Responses of renal inner medullary collecting duct cells (IMCD) to osmotic stress.

Maurice B. Burg

NHLBI, Bethesda, MD 20897-1603, maurice_burg@nih.gov

Accompanying urinary concentration and dilution, IMCD cells are exposed to concentrations of NaCl and urea that are variable and often very high (total $1700 mosm), yet the cells survive and function. Accumulation of compatible and counteracting organic osmolytes and activation of heat proteins were previously identified as important factors. We have been further investigating the mechanisms involved, using cultures of mouse IMCD cells that are either early passage or are immortalized with SV40 (mIMCD3). With mIMCD3 cells, a step increase in osmolality from 300 to a total of 500-600 mosm stops proliferation for ~18 h, followed by renewed growth at the original rate. Increase to ~700 mosm kills most cells by apoptosis. Elevating to 500-600 mosm with NaCl (but not with urea) causes DNA double strand breaks and activates p53, a tumor suppressor protein, causing the cell cycle delay. Suppression of p53 abrogates the cell cycle delay and cells that proceed to replicate damaged DNA become apoptotic. At ~700 mosm (but not at 500-600) there is rapid decrease in mitochondrial PD and exit of NADPH from the mitochondria followed by cell death. Early passage IMCD cells survive linear (over 18 hours, like in vivo), but not step, increases to ~1700 mosm. The difference presumably is that a step increase rapidly increases intracellular ionic strength, which damages DNA, whereas linear increase gives time for organic osmolytes to accumulate, minimizing intracellular ionic strength and DNA damage.

Volume regulation during dehydration of East-African desert beetles

Karl Erik Zachariassen

Department of Zoology, Norwegian University of Science and Technology (NTNU), 7491 Trondheim, Norway

In arid areas in East Africa dietary water is not available during the dry seasons. Since the dry seasons may last for many months, sometimes for more than a year, insects living in these areas may become severely dehydrated and in extreme cases lose more than 80% of their body water.
The water loss takes place mainly at the expense of the extracellular fluid, i.e. the haemolymph volume drops to zero while the cell volume is only moderately reduced.
The protection of cell volume at the expense of the haemolymph requires that the haemolymph solutes are transferred to cellular compartments, sequestered within the body in an osmotically inactive state or excreted from the body.
In the predatory carabid beetles Na is excreted, but in these beetles Na can easily be replaced from the diet. In the tenebrionids, which feed on low Na detritus, Na from the haemolymph is deposited in fat body or gut tissue. Free amino acids are moved from the haemolymph to the cells, but a substantial amount seems also to be polymerized into proteins. As the beetles become rehydrated, the amino acids are rapidly depolymerized and added to the body fluids.
Another factor that might contribute to stabilize cellular volume is a rapid increase in the osmotic activity of intracellular proteins as the cellular water content is reduced.

Protective properties of organic osmolytes in deep-sea fish and vestimentiferans

Paul H. Yancey, Wendy Blake, James Conley

Biology Department, Whitman College, Walla Walla, WA 99362 USA, yancey@whitman.edu

Hydrostatic pressure (1 atm per 10m depth) perturbs protein stability and function; many proteins in deep-sea organisms have evolved resistance to this, but many still retain sensitivity. Recently our laboratory has found unusual osmolyte levels in deep-sea animals. Trimethylamine N-oxide (TMAO), usually under 100 mmol/kg wet wt. in most shallow animals, increases with depth in muscles of teleost fishes and decapod crustaceans, reaching 300 mmol/kg at 3000m. The giant vestimentiferan Riftia (from 2500m, near hydrothermal vents) contains the unusual osmolyte hypotaurine at 125 mmol/kg. The animal experiences both high pressure and rapid temperature changes from 5 to 25°C, which could perturb proteins. We tested our hypothesis that these osmolytes counteract pressure and/or temperature effects. With proteins from abyssal grenadier fish at 300-500 atm, 250 mM TMAO fully offset pressure inhibition of stability and cofactor binding of lactate dehydrogenase and of actin polymerization actin. Glycine, a common shallow-water osmolyte, was not protective. To test the universality of TMAO protection, yeast were cultured, pressurized and plated. For 1 hr at 700 atm, 3.5 hr at 700 atm, and 17 hr at 300 atm, 150 mM TMAO generally doubled the number of surviving cells. For Riftia malate dehydrogenase incubated at 300 atm for 24 hr, 125 mM hypotaurine only partly offset a 32% loss of activity at 5, but fully offset a 35% loss at 25°C, thus showing greater protection at the higher temperature.
Funded by the National Science Foundation and M.J. Murdock Charitable Trust.

Calcium homeostasis in crustaceans: evolutionary considerations

Michele Wheatly and Flavia P. Zanotto

Wright State University and University of Sao Paulo

Calcium homeostasis in crustaceans is influenced by their natural molting cycle that periodically requires replacement of the calcified exoskeleton in order for growth to occur. Whole body Ca balance transitions from intermolt (zero net flux) to premolt (net efflux) and postmolt (net influx at the rate of 2 mmol/kg/h). As such, molting provides a convenient model to study up- and down regulation of epithelial Ca transporting proteins (such as Ca pumps and exchangers), the genes that encode them, and the steroid hormone (ecdysone) that putatively regulates the genes. Species residing in either fresh water or in terrestrial environments are more limited in their Ca availability than are marine species. Further the advance towards terrestriality is accompanied by decreased reliance upon branchial Ca uptake and increased reliance upon digestive uptake. This plenary lecture will correlate Ca handling strategies with environment in a range of species through examining extracorporeal, extracellular and intracellular Ca compartments. Methodological approaches will include systems physiology, epithelial transport (gills, antennal gland, hepatopancreas and hypodermis), subcellular homeostasis and molecular regulation. Flow cytometry and polyclonal antibodies will be introduced as novel techniques to assist with the elusive stages of pre- and postmolt.
Supported by NSF grant IBN 9870374 to MGW and FAPESP grant 98/09756-9 and a CNPq-NSF international cooperation grant to FPZ.