THE LESSON FROM SNAKE VENOM: ION TRANSPORT AND GLYCOLYSIS ARE COUPLED THROUGH THE PUMPING RATE OF THE NA/KATPASE

FERNANDES DE LIMA,V.M.1; HANKE,W.2; CAMILLO,M.A.3; SPENCER,P.3 AND NASCIMENTO,N. 3

1- ANVISA/Centro de Biologia Molecular  IPEN-CENEN/São Paulo; 2- Membrane Physiology Division, Institute of Zoophysiology, 230 Hohenheim University, Stuttgart, Germany; 3- Centro de Biologia Molecular, Instituto de Pesquisa Nuclear (IPEN) CENEN/ São Paulo, Brasil  

Electrochemical waves are very commom in biology and in excitable tissue (muscle and brain) they are the key to understand their physiology. The in vitro retina model provides a very usefull experimental tool to investigate the spread of exciation waves in central nervous system(CNS). One of the reasons is the marked intrinsic optical signal (IOS) associated with retinal waves. The optical profile of excitation waves in retinae, contain information about the state of membrane channels, transporters (pumps) and the rate of ATP synthesis through glycolsysis in the following way: the rise of the first peak (light scatter) is associated with eletrochemical gradients expenditure, its recovery with increased pumping rate in order to restore these gradients  with the consequent ATP breakdown. The rise of the second peak and its amplitude is associated with the increase in the metabolic glycolysis and lactic acid output toward the extracellular space by the glial cells. We have just found that two of the three main toxins of the rattlesnake venom (Crotalus durissus terrificus), gyroxin and crotamine have the Na/KATPase has a target and that by slowing down the ion transport, they modulate glycolysis. The effect in the metabolism is similar to lowering the temperature of the preparation from 30 to 20 degrees Celsius. This finding confirm the reports about the receptor role of the sodium pump and rises some interesting questions: these two protein have no homology either in primary or terciary structure, and yet, they share a comom target. Could each one have a different isoform of the pump as the main target? Could these toxins mimic a endogenous glycoside modulation? The exquisite sensisitivity of the tissue to ouabain, appears to indicate that this is true. Furthermore, the third main toxin, crotoxin, also modulates the retinal glial pump, but in different way: it brings “spontaneous” waves with a lacking second optical component, a situation similar to accelerating the pump to its maximun rate before the wave such that the frontwave cannot accelerate it further.  This effect on the optical profile is similar to rising the potassium in the bath solution to 20 mEq/l, what brings the glial pump to its maxium rate. Gyroxin in high dosis kills the tissue; ouabain, a cardiac glycosideo, also kills. This toxic effect  is a consequence of excitotoxicity- prolonged depolarization and high calcium activity within cells leds to lysis. However, ouabain in the retina also kills in low dosis ( 10 nM). This toxic effect looks different at macroscopic level,  probably envolving a different mechanism. In contrast, gyroxin at low dosis is not toxic and appears even protective to the tissue. The same is true for crotamine even at high concentration. Excitotoxicity also spreads in the tissue, but the spread is not wavelike, it “jumps” ahead and invades quiescent tissue in patches at different velocities. By using a small drop (50  ml) of concentrated solution we could follow the spread of excitotoxic response in the presence of high dosis of ouabain and gyroxin and could compare the tissue response to both substances. The spread is similar but the final outcome differs: oubain toxicity is stronger than the gyroxin toxicity. For the first time non-wavelike spread of excitation is documented in detail. This finding will be of interest to the theorists in the field.