.-.sub.3] into an HC[O.sup.-.sub.3]-rich (125 mM) luminal fluid after secretin stimulation (Ishiguro et al. 1998. J. Physiol. 511:407-422). Under these conditions intracellular HC[O.sup.-.sub.3] is ~20 mM and intracellular potential is approximately -60 mV, indicating that there is a luminally directed electrochemical gradient for HC[O.sup.-.sub.3] (Ishiguro et al. 2002. J. Gen. Physiol. 120:617-628). To examine whether apical HC[O.sup.-.sub.3] transport is mediated by an anion conductance, we measured changes in intracellular pH ([pH.sub.i]) when the cells were de- or hyperpolarized by manipulation of extracellular K+ ([K.sup.+].sub.B). The isolated ducts were superfused with HC[O.sup.-.sub.3]/ [CO.sub.2]-free HEPES-buffered solution and luminally perfused with 125 mM H[O.sup.-.sub.3], 24 mM [Cl.sup.-] , and 5% [CO.sub.2] [pH.sub.i] was measured with BCECF. When [[K.sup.+].sub.B] was raised from 5 to 70 mM, [pH.sub.i] in the unstimulated ducts changed only slightly. In the presence of dbcAMP, the depolarization caused a large increase in [pH.sub.i] from 6.83 [+ or -] 0.11 to 7.32 [+ or -] 0.09 (mean [+ or -] SEM, n = 4, P < 0.01). When [[K.sup.+]].sub.B] was reduced from 5 to 1 mM, [pH.sub.i] decreased by 0.11 [+ or -] 0.01 (P < 0.05). Under [Cl.sup.-]-free conditions, when [[K.sup.+].sub.B] was reduced from 5 to 1 mM and then raised to 70 mM in the presence of dbcAMP, [pH.sub.i] decreased from 7.15 [+ or -] 0.06 (n = 4) to 7.06 [+ or -] 0.07 and then increased to 7.54 [+ or -] 0.16 (P < 0.05). In summary, de- and hyperpolarization caused changes in [pH.sub.i] that most probably reflected the influx and efflux of HC[O.sup.-.sub.-] across the apical membrane. These HC[O.sup.-.sub.-] movements were not dependent on the presence of [Cl.sup.-] and may be attributed to the presence of a significant HC[O.sup.-.sub.3] conductance at 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 (PAC1) 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. [Ala4]- and [Val5]PACAP-27 were more potent than PACAP-27 in stimulating the gallbladder. In contrast, [Ala4, Val5]- and [Ala4, Val5, Asn9]PACAP-27 induced relaxation similarly to VIP. [Asn9]-, [Thr11]- or [Leu13]PACAP-27 had 20-70% contractile activity of PACAP-27, whereas [Asn24,Ser25,Ile26]PACAP-27 showed no change in the activity. All VIP analogs, including [Gly4,Ile5,Ser9] VIP, induced relaxation. In the presence of a PAC1 receptor antagonist, PACAP(6-38), the contractile response to PACAP-27 was inhibited and relaxation became evident. RT-PCR analysis revealed abundant expressions of PAC1 receptor, "hop" splice variant, and VPACI and VPAC2 receptor mRNAs in the guinea pig gallbladder. In conclusion, PACAP-27 induces contraction of the gallbladder via PAC1/hop receptors. Gly4 and Ile5 are the key N1-12-terminal residues of PACAP-27 that distinguish PAC1/hop receptors from VPAC1/VPAC2 receptors. However, both the NH2-terminal and α-helical regions of PACAP-27 are required for initiating gallbladder contraction. [ABSTRACT FROM AUTHOR]

Aberrant HCO3- transport is a hallmark of cystic fibrosis (CE) and is associated with aberrant Cl-dependent HCO3- transport by the cystic fibrosis transmembrane conductance regulator (CFTR). We show here that HCO3- current by CFTR cannot account for CFTR-activated HCO3- transport and that CFTR does not activate AE1-AE4. In contrast, CFTR markedly activates Cl- and OH-/HCO3- transport by members of the SLC26 family DRA, SLC26A6 and pendrin. Most notably, the SLC26s are electrogenic transporters with isoform-specific stoichiometries. DRA activity occurred at a Cl-/HCO3- ratio &ges;2. SLC26A6 activity is voltage regulated and occurred at HCO3-/Cl- &ges;2. The physiological significance of these findings is demonstrated by interaction of CFTR and DRA in the mouse pancreas and an altered activation of DRA by the R117H and GS51D mutants of CFTR. These findings provide a molecular mechanism for epithelial HCO3- transport (one SLC26 transporter-electrogenic transport; two SLC26 transporters with opposite stoichiometry in the same membrane domain-electroneutral transport), the CE-associated aberrant HCO3- transport, and reveal a new function of CFTR with clinical implications for CE and congenital chloride diarrhea. [ABSTRACT FROM AUTHOR]

The SLC26 transporters are a family of mostly luminal Cl-- and HCO3- transporters. The transport mechanism and the Cl-/HCO3- stoichiometry are not known for any member of the family. To address these questions, we simultaneously measured the HCO3- and Cl- 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 2Cl-/1HCO3- exchanger. The membrane potential modulated the apparent affinity for extracellular Cl-- of Cl-/HCO3- exchange by slc26a3. Interestingly, the replacement of Cl-- with NO3- or SCN- uncoupled the transport, with large NO3- and SCN- currents and low HCO3- transport. An apparent uncoupled current was also developed during the incubation of slc26a3-expressing oocytes in HCO3-buffered Cl---free media. These findings were used to develop a turnover cycle for Cl-- and HCO3- transport by slc26a3. Cl-- and HCP3- flux measurements revealed that slc26a6 mediates a 1Cl-/2HCO3- exchange. Accordingly, holding the membrane potential at 40 and -100 mV accelerated and inhibited, respectively, Cl---mediated HCO3- influx, and holding the membrane potential at -100 mV increased HCO3--mediated Cl-- influx. These findings indicate that slc26a6 functions as a coupled 1Cl-/2HCO3- exchanger. The significance of isoform-specific Cl-- and HCO3- transport stoichiometry by slc26a3 and slc26a6 is discussed in the context of diseases of epithelial Cl-- absorption and HCO3- secretion. [ABSTRACT FROM AUTHOR]