Pulmonary surfactant, secreted via exocytosis of lamellar bodies (LB) by alveolar type II (AT II) cells, maintains low alveolar surface tension and is therefore essential for normal lung function. Here we describe real-time monitoring of exocytotic activity in these cells by visualizing and quantifying LB fusion with the plasma membrane (PM). Two approaches were used. First, f luorescence of LysoTracker Green DND-26 (LTG) in LB disappeared when the dye was released after exocytosis. Second, phospholipid staining by FM 1-43 resulted in bright f luorescence when this dye entered the LB through the fusion pore. Both processes were restricted to and colocalized with LB and occurred simultaneously. In AT II cells, FM 1-43 offered the unique advantage to independently define the moment and cellular location of single exocytotic events as well as the amount of material released, and to monitor its extracellular fate. Furthermore, both dyes could be used in combination with fura-2. The results indicate considerable diversity in the dynamics of LB exocytosis. In the majority of cells stimulated with ATP and isoproterenol, the first fusion of LB coincided with the rise of [Ca 2؉ ] i , but subsequent response of other LB in the same cell considerably outlasted this signal. In other cells, however, the onset of exocytosis was delayed by several minutes. After LB fusion, release of surfactant from LB into an aqueous solution was slow. In summary, stimulated exocytosis in AT II cells occurs at a much slower rate than in most other secretory cells but is still a more dynamic process than predicted from conventional measurements of surfactant released into cell supernatants.

Ion channels in Madin-Darby canine kidney cells serve transepithelial chloride transport and probably cell volume regulation. Three distinct potassium channels and one anion channel have been revealed by patch clamp studies in Madin-Darby canine kidney cells. The potassium channels are activated by an increase in intracellular calcium activity. A number of hormones activate the potassium channels by an increase in intracellular calcium activity. However, under certain conditions the hormones hyperpolarize the cell membrane without increasing intracellular calcium activity sufficiently to activate the calcium-sensitive potassium channels. Thus, the hormones may activate potassium channels via another, as yet undefined, intracellular mechanism. The anion channel is stimulated by cAMP. Another factor modifying channel activity is cell volume: cell swelling leads probably to subsequent activation of potassium and anion channels. The net result is a variable transient hyperpolarization followed by a sustained depolarization of the cell membrane.

Extracellular ATP has been shown to stimulate transepithelial chloride transport in confluent Madin-Darby canine kidney (MDCK) cell layers and to enhance potassium conductance in subconfluent MDCK cells. The present study has been performed to test for the effect of extracellular ATP on channel activity in patches from subconfluent MDCK cells. Within 8 s, addition of extracellular ATP (10 mumol/l) leads to a sustained, but fully reversible, appearance of potassium-selective channels in cell-attached patches [increase of open probability from 0.03 +/- 0.02 (n = 10) to 0.50 +/- 0.07 (n = 6)]. With the use of pipettes filled with 145 mmol/l KCl, inwardly rectifying property of the channels is disclosed with a single-channel conductance of 65.7 +/- 3.1 pS (n = 9) at zero potential difference between pipette and bath and with a reversal potential of 75.4 +/- 2.0 mV (n = 5; pipette negative vs. reference in the bath). The open probability of the channels is not significantly modified by altering pipette potential from -50 mV, pipette positive, to 50 mV, pipette negative. At extracellular calcium activities of less than 10 nmol/l, ATP leads to a transient activation of channels. In conclusion, extracellular ATP activates inwardly rectifying potassium channels in the cell membrane of subconfluent MDCK cells. A sustained activation of the channels requires the presence of extracellular calcium and is probably mediated by increases in intracellular calcium.

The present study has been performed to test for the effect of intracellular calcium and of serotonin on the channel activity in patches from subconfluent MDCK-cells. In inside-out patches, inwardly rectifying potassium-selective channels are observed with open probabilities of 0.01 +/- 0.01, 0.24 +/- 0.03 and 0.39 +/- 0.07, at 100 nmol/liter, 1 mumol/liter or 10 mumol/liter calcium activity, respectively. The single-channel slope conductance is 34 +/- 2 pS, if the potential difference across the patch (Vp) is zero, and approaches 59 +/- 1 pS, if Vp is -50 mV, cell negative. In the cell-attached mode, little channel activity is observed prior to application of serotonin (open probability = 0.03 +/- 0.03). If 1 mumol/liter serotonin is added to the bath perfusate, the open probability increases rapidly to a peak value of 0.34 +/- 0.04 within 8 sec. In continued presence of the hormone, the open probability declines to approach 0.06 +/- 0.02 within 30 sec. At zero potential difference between pipette and reference in the bath (i.e., the potential difference across the patch is equal to the potential difference across the cell membrane), the single-channel conductance is 59 +/- 4 pS. In conclusion, inwardly rectifying potassium channels have been identified in the cell membrane of subconfluent MDCK-cells, which are activated to a similar extent by increase of intracellular calcium activity to 1 mumol/liter and by extracellular application of 1 mumol/liter serotonin.

The present study has been performed to test for the effect of hypotonic extracellular fluid on the electrical properties of Madin Darby canine kidney (MDCK)-cells. The volume of suspended MDCK-cells is 1,892 +/- 16 fl (n = 8) in isotonic (298.7 mosmol/l) extracellular fluid. Exposure of the cells to hypotonic (230.7 mosmol/l) extracellular fluid is followed by cellular swelling to 2,269 +/- 18 fl (n = 4) and subsequent volume regulatory decrease to 2,052 +/- 22 fl (n = 4) within 512 s. Volume regulatory decrease is abolished by quinidine (1 mmol/l) and by lipoxygenase inhibitor nordihydroguaiaretic acid (50 mumol/l). The potential difference across the cell membrane averages -53.6 +/- 0.9 mV (n = 49) in isotonic extracellular perfusates. Reduction of extracellular osmolarity depolarizes the cell membrane by +25.7 +/- 0.8 mV (n = 67), reduces the apparent potassium selectivity of the cell membrane, from 0.55 +/- 0.07 (n = 9) to 0.09 +/- 0.01 (n = 26), and increases the apparent chloride selectivity from close to zero to 0.34 +/- 0.02 (n = 21). Potassium channel blocker barium (1 mmol/l) depolarizes the cell membrane by +15.2 +/- 1.1 mV (n = 13). In the presence of barium, reduction of extracellular osmolarity leads to a further depolarization by +14.0 +/- 1.4 mV (n = 12). Addition of chloride channel blocker anthracene-9-COOH (1 mmol/l) leads to a hyperpolarization of the cell membrane by -6.7 +/- 2.2 mV (n = 11). In the presence of anthracene-9-COOH, reduction of the extracellular osmolarity leads to a depolarization by +22.4 +/- 1.7 mV (n = 11).(ABSTRACT TRUNCATED AT 250 WORDS)