In the present study we used the Na)+)-sensitive fluorescent dye SBFI and optical measurement of endpiece volume to investigate the transport of Na+ in sheep parotid secretory cells. Sheep parotid endpiece cells bathed in a HCO3(-)-free Cl(-)-rich solution had a resting intracellular Na+ concentration ([Na+]i) of 17 +/- 2 mmol/l (n = 39). Exposure of the cells to a 2-min pulse of acetylcholine (ACh) (3 x 10(-7) mol/l) in a HCO3(-)-free bathing solution produced no change in [Na+]i or in cell volume. Changing from a Cl(-)-containing HCO(3-)-free bath solution to a Cl- solution containing 25 mmol/l HCO3- caused the endpieces to swell by 8 +/- 2% (n = 11) and the [Na+]i to increase by 10 +/- 2 mmol/l (n = 14). Subsequent exposure of the cells to ACh led to shrinkage of the cells by 12 +/- 2% from the volume in the HCO3(-)-containing solution prior to ACh exposure, with the maximum decrease occurring after 29 +/- 7 s (n = 9). This shrinkage was accompanied by a rapid and transient increase in [Na+]i, the [Na+]i reaching a peak at 70 +/- 5 mmol/l above the unstimulated level (n = 9). Substitution of gluconate for Cl- did not significantly alter the effects of HCO3- on unstimulated [Na+]i or endpiece volume, nor did it significantly inhibit the effects of ACh on these two parameters when HCO3- was present.(ABSTRACT TRUNCATED AT 250 WORDS)

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The relevant influx pathway for stimulated Ca2+ entry into epithelial cells is largely unknown. Using flufenamate (Flu) and Gd3+, both known pharmacological blockers of non-selective cation currents in other epithelial preparations, we tested whether the stimulated Ca2+ entry in CFPAC-1 cells was inhibited by these agents. Transmembraneous Ca2+ influx into CFPAC-1 cells was stimulated by either ATP (10(-4) and 10(-5) mol/l), carbachol (CCH, 10(-4) mol/l) or thapsigargin (TG, 10(-8) mol/l). Three different experimental approaches were used. (1) Because the plateau phase of an agonist-induced [Ca2+]i transient reflects Ca2+ influx into these cells, we investigated the influence of Flu and Gd3+ on the level of the stimulated [Ca2+]i plateau. (2) The fura-2 Mn(2+)-quenching technique was used to visualise divalent cation entry and monitor its inhibition. (3) During the "refilling period" after agonist-induced discharge of the intracellular pools the putative influx inhibitors Flu and Gd3+ were given and subsequently the filling state of the agonist-sensitive intracellular stores tested. The results from the first experimental approach showed that both Flu and Gd3+ were potent inhibitors of the stimulated Ca2+ entry in CFPAC-1 cells. Flu reversibly decreased the ATP-induced [Ca2+]i plateau in a concentration dependent manner, with an IC50 value of 33 mumol/l (n = 6). Similar results were obtained for the CCH- (n = 5) and the TG-induced (n = 5) [Ca2+]i plateau. Gd3+ concentration dependently inhibited the stimulated Ca2+ plateau. A complete block of the ATP-induced [Ca2+]i plateau was seen at 0.5 mumol/l (ATP 10(-5) mol/l, n = 8).(ABSTRACT TRUNCATED AT 250 WORDS)

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HT29 cells were preincubated with forskolin (10(-5) mol/l, FORHT) or phorbol 12-myristate 13-acetate (PMA) (10(-7) mol/l, PMAHT) for 20 h, which has been shown previously and is also shown here, to upregulate and downregulate, respectively, the expression of the cystic fibrosis transmembrane conductance regulator (CFTR). CFPAC-1 cells underwent the same protocols. HT29 cells were examined by slow (SWC) and fast (FWC) whole-cell patch-clamp techniques. The results of SWC and FWC were indistinguishable and were pooled. CFPAC-1 cells were examined with FWC. The membrane voltage (V) of FORHT was -41.8 +/- 1.4 mV (n = 77) and that of PMAHT was -43.6 +/- 2.4 mV (n = 76). The conductance (G) of FORHT (9.4 +/- 0.9 nS, n = 77) was significantly larger than that of PMAHT (3.7 +/- 0.4 nS, n = 76). Acute application of forskolin (10(-5) mol/l, FOR) plus 0.5 mmol/l 8-(4-chlorophenylthio)-cAMP (cAMP) depolarized V by 12 (FORHT) and 8 (PMAHT) mV, respectively. The acute increase of G by FOR plus cAMP was by 7.6 +/- 1.9 nS for FORHT (n = 22) and only 2.2 +/- 1 nS for PMAHT (n = 13). ATP (10(-4) mol/l) depolarized V in both types of cells. It enhanced G by 16.7 +/- 4.1 nS in FORHT (n = 14) and significantly less (by 5.5 +/- 1.2 nS, n = 14) in PMAHT. Also the G increase lasted longer in FORHT. Neurotensin (NT, 10(-8) mol/l) also had a stronger and longer lasting effect in FORHT.(ABSTRACT TRUNCATED AT 250 WORDS)