No abstract

The potential difference (PD)-dependent component of transcellular Mg(2+) uptake in sheep rumen epithelium was studied. Unidirectional (28)Mg(2+) fluxes were measured at various transepithelial PD values, and the unidirectional mucosal-to-serosal (28)Mg(2+) flux (J(Mg)(ms)) was correlated with the PD across the apical membrane (PD(a)) determined by mucosal impalement with microelectrodes. PD(a) was found to be -54 +/- 5 mV, and J(Mg)(ms) was 65.9 +/- 13.8 nmol. cm(-2). h(-1) under short-circuit conditions. Hyperpolarization of the ruminal epithelium (blood-side positive) depolarized PD(a) and, most noticeably, decreased J(Mg)(ms). Further experiments were performed with cultured ruminal epithelial cells (REC). With the aid of the fluorescence probe mag-fura 2, we measured the intracellular free Mg(2+) concentration ([Mg(2+)](i)) of isolated REC under basal conditions at various extracellular Mg(2+) concentrations ([Mg(2+)](e)) and after alterations of the transmembrane voltage. Basal [Mg(2+)](i) was 0.54 +/- 0.08 mM. REC suspended in media with [Mg(2+)](e) between 0.5 and 7.5 mM showed an increase in [Mg(2+)](i) that was dependent on [Mg(2+)](e) and that exhibited a saturable component (Michaelis-Menten constant = 1.2 mM; maximum [Mg(2+)](i) = 1.26 mM). Membrane depolarization with high extracellular K(+) (40, 80, or 140 mM K(+)) and the K(+) channel blocker quinidine (50 and 100 microM ) resulted in a decrease in [Mg(2+)](i). On the other hand, hyperpolarization created by K(+) diffusion (intracellular K(+) concentration > extracellular K(+) concentration) in the presence of valinomycin induced a 15% increase in [Mg(2+)](i). None of the manipulations had any effect on intracellular Ca(2+) concentration and intracellular pH. The results support the assumption that the membrane potential acts as a principal driving force for Mg(2+) entry in REC and suggest that the pathway for this electrodiffusive Mg(2+) uptake across the luminal membrane is a channel or a carrier.

SUMMARY Secretion in blowfly salivary glands is induced by the neurohormone serotonin and powered by a vacuolar-type H+-ATPase (V-ATPase)located in the apical membrane of the secretory cells. We have established a microfluorometric method for analysing pH changes at the luminal surface of the secretory epithelial cells by using the fluorescent dye 5-N-hexadecanoyl-aminofluorescein (HAF). After injection of HAF into the lumen of the tubular salivary gland, the fatty acyl chain of the dye molecule partitions into the outer leaflet of the plasma membrane and its pH-sensitive fluorescent moiety is exposed at the cell surface. Confocal imaging has confirmed that HAF distributes over the entire apical membrane of the secretory cells and remains restricted to this membrane domain. Ratiometric analysis of HAF fluorescence demonstrates that serotonin leads to a reversible dose-dependent acidification at the luminal surface. Inhibition by concanamycin A confirms that the serotonin-induced acidification at the luminal surface is due to H+ transport across the apical membrane via V-ATPase. Measurements with pH-sensitive microelectrodes corroborate a serotonin-induced luminal acidification and demonstrate that luminal pH decreases by about 0.4 pH units at saturating serotonin concentrations. We conclude that ratiometric measurements of HAF fluorescence provide an elegant method for monitoring V-ATPase-dependent H+transport in the blowfly salivary gland in vivo and for analysing the spatiotemporal pattern of pH changes at the luminal surface.

Cockroaches have acinar salivary glands. The acini consist of peripheral cells specialized for electrolyte and water transport and central cells contributing proteinaceous components to the saliva. Salivary duct cells probably modify the primary saliva. The acinar cells in Nauphoeta cinerea had been shown to be electrically coupled and dye-coupled. Since intercellular communication via gap junctions between acinar cells is difficult to reconcile with previous findings that dopamine and serotonin selectively stimulate the secretion of either protein-free or protein-rich saliva in Periplaneta americana, we investigated whether dye-coupling occurs between both acinar cell types and between duct cells. We iontophoretically loaded Lucifer yellow into impaled cells and tested for dye-coupling by confocal laser scanning microscopy. We found that: (1) peripheral and central cells within an acinar lobulus of the gland in P. americana are dye-coupled; and (2) salivary duct cells are dye-coupled.

The effects of clamping the transepithelial potential difference (PDt; mucosa reference) have been studied in sheep rumen epithelium. Pieces of ruminal epithelium were examined in Ussing chambers, in a part of the experiments combined with conventional intracellular recordings. After equilibration, the tissue conductance ( G t) was 2.50 ± 0.09 mS/cm2, the potential difference of the apical membrane (PDa) was −47 ± 2 mV, and the fractional resistance of the apical membrane (f R a) was 68 ± 2% under short-circuit conditions. Hyperpolarization of the tissue (bloodside positive) depolarized PDa, decreased f R a, and increased G tsignificantly. Clamping PDt at negative values caused converse effects on PDa and f R a. All changes were completely reversible. The determination of individual conductances revealed that the conductance of the apical membrane increased almost linearly with depolarization of PDa. The PD-dependent changes were significantly reduced by total replacement of Na. These observations support the assumption of a PD-dependent conductance in the apical membrane that permits enhanced apical uptake of Na even at depolarized PDa. This mechanism appears to be important for the regulation of osmotic pressure in forestomach fluid.