Assay system for anti-stress agents

Details Assay system for anti-stress agents Assay system for anti-stress agents

1996-01-19

2001-05-15

Venkat, Jyothsna (Department: 1627)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

C435S025000, C435S026000, C435S190000, C436S501000, C436S164000

Reexamination Certificate

active

06232060

ABSTRACT:

BACKGROUND
Progress in the discovery and development of therapeutic agents for the treatment of hypoxic stress is limited by: 1) the absence of model systems in which to conduct studies into the cellular mechanism of injury; and 2) the inability to effectively screen putative lead compounds as potential therapeutic agents. Physiologic model systems are limited primarily by the poor mechanistic resolution, inconvenience, and limited ability to screen large numbers of compounds to develop lead molecules. Primary or transformed cell (culture) models are limited by alterations in cell energy metabolism secondary to transformation processes (reversion to alternative fuel substrates/pathways, such as glycolysis being substituted by oxidative phosphorylation). Also, there is a generalized lack of fidelity when compared to their primary derived tissue.
The preponderance of published data investigating the effects of hypoxia have been performed on whole animals, isolated organs, or primary cells. Tissues that succumb to the effects of hypoxia are characterized by high rates of oxidative metabolism and high cellular energy demand. Cells adapted to culture conditions are typically not ideal models for the study of the effects of hypoxia. They routinely possess fewer mitochondria and derive energy needs primarily by glycolysis, i.e., anaerobic metabolism. Hence, these cell types are resistant to the effects of hypoxia.
Previous studies employing primary renal cells rabbit proximal tubules), a highly oxidative tissue, demonstrated that both alanine and glycine effect considerable cytoprotection to the effects of tissue anoxia (Garza-Quintero et al. (1990)
Am. J. Phys.
258; Renal
Elect. Phys.
27:F1075-F1083). The cytoprotective effect of fructose 1,6-bisphosphate to the effects of hypoxia has also been described (Markov et al., (1980)
Am. Heart. J.
100:639-646).
SUMMARY OF THE INVENTION
Methods and compositions are provided for screening cytoprotectant compositions for use in alleviating stress, such as hypoxia. A rat hepatoma cell line, WIF-B, is found to respond to mitochondrial inhibitors by releasing extracellularly lactate hydrogenase (LDH), as well as lactate, within a convenient period of time. Known cytoprotectants are found to reduce the rate of LDH release. The WIF-B cells are cultured in accordance with a convenient schedule, transferred to the assay medium and the mitochondrial inhibitor added. A cytoprotectant candidate may be added at any convenient time thereafter and the rate of LDH release into the medium is monitored. Reduction in LDH release is indicative of cytoprotection.


REFERENCES:
patent: 5198336 (1993-03-01), Knobeloch et al.
Marsh et al., Hepatology., 17(1), pp. 91-98, 1993.*
Jackson M J et al., Clinical Science, 80(6), pp. 559-564, Jun. 1991.*
Ihrke et al., “WIF-B Cells: An In Vitro Model for Studies of Hepatocyte Polarity,” The J. of Cell Biology (1993), 123:1762-1775.
Shanks et al., “An Improved Polarized Rat Hepatoma Hybrid Cell Line,” J. of Cell Science (1994), 107:813-825.

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