	What does it all mean?!!! The first portion of the experiment assessed how readily CO₂ entered into the oocyte. As the CO₂ passed through the membrane, it underwent a reaction that leads to an increase in acidity within the cell. The reaction looks like this… CO₂ + H₂O => H₂CO₃ => H⁺ + HCO₃^- The more H⁺ that gets produced within the cell, the lower the pH (or base concentration) will be; because the only way to generate this reaction is to supply CO₂, the rate of pH reduction is a terrific indicator of CO₂ absorption. The first tests conducted determined the effect of CO₂/HCO₃^- solution exposure on two types of oocyte. Oocytes injected with cRNA encoding AQP1(used to express water channels suspected of also facilitating CO₂ permeability) expression acted as the variable group, and oocytes injected with water acted as the control. Under these circumstances, the rates of pH decrease, which is indicative of CO₂ permeability, for both the variable and the control were very similar. The rate of acidification for the water injected oocyte (the control subject) was 11.3 x 10-4 pH units/sec. The rate of decrease for the AQP1 expressed oocyte without added CA was higher by a rather unremarkable amount: 13.3 x 10-4 pH units/sec. However… If Carbonic Anhydrase (CA)- an enzyme that acts as a catalyst to speed up the diffusion reaction of CO₂ mentioned- above is added, conversion of CO₂ to H₂CO₃ is substantially increased. When an oocyte injected with CA was exposed to a CO₂/HCO₃^- solution, the decreases in pH were measured at 54 and 44 x 10-4 pH units/sec. These rates are both about four times faster than the rate recorded for the oocyte expressing AQP1 solely. Now that's fast!!! Furthermore… Optimal conditions for CO₂ permeability are achieved when an AQP1 expressing oocyte is injected with CA. Upon introduction to a CO₂/HCO₃^- solution, pH rate of decrease is phenomenal. The initial rate of decrease was reported at 75 x 10-4 pH units/sec., topping all previous rates for pH decrease. Thus… In order to fully realize the effect that the presence of CA had on CO₂ permeability, a CA inhibitor called Ethoxzdamide was introduced. This inhibitor reduced the effects of CA injections substantially. For the H₂O/CA oocyte originally decreasing in pH at a rate of 54 x 10-4 pH units/sec, Ethoxzdamide reduced this to 10 x 10-4 pH units/sec. For the AQP1/ CA oocyte originally decreasing at 74 x 10-4 pH units/sec, Ethoxzdamide reduced this rate to 17 x 10-4 pH units/sec. Finally… In summary, the results showed that CA/AQP1 oocytes decreased in pH, by the admission of CO₂, at a fantastic rate: 74 x 10-4 pH units/sec., H₂O/CO₂ oocytes decreased in pH at a rate of 54 x 10-4 pH units/sec., AQP1 oocytes reduced in pH at a rate of 13.3 x 10-4 pH units/sec., and the control H₂Ooocytes reduced in pH at a rate of 11.3 x 10-4 pH units/sec. Although there are several possible explanations for the observed response of cell pH to the presence of AQP1, the most likely (and one supported by more recent evidence: Cooper and Boron, Am. J. Physiol. In press) is that AQP1 expression caused this acidification.


	Any Questions??? Q. What is a catalyst and why is Carbonic Anhydrase considered one? A. A catalyst is a material, (in biology, typically an enzyme), that helps to speed a reaction by lowering the amount of energy required to initiate it. This is referred to as lowering activation energy. The Carbonic Anhydrase helps to speed the reaction of CO₂ and H₂Oto completion. This has the effect of producing more H⁺ and HCO₃^-, and also works to keep the accumulation of CO₂ near the intracellular surface at a minimum. This insures that the gradient driven influx is maintained. Q. What is pH and how is it measured? A. A solution's acidity or alkalinity is expressed on a pH scale, which runs from 0 to 14, 7 being neutral. This scale is based on the concentration of H⁺ in moles per liter: pH = -log [H+]. A solution that has more H⁺ than OH- is an acidic solution and has a pH lower than 7. This is why, in the experiment, a measure of pH can be used to determine a relative rate of CO₂ permeability. More CO₂ into the cell means that more H⁺ will be produced in the reaction catalyzed by Carbonic Anhydrase, and more H⁺ means a lower pH levels. Thus, a more rapid pH decrease indicates more rapid CO₂ permeability.