Lecture 2 Salt Reabsorption Flashcards
What kind of epithelium is the frog skin a good model for
Tight epithelium.
Which specific epithelial tissues can frog skins be a suitable model for
Renal collecting duct distal colon salivary gland sweat duct and airway surface epithelium
Describe the Ussing chamber technique to investigate the presence of an epithelial Na+ channel in frog skin
The Ussing chamber consists of two chambers. The target tissue is mounted between these two chambers (can be fresh tissue or sheet of primary/standard cell lines). There are then two electrodes to measure the transepithelial potential and two additional electrodes (current injection electrodes) that allow you to inject a current of known magnitude
In the initial studies that lead to the identification of the epithelial Na+ channel what technique was combined with the Ussing chamber approach in order to study Na+ movement across the frog skin
As well as using the Ussing chamber radioactive 24Na was added into chamber 1 of the Ussing chamber. This enabled scientists to directly measure how much 24Na was being transported across the epithelia. 24Na was added in chamber 1 and then the solution in chamber 2 was sampled. The reverse was also carried out to determine leak-back.
What conditions were used in the initial Ussing chamber frog skin experiment and the assumptions made
The same (Krebs) solution with the same concentration of solutes were added to both chambers either side of the frog skin. As the concentrations of the ions were the same there would be no net movement of charge and no initial potential would be generated
In the initial Ussing chamber experiments used to determine an epithelial Na+ channel if the frog skin was left and there was subsequent transport of ions then a potential would be generated across the epithelia. What would the size of this transepithelial potential depend on
The size of the potential generated would depend on how much transport was going on and also how much was leaking back
Below is a graph of the data obtained from the early experiments on frog skin that combined the Ussing chamber technique with 24Na tracing to determine the presence of an epithelial Na+ channel. Discuss what these results show
The bar graph shows that the total current (the short circuit current) was measured at around 135mC cm-2 h-1 using the recording electrodes present in the Ussing chamber setup. The other two bars represent the results obtained from the 24Na tracer experiments. These results show that Na+ influx massively predominates of Na+ efflux. In addition 24Na influx corresponded to a current density of around 135mC cm-2 h-1 this is the same value as the recording of the short circuit current. This in turn implies that Na+ influx is the dominant process with very little leak back and that solely the transport of Na+ across the epithelium is sufficient to cause the short circuit current. Hence the frog skin is a Na+ absorbing epithelium with Na+ channels being the only channels present in the membrane.
How were the 24Na tracing data used to give a current density which could then be compared with the Ussing chamber recordings
The 24Na tracing experiments gave a value of the number of picomoles per second of Na+ entering chamber 2 (inside the frog) from chamber 1 (outside the frog). These number of Na+ per second could then be used to calculate the corresponding current. This current could then be used to calculate the current density which would allow the comparison of 24Na movement with the short circuit current being generated. This would allow the assessment of the degree of contribution of 24Na movement to the short circuit current. It was found that 24Na influx alone could entirely account for the short circuit current recorded by the Ussing chamber. Subtracting the 24Na efflux current from the influx current generated a value that was the same as the short circuit current.
As Na+ transport across the frog skin epithelia could entirely account for the short circuit current what could be inferred about its transport
Na+ is the only actively transported ion in this epithelium
The Ussing chamber and 24Na tracing experiments revealed that Na+ is the only actively transported ion in the frog skin however the epithelial Na+ channel is not an active transporter of Na+ ions. What does this actually mean in terms of Na+ transport
ENaC isn’t actively transporting Na+ into or out of the cell. The component that contributes to the overall net active transport of Na+ is the basolateral Na+/K+ATPase which is pumping Na+ out of the epithelial cells into the extracellular solution.
What is the limitation of the Ussing chamber approach to study the movement of ions across epithelia
The Ussing chamber technique can only be used to measure net transport of ions over an epithelium
You need the active transport of an ion to result in an overall net movement of ions across an epithelium T or F
T
Describe the hypothesis and subsequent electrophysiology experiments carried out once it had been determined that there was a net movement of Na+ across the frog skin epithelia
It was hypothesised that on the apical membrane of the frog skin there would be a Na+ channel this wouldn’t be voltage-dependent. Subsequent electrophysiological experiments looked at the impact of changing the extracellular concentration of Na+. This would in turn change the Nernst potential for Na+ and lead to a subsequent change in the apical membrane potential if it was permeable to Na+. This was indeed observed hence indicating that there was an apical Na+ channel in the membrane of the frog skin epithelium
To characterise the epithelial Na+ channel the effects of amiloride were investigated in human colonic biopsy cells. Discuss the properties of human colonic epithelia
The human colon is a Na+ absorbing epithelium that is not particularly tight. There are lots of other transport pathways going on. Hence the colon has a much lower Vte than frog skin due to leak back and the presence of a number of additional channels.
What is the difference in transepithelial potential seen in frog skin compared to colon epithelium
Frog skin transepithelial potential = -50mV Colon transepithelial potential = -1mV
Below are the results of patch clamp experiments carried out by Meyer and Sackman on the ENaC channel. Describe what these results show
These traces show that the downward deflections representing closing of the channel decrease as the Na+ concentration in the extracellular solution decreases. This means that overall currents are larger when there is a high Na+ concentration.
