Characterisation of penicillin G uptake in human small intestinal brush border membrane vesicles

Abstract

BACKGROUND—Many β lactams are well absorbed by the small intestine, although the reasons for this are poorly understood. 
AIMS—To characterise the uptake of penicillin G into human small intestinal brush border membrane vesicles (BBMV) and to compare the uptake characteristics to those of rabbit BBMV. 
METHODS AND RESULTS—Uptake of penicillin G was studied in human BBMV. Penicillin G was actively transported into the lumen of BBMV via an H⁺ dependent, Na⁺ independent uptake system. The carrier mediated process was saturable and adhered to Michaelis-Menten kinetics (V_max 52 nmol penicillin G per mg protein per 30 seconds, K_m 13.9 mM). These results are similar to those previously reported in rabbit BBMV. 
CONCLUSIONS—It is suggested that penicillin G can be used as a model molecule for peptide and β lactam transport studies as it is cheap and readily available in isotopically labelled form. Furthermore, rabbit BBMV may be used as an acceptable substitute for human BBMV for the study of penicillin transport. 



Keywords: brush border membrane vesicles; β lactam; antibiotic uptake; oral administration; pH gradient

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Figure 1 .

Time course of glucose uptake in hBBMV. Vesicles were loaded with 5 mM HEPES/Tris, 0.1 mM MgSO₄, 100 mM mannitol, 100 mM KCl at pH 7.1. The assay mixture contained 100 mM NaCl or 100 mM KCl, 100 mM mannitol, 0.1 mM glucose, and 5 mM HEPES/Tris pH 7.1. Samples were incubated at 37°C. Results represent the average of three experiments carried out in triplicate. Error bars represent standard deviation. 


Figure 2 .

Time course of penicillin G uptake in hBBMV. Vesicles were loaded with 5 mM HEPES/Tris, 0.1 mM MgSO₄, 100 mM mannitol, 100 mM KCl at pH 7.1. The assay mixture contained 100 mM KCl, 100 mM mannitol, 0.1 mM penicillin G, and 5 mM MES/Tris pH 5.0, or 5 mM HEPES/Tris for pH 7.1, respectively. Samples were incubated at 37°C. Results represent the average of three distinct experiments carried out in triplicate. Error bars represent standard deviation. 


Figure 3 .

Effect of osmolarity on penicillin G uptake in hBBMV. Vesicles were loaded with 50 mM HEPES/Tris, 0.1 mM MgSO₄, 50 mM mannitol, 100 mM KCl at pH 7.1. The external buffer contained 100 mM KCl, 50-550 mM mannitol, 50 mM MES/Tris pH 5.0 and 0.1 mM penicillin G. Samples were incubated for 30 minutes at 37°C. The results represent the average of two distinct experiments carried out in triplicate. Error bars represent standard deviation. Intercept on y axis represents the amount of penicillin G associated with the membrane (12%); r=0.791. 


Figure 4 .

Eadie-Hofstee plot of penicillin G uptake in hBBMV. Vesicles were loaded with 50 mM HEPES/Tris, 0.1 mM MgSO₄, 50 mM mannitol, 100 mM KCl at pH 7.1. The external buffer contained 100 mM KCl, 0-50 mM mannitol (to correct for penicillin G addition), 50 mM MES/Tris pH 5.0, and 0.1-50 mM penicillin G. Samples were incubated for 30 seconds at 37°C. Results represent the average of three distinct experiments carried out in triplicate. Error bars represent standard deviation. V_max = 52 nmol/mg protein/30 sec; K_m = 13.9 mM; r=0.924.