Extracellular adenosine 5'-triphosphate (ATP) has been described to act as a regulator in many cells and tissues, including epithelia, and in the gastrointestinal tract ATP is one of the substances involved in non-cholinergic non-adrenergic control. However, very little is known about the effect of ATP on pancreatic ducts, which normally secrete bicarbonate-rich fluid in response to secretin. Hence, the aim of our present study was to test the effect of ATP and other nucleotides on intracellular Ca2+ activity ([Ca2+]i) of pancreatic ducts, and thereby get information about purinergic receptors that might play a role in the regulation of pancreatic bicarbonate transport. Native intralobular ducts were obtained from rat pancreas and [Ca2+]i in 10-20 cells was measured using the fura-2 method. ATP (10(-4) mol/l) evoked a characteristic biphasic Ca2+ transient in duct cells. Nucleotides, used to classify the P2 receptors, acted with the following potency on the peak Ca2+ in many ducts: uridine 5'-triphosphate (UTP) >/= ATP >inosine 5'-triphosphate >/= 2-methylthio-ATP > beta,gamma-methyl-ATP > adenosine. However, although the peak [Ca2+]i responses to ATP and UTP were similar, the plateau [Ca2+]i was nearly doubled with UTP. Moreover, in about one-third of the ducts studied, UTP had no effect on cell Ca2+, while the response to ATP was normal. In further experiments we found that removal of extracellular Mg2+ increased the peak [Ca2+]i evoked in response to ATP. 2'&3'-O-(4-benzoylbenzoyl) ATP (BzATP) evoked a monophasic and slower increase in [Ca2+]i, which was inhibited by removal of extracellular Ca2+, or by addition of 4,4'-diisothiocyanatostilbene-2, 2'-disulphonic acid (DIDS). Taken together, our data indicate that there are two types of purinergic receptors on pancreatic ducts through which ATP can act. These are pharmacologically known as P2U and P2Z receptors and may correspond to P2Y2 and P2X7 receptors.
In epithelia, extracellular nucleotides are often associated with regulation of ion transporters, especially Cl ؊ channels. In this study, we investigated which purinoceptors are present in native pancreatic ducts and how they regulate ion transport. We applied whole-cell patch-clamp recordings, intracellular Ca
SUMMARY1. Active Cl-currents were studied in short-circuited toad skin epithelium in which the passive voltage-activated Cl-current is zero. Under visual control doublebarrelled microelectrodes were used for impaling principal cells from the serosal side, or for measuring the pH profile in the solution bathing the apical border.2. The net inward (active) 36CP-flux of 27+8 pmol s-1 cm-2 (16) (mean + s.E.M (number of observations)) was abolished by 2 mM-CN-(6-3 ± 3-5 pmol s-1 cm-2 (8)). The active flux was maintained in the absence of active Na+ transport when the latter was eliminated by either 100 ,um-mucosal amiloride, replacement of mucosal Na+ with K+, or by 3 mM-serosal ouabain.3. In Ringer solution buffered by 24 mM-HCO3,-5 % CO2 mucosal amiloride reversed the short circuit current (ISC). The outward ISc was maintained when gluconate replaced mucosal Cl-, and it was reversibly reduced in C02-free 5 mM-Trisbuffered Ringer solution (pH = 7 40) or by the proton pump inhibitor oligomycin.These observations indicate that the source of the outward ISc is an apical proton pump.4. Amiloride caused principal cells to hyperpolarize from a basolateral membrane potential, Vb, of -73 + 3 (22) to -93 +1 mV (26), and superfusion with C02-free Trisbuffered Ringer solution induced a further hyperpolarization (Vb =-101 +1 mV (26)) which could be blocked by Ba2+. The C02-sensitive current changes were null at Vb = EK (potassium reversal potential, -106 + 2 mV (55)) implying that they are carried by K+ channels in the basolateral membrane. Such a response cannot account for the inhibition of the outward ISc which by default seems to be located to mitochondria-rich (MR) cells.5. In the absence of mucosal Cl-a pH gradient was built up above MR cells with pH = 7-02 + 0 04 (42) and pH increasing to 7X37 + 0X02 (10) above principal cells (pH = 7
Pancreatic ducts secrete HCO3(-), but transport mechanisms are unresolved and possibly vary between species. Our aim was to study the intracellular pH (pHi) regulation and thus H+/HCO3- transport in rat pancreatic ducts. Of particular interest was the Na+/HCO3(-) cotransporter, thought to be important in HCO3(-) -transporting epithelia. pHi was measured with BCECF in freshly isolated intralobular ducts. A reduction in extracellular Na+ concentration or application of HOE 694 (1 microM) decreased pHi by 0.1 to 0.6 pH units, demonstrating Na+/H+ exchanger activity. A reduction in extracellular Cl- concentration or addition of H2DIDS (10 microM) increased pHi by 0.1 to 0.5 pH units, demonstrating Cl-/ HCO(3)- (OH ) exchanger activity. In experimental acidosis, extracellular HCO3(-)/CO2 buffer did not increase the rate of pHi recovery, indicating that provision of HCO3(-) by the Na+/HCO3(-) cotransporter was not apparent. Most importantly, Na+/HCO3(-) cotransport was not stimulated by secretin (1 nM). In contrast, in experimental alkalosis the pHi recovery was increased in HCO3(-)/CO2 buffer, possibly due to Na+/HCO3(-) cotransport in the efflux mode. Secretin (1 nM) and carbachol (1 microM) stimulated HCO3(-) efflux, which can account for the observed HCO3(-) concentrations in rat pancreatic juice. Acetate and HCO3(-) buffers were handled similarly, indicating similar transport mechanisms in pancreatic ducts.
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