Patch clamp experiments were performed on two human osteosarcoma cell lines (MG-63 and SaOS-2 cells) that show an osteoblasticlike phenotype to identify and characterize the specific K channels present in these cells. In case of MG-63 cells, in the cell-attached patch configuration (CAP) no channel activity was observed in 2 mM Ca Ringer (control condition) at resting potential. In contrast, a maxi-K channel was observed in previously silent CAP upon addition of 50 nM parathyroid hormone (PTH), 5 nM prostaglandin E2 (PGE2) or 0.1 mM dibutyryl cAMP + 1 microM forskolin to the bath solution. However, maxi-K channels were present in excised patches from both stimulated and nonstimulated cells in 50% of total patches tested. A similar K channel was also observed in SaOS-2 cells. Characterization of this maxi-K channel showed that in symmetrical solutions (140 mM K) the channel has a conductance of 246 +/- 4.5 pS (n = 7 patches) and, when Na was added to the bath solution, the permeability ratio (PK/PNa) was 10 and 11 for MG-63 and SaOS-2 cells respectively. In excised patches from MG-63 cells, the channel open probability (Po) is both voltage- (channel opening with depolarization) and Ca-dependent; the presence of Ca shifts the Po vs. voltage curve toward negative membrane potential. Direct modulation of this maxi-K channel via protein kinase A (PKA) is very unlikely since in excised patches the activity of this channel is not sensitive to the addition of 1 mM ATP + 20 U/ml catalytic subunit of PKA. We next evaluated the possibility that PGE2 or PTH stimulated the channel through a rise in intracellular calcium. First, calcium uptake (45Ca2+) by MG-63 cells was stimulated in the presence of PTH and PGE2 an effect inhibited by Nitrendipine (10 microM). Second, whereas PGE2 stimulated the calcium-activated maxi-K channel in 2 mM Ca Ringer in 60% of patches studied, in Ca-free Ringer bath solution, PGE2 did not open any channels (n = 10 patches) nor did cAMP + forskolin (n = 3 patches), although K channels were present under the patch upon excision. In addition, in the presence of 2 mM Ca Ringer and 10 microM Nitrendipine in CAP configuration, PGE2 (n = 5 patches) and cAMP + forskolin (n = 2 patches) failed to open K channels present under the patch. As channel activation by phosphorylation with the catalytic subunit of PKA was not observed, and Nitrendipine addition to the bath or the absence of calcium prevented the opening of this channel, it is concluded that activation of this channel by PTH, PGE2 or dibutyryl cAMP + forskolin is due to an increase in intracellular calcium concentration via Ca influx.

1. Macula densa (MD) cells are located within the thick ascending limb (TAL) and have their apical surface in contact with tubular fluid and their basilar region in contact with the glomerulus. These cells sense changes in luminal fluid sodium chloride concentration ([NaCl]) and transmit signals resulting in changes in vascular resistance (tubuloglomerular feedback) and renin release. 2. Current efforts have focused on understanding the cellular transport mechanisms of MD cells. Progress in this area has benefited from the use of the isolated perfused TAL-glomerular preparation, which permits direct access to MD cells. 3. Using microelectrodes to measure basolateral membrane potential (VBL) of MD cells, it was found that VBL was very sensitive to changes in luminal fluid [NaCl]. As [NaCl] was elevated from 20 to 150 mmol/L, VBL was found to depolarize by over 30 mV. 4. Basolateral membrane potential measurements were also used to identify an apical Na+:2Cl-:K+ cotransport pathway in MD cells that is the major pathway for NaCl entry into these cells. 5. Other work identified a basolateral chloride channel that is presumed to be responsible for changes in VBL during alterations in luminal [NaCl]. This channel, which is the predominant conductance across the basolateral membrane, may be regulated by intracellular Ca2+ and cAMP. 6. An apical Na+:H+ exchanger in MD cells was detected by measuring changes in intracellular pH using the fluorescent probe 2',7'-bis-(2-carboxyethyl)-5(and-6) carboxyfluorescein. 7. Using patch-clamp techniques, a high density of pH- and Ca(2+)-sensitive K+ channels was observed at the apical membrane of MD cells. 8. Other studies found that, at the normal physiological conditions prevailing at the end of the TAL (luminal [NaCl] of 20-60 mmol/L), reabsorption mediated by MD cells is very sensitive to changes in luminal [NaCl].