In the retina of the eye, there are equal amounts of endothelial cells and cells
ID: 63612 • Letter: I
Question
In the retina of the eye, there are equal amounts of endothelial cells and cells called pericytes. The two types of cells are associated with one another and may communicate with each other. You work in a lab that is particularly interested in pericytes because it had been observed that pericyte damage and thickening of basement membrane occur during the early stages of diabetes retinopathy, which ultimately leads to blindness. Your lab is specifically interested in the factors responsible for causing pericyte damage. Your lab noted that cells in the kidney that were similar in function to the pericyte possessed a sodium-coupled glucose transport (SGLT), and hypothesized that such a transporter, if it existed, would facilitate and perhaps regulate the entry of glucose into the cell.
1. To start investigating whether pericytes have a SGLT, you culture pericytes and endothelial cells separately and measure their ability to take up glucose. Glucose uptake was measured both in the presence (NaCl) and absence of sodium (Choline Chloride-this maintains osmolarity.) The results are shown in the following Figure. What is your interpretation of the results?
2. Next, the investigators measured the effect of increasing sodium concentration on glucose uptake in both pericytes ad endothelial cells. The results are shown in the figure below. What is your interpretation of these results? What information does the shape of the curves convey?
3. Use the information in the figure to estimate the Km ad Vmax for glucose uptake by pericytes in the presence and absence of sodium. What have you learned about how this transporter works by these values?
4. The activity of the SGLT in pericytes was investigated in the presence of inhibitors. Radioactively labeled glucose was added to cultured pericytes, in the absence (control) or presence of several potential inhibitors. The transport experiments were carried out both in the presence and absence of sodium ions. The results are shown in the figure below.
a.) What is the effect of sugars such as galactose and 2-deoxyglucose on glucose transport?
b.) What is the effect of 0.2 mM phlorizin on glucose transport?
5. Has your lab demonstrated convincingly that a SGLT exists in pericytes? What about endothelial cells? Explain, using the data to support your answers.
Explanation / Answer
1.
Pericytes express sodium-coupled SGLT as absence of sodium in the form of NaCl decreased glucose uptake compared to one with sodium chloride. No change in glucose uptake in presence or absence of sodium chloride demostrates Endothelial cells don't express SGLT.
2.
Glucose uptake is independent of Sodium chloride concentration in Endothelial cells.
Glucose uptake is enhanced with increasing concentration of sodium chloride which then reaches a threhold after which there is no net increase. The transporter works much like first order enzyme kinetics.
3.
A plot of 1/V0 versus 1/[S], called a Lineweaver-Burk or double-reciprocal plot, yields a straight line with an intercept of 1/Vmax and a slope of KM/Vmax. The intercept on the x-axis is -1/KM.
Vmax in presence of sodium ions = 1 / .025 = 40 mM / hr ; Km = -1 / - 0.25 = 4 mM
Vmax in absence of sodium ions = 1 / 0.05 = 20 mM / hr; so Km = -1 / - 0.25 = 4 mM
The sodium ions are non-competitive activators of SGLT because Km is unchanged, but Vmax is incresed.
4.
a) galactose and 2-deoxyglucose shows no significant effect on glucose transport in pericytes in presence or absence of NaCl.
b) 0.2 mM phlorizin is specific inhibitor of SGLT mediated glucose transport.
5.
Yes, our lab results demonstrate that SGLT exists in pericytes and not in endothelial cells. Moreover, our data shows that sodium ions are competitive activators of this transporter and enhance the glucose transport almost 2 fold. No effect of galactose or 2-deoxyglucose on glucose transport by this transporter shows that the SGLT has high affinity for glucose or is very specific transporter of glucose.