The tonoplast of isolated vacuoles from photoautotrophic suspension cells of Chenopodium rubrum L. was studied by means of the patch-clamp technique. In a symmetrical K+ concentration of 46 mM, similar to in vivo conditions, the tonoplast displayed a membrane potential near zero and a linear current-voltage relationship with a mean slope of 1.0 S/M2. ATP at 2 mM hyperpolarized the tonoplast (vacuole positive) by 15-20 mV and, in a parallel experiment, acidified the vacuole (outside pH 7.0) to pH 5.0, as monitored by accumulation of acridine orange. Analysis of the voltageclamp current indicates a 2-fold, ATP-dependent increase of the membrane capacitance, from 4 to 8 mF/m2, and an ATP-independent, unidentified ion channel having a mean opening time of about 5 msec and a conductivity of 0.5-1.0 pS. Considerable attention currently is devoted to the energetics of substrate transport across the tonoplast, the membrane separating cytoplasm and vacuole in plant cells (1). A membrane ATPase is thought to pump protons into the vacuole and thus to energize the tonoplast by a pH gradient and possibly by a membrane potential. By applying the patch-clamp technique to isolated vacuoles, we have studied the electrical properties of the tonoplast and found that the latter features an ATP-dependent electrogenic pump that creates a small membrane potential but a substantial pH gradient, revealed by parallel experiments with the pH probe acridine orange. MATERIALS AND METHODSPhotoautotrophic and phytohormone-independent suspension cells derived from hypocotyl cells of Chenopodium rubrum L. (2) were cultured as described (3). For protoplast isolation, cells from the exponential growth phase were used (for the growth pattern of the culture, see ref. 4). Cells from 6-day-old cultures were harvested from the suspension by filtration through a 15-,um-pore nylon net (Thoma, Mossingen, F.R.G.). Fifteen grams of cells (fresh weight) was suspended in 50 ml of medium I [20 mM 2-(N-morpholino)ethanesulfonic acid (Mes)/KOH, pH 5.3/0.3 M mannitol/2 mM CaCl2/10 mM KCl/1 mM DL-dithiothreitol/5 mM MgCl2/0.5% bovine serum albumin]. Fifty milliliters of medium I containing 2.5 g of cellulase TC from Trichoderma reesi (Serva, Heidelberg) and 2.5 g of pectinase 5S from Aspergillus niger (Serva) was added to the cell suspension and incubated on a gyratory shaker (120 rpm); after 90 min, there was no more Calcofluor white-staining (5) detectable at the cell surface (Calcofluor white ST solution was a gift from U. Seitz, Tubingen, F.R.G.). Protoplasts were harvested by centrifugation (100 x g, 15 min) and washed twice with medium II (20 mM Mes/KOH, pH 6/0.3 M mannitol/25 mM KCl/1 mM DL-dithiothreitol/0.5 mM MgCl2/0.1% bovine serum albumin); yield of protoplasts was 81%. For further purification, the protoplasts in medium II were loaded on a step gradient of 10, 7.5, 5, and 2.5% (wt/wt) Ficoll 400 in medium II and centrifuged (100 x g, 15 min). Spherical protoplasts (60o of the crude protoplasts) banding at the 7.5/5% (wt/wt) Ficoll 400 interfa...

Aldosterone stimulates Na+-absorption in rabbit distal colon. Due to circadian variations in plasma aldosterone level, Na+-transport varies in this epithelium. In vitro measurements (Ussing-chambers) yielded a trans-epithelial voltage (Vt) of 13 +/- 1.6 mV for low-transporting epithelia (LT) and 25.7 +/- 2 mV for high-transporting epithelia (HT). However, the comparison of transepithelial conductance (Gt) in LT epithelia (2.73 +/- 0.21 mS/cm2) and HT epithelia (2.96 +/- 0.41 mS/cm2) revealed no difference. Colons from both groups were stimulated by exogenous aldosterone (4 h prior to experiment). The transepithelial values changed as follows: LT epithelia showed a significantly increased Vt (26.1 +/- 4 mV) and Gt (3.74 +/- 0.23 mS/cm2), whereas in HT epithelia both parameters remained unchanged. Transepithelial amiloride-sensitive conductance was higher in HT than in LT. However, only in LT epithelia aldosterone increased this conductance. To get a more detailed view of the action of aldosterone, we used intracellular microelectrodes to calculate the resistances of apical (Ra), basolateral (Rbl) and paracellular (Rp) pathway. The calculation of the resistances was based on a lumped equivalent circuit model and changes in Ra were induced by 50 microM/l mucosal amiloride. Comparison of the control tissues revealed a lower Rbl only in HT tissues. In both groups stimulation by exogenous aldosterone led to a marked decrease of Ra. Furthermore Rbl was reduced to the same value as in HT control tissues. A leak resistance (Rl) was found, which was modulated by aldosterone in LT- and in HT epithelia. Differences in amiloride-sensitive transepithelial conductance between both epithelia groups could be explained by a regulation of Rl.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of the paracellular pathway in rabbit distal colon by the hormone aldosterone was investigated in vitro in Ussing chambers by means of transepithelial and microelectrode techniques. To evaluate the cellular and paracellular resistances an equivalent circuit analysis was used. For the analysis the apical membrane resistance was altered using the antibiotic nystatin. Under control conditions two groups of epithelia were found, each clearly dependent on the light: dark regime. Low-transporting epithelia (LT) were observed in the morning and high-transporting epithelia (HT) in the afternoon. Na+ transport was about 3-fold higher in HT than in LT epithelia. Incubating epithelia of both groups with 0.1 mumol.l-1 aldosterone on the serosal side nearly doubled in LT epithelia the short circuit current and transepithelial voltage but the transepithelial resistance was not influenced. Maximal values were reached after 4-5 h of aldosterone treatment. In HT epithelia due to the effect of aldosterone all three transepithelial parameters remained constant over time. Evaluation of the paracellular resistance revealed a significant increase after aldosterone stimulation in both epithelial groups. This increase suggests that tight junctions might have been regulated by aldosterone. The hormonal effect on electrolyte transport was also dependent on the physiological state of the rabbit colon. Since net Na+ absorption in distal colon is, in addition to transcellular absorption capacity, also dependent on the permeability of the paracellular pathway, the regulation of tight junctions by aldosterone may be a potent mechanism for improving Na+ absorption during hormone-stimulated ion transport.

We investigated the basic bioelectrical properties and the transport of Na+, Rb+, and Cl- in the isolated cecal epithelium of the rabbit. Intracellular microelectrode recordings provided first estimates of the individual membrane potentials and resistances. The cecal epithelium was found to be a moderately tight epithelium with a high transepithelial potential and a relative high paracellular resistance. It has, however, a low transepithelial resistance caused by a high rate of transcellular ion transport and a high transcellular conductance. Under short-circuit conditions Na+ and Cl- are absorbed, whereas Rb+ is secreted. Na+ absorption seems to be mediated via an electrogenic mechanism with low amiloride sensitivity and is partially linked to Cl- transport. Cl- absorption is not influenced by secretagogues, and Rb+ secretion occurs via a transcellular route. A basolateral K+ conductance was also found. The results show rabbit cecum as an epithelium with a high electrolyte transport capacity, distinctly different from the neighboring proximal colon, and suited to absorb the large quantities of electrolytes that are present in the luminal liquid.

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