LONG-TERM SHAKE SUSPENSION AND MEMBRANE-VESICLES OF MEDULLARY THICK ASCENDING LIMB - TECHNICAL NOTE

Cultured medullary thick ascending limb (MTAL) cells may lack some of the main carriers of fresh MTAL cells, such as apical Na+-K+(NH4+)-2Cl (-) cotransporter (BSC-1) and Na+/H+ exchanger (NHE-3). We have develo ped a technique to maintain rat MTALs several hours in suspension and in a good state of viability. Medullary thick ascending limbs were sus pended in a 1:1 mixture of Ham's nutrient mixture F-12 and Dulbecco's modified Eagle's essential medium (HDMEM) supplemented with 25 mM HCO3 - and gassed with 95% O-2/5% CO2; the resulting mixture was placed in a rotary shaking water bath at 37 degrees C for 16 hours. As seen by e lectron microscopy, MTALs from the HDMEM-suspension retained a virtual ly normal tubular organization. Na+-K+(NH4+)-2Cl(-) cotransport activi ty and NHE consistent with both apical NHE-3 and basolateral NHE-1 act ivities were underscored both in intact cells by intracellular pH meas urements and in a membrane fraction enriched in apical and basolateral membranes by Na-22(+) uptake experiments. These results demonstrate t hat freshly harvested MTALs can be maintained in a well differentiated state for at least 16 hours; this preparation should make long-term i n vitro studies of MTAL transport regulations possible.