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John (Rick) Ash

Professor of Neurobiology and Anatomy

B.S. University of Illinois, Urbana

Ph.D. Stanford University

Research

References

ash@neuro.utah.edu

 

Research

Solute transporters play critical and complex roles in the physiology of all organisms. We are studying two types of transporters: amino acid carriers and ion pumps. We entered the amino acid transport field by uncovering several novel regula­tions of glutamate transport in human and rodent cell lines. Glutamate is the major excitatory neuro­transmitter in the mammalian brain and under­standing the mechanisms and regulation of its transport is important for neurobiologists. We have also uncovered a link between glutamate uptake and the synthesis of glutathione, a key cellular anti-oxidant that plays a role in cancer biology.

We used tritium suicide selection to isolate Chinese hamster ovary cells with mutations in a sodium-dependent glutamate and aspartate uptake system. Unexpectedly, these mutants were also deficient in cystine uptake and glutathione synthesis. Analysis of these cells revealed that linkages among three transport systems are critical for anti-oxidant synthesis. A second class of mutants has been selected that suffers abnormal regulation of several different trans­port systems. These regula­tory muta­tions are being analyzed by a variety of approaches to estimate the number of genes whose transcription is regulated, and to identify some of the genes involved.

The second project focuses on the Na,K-ATPase, or sodium pump. This enzyme produces electrochemical gradients that energize several key processes, including amino acid transport. Some well-known inhibitors of the sodium pump, such as ouabain and digitalis, are used medically to increase blood pressure and recently it has been discovered that our adrenal glands synthesize a ouabain-like compound. This suggests that we may regulate pump activity via endogenous hormonal regulation, perhaps to modulate blood pressure or cardiac output. Understanding this potential regulatory system in higher animals is proving difficult.

The sodium pump has been well studied in vertebrates, but has received less attention in lower animals. We decided to investigate the sodium pump in a very primitive organism, the planarian. We have discovered that a fresh water planarian maintains at least two pump isoforms that differ in ouabain affinity (see figure). Future work will probe the nature and tissue distribution of pump isoforms, the role of endogenous inhibitors in regulating pump activity, and the possible role of the pump as a signal initiator in a Src signaling pathway. Planaria offer an early evolutionary example of sodium pump genes and proteins as well as providing a simple model system for understanding the physiological roles this enzyme plays in specific cell types and for an entire organism.

 

Ouabain binding to membranes purified from whole planaria can be fit to a model combining two saturable systems. The Kms of the systems, ca. 100 nM and 2 µM, are 20-fold different, suggesting that they could be independently regulated by an endogenously produced ouabain-like compound.

References