INTRACELLULAR RESPONSES OF PRODUCTIVE HYBRIDOMAS SUBJECTED TO HIGH OSMOTIC-PRESSURE

It has previously been found that hybridoma cells under hyperosmotic s tress produce higher amounts of antibody. This study identified the ce llular processes and mechanisms that occur during this event. In studi es of hybridomas adapted to osmolarities ranging between 300 and 450 m Osm (using NaCl), antibody production increased to a saturation level while cell growth decreased progressively. At 500 mOsm, lower cell num bers and markedly decreased productivity resulted. Sucrose and KCI wer e found to induce similar trends, except to different extents. Several important changes in cellular responses were observed. Elevation of o smolarity with NaCl from 300 to 350 mOsm causes an increase of zwitter ionic amino acid uptake, which occurred via Na+-dependent transport sy stems. In particular, system A was enhanced by 1.86-fold, but no enhan cement was observed for Na+- independent transport systems. In additio n, amino acids reactive with Na+-dependent transport systems were obse rved to be abundant within osmotically stressed hybridomas in the midd le and late exponential stages. Sucrose and KCl caused similar uptake effects, but to a lesser degree, as long as sodium ions were present i n solution. Specific consumption rates of glucose and glutamine increa se by 19% and 20%, respectively, under high osmolarity treatment. Thes e increases were confirmed by the 5% to 10% increase in cellular metab olic activity. At 350 mOsm, growth rate was slower compared with the 3 00-mOsm culture, which was reflected by the lower DNA concentration. S tressed cultures contained enhanced levels of total RNA, of which simi lar to 80% is ribosomal RNA (rRNA). Higher rRNA content could in turn increase the translation rates of proteins. This was reflected in the accumulation of both dry cell weight and total cellular protein at lin ear rates of 0.42 mu g/10(6) cells/mOsm and 0.21 mu g/10(6) cells/mOsm , respectively, with increasing osmolarity between 300 and 450 mOsm. O verall, hybridomas increased their metabolic activities and am ino aci ds uptake via the Na+-dependent symports to compensate for the osmotic ally elevated external environment. These effects contribute directly and indirectly to the increased cell mass consisting of a larger pool of amino acids, RNA, cellular proteins, and secreted antibody product. (C) 1995 John Wiley & Sons, Inc.