Characterization of calcium transport by basolateral membrane vesicles of human small intestine

1988 ◽  
Vol 255 (4) ◽  
pp. G482-G489 ◽  
Author(s):  
K. Kikuchi ◽  
T. Kikuchi ◽  
F. K. Ghishan
Keyword(s):  
Small Intestine ◽  
Osmotic Shock ◽  
Basolateral Membrane ◽  
Membrane Vesicles ◽  
Ionophore A23187 ◽  
Basolateral Membrane Vesicles ◽  
Michaelis Constant ◽  
Exchange System ◽  
Human Small Intestine

The present studies investigated the mechanism of Ca2+ transport across basolateral membrane vesicles (BLMVs) prepared from human small intestine. Ca2+ uptake represented transport into the intravesicular space as evident by osmolality study and by the demonstration of Ca2+ efflux from the intravesicular space by Ca2+ ionophore A23187. Ca2+ uptake was stimulated by Mg2+-ATP. Kinetic parameters for ATP-dependent Ca2+ uptake revealed a Michaelis constant (Km) of 0.02 +/- 0.01 microM and a maximum rate of uptake (Vmax) of 1.00 +/- 0.03 nmol.mg protein-1.min-1.Ca2+ uptake in the absence of Mg2+ was inhibited by 75%. The Km of ATP concentration required for half-maximal Ca2+ uptake was 0.50 +/- 0.1 mM. Calmodulin (10 micrograms/ml) increased Vmax to 1.62 +/- 0.02 nmol.mg protein-1.min-1 (P less than 0.001). Km values were 0.017 +/- 0.001 microM, which was not significantly different from control values. Basolateral membranes depleted of calmodulin by EDTA osmotic shock decreased ATP-dependent Ca2+ uptake by 65%. Trifluoperazine, an anticalmodulin drug, inhibited ATP-dependent Ca2+ uptake by 50%, while no inhibition was noted in calmodulin-depleted membranes. Efflux of Ca2+ in the BLMVs was stimulated by trans-Na+. Na+-dependent Ca2+ uptake was saturable with respect to Ca2+ concentration and exhibited a Km of 0.09 +/- 0.03 microM and a Vmax of 1.08 +/- 0.01 nmol.mg protein-1.min-1. These results are consistent with the existence of a Na+-Ca2+ exchange system and ATP and Mg2+-dependent, calmodulin-regulated Ca2+, transport mechanism in BLMVs of human enterocytes.

scholarly journals Preparation and characterization of basolateral plasma-membrane vesicles from sheep parotid glands. Mechanisms of phosphate and d-glucose transport

1991 ◽  
Vol 279 (3) ◽  
pp. 843-848 ◽  
Author(s):  
S Vayro ◽  
R Kemp ◽  
R B Beechey ◽  
S Shirazi-Beechey
Keyword(s):  
Basolateral Membrane ◽  
Membrane Vesicles ◽  
Acinar Cells ◽  
Tissue Homogenate ◽  
Basolateral Membrane Vesicles ◽  
Parotid Glands ◽  
Plasma Membrane Vesicles ◽  
Basolateral Plasma Membrane ◽  
Sheep Parotid

A procedure is described for the preparation of basolateral membrane vesicles from the acinar cells of the sheep parotid gland. The ouabain-sensitive K(+)-activated phosphatase activity was enriched 30-fold over the tissue homogenate; 45% of this activity was recovered in the final membrane fraction. The presence of membranes from other organelles was negligible. Evidence is presented for the location of Na(+)-dependent symporters for phosphate and D-glucose on the basolateral membrane.

Stimulation of basolateral Na(+)-HCO3- cotransporter by angiotensin II in rabbit renal cortex

1993 ◽  
Vol 265 (2) ◽  
pp. F195-F203 ◽  
Author(s):  
S. Eiam-Ong ◽  
S. A. Hilden ◽  
C. A. Johns ◽  
N. E. Madias
Keyword(s):  
Renal Cortex ◽  
Basolateral Membrane ◽  
Enzyme Reaction ◽  
Membrane Vesicles ◽  
Independent Effect ◽  
Ang Ii ◽  
Basolateral Membrane Vesicles ◽  
Michaelis Constant ◽  
Direct Stimulation ◽  
Stimulation Of

Angiotensin (ANG) II is now recognized as a powerful direct controller of Na+ reabsorption in the proximal convoluted tubule, a property that predominantly reflects stimulation of the transepithelial NaHCO3 flux. Numerous studies have established that this effect of ANG II represents stimulation of the apical Na+/H+ exchanger, but a single microperfusion study has also suggested direct stimulation of the basolateral Na(+)-HCO3- cotransporter. We have carried out studies in basolateral membrane vesicles from rabbit renal cortex to examine directly whether ANG II exerts an independent effect on the Na(+)-HCO3- cotransporter. Preincubation of vesicles with ANG II (10(-11) to 10(-9) M) for 15 min enhanced the activity of the cotransporter, the greatest effect occurring at 10(-11) M (41 +/- 1.1%, P < 0.005). This stimulation reflected an increase in the maximal enzyme reaction velocity of the cotransporter but no change in the Michaelis constant for Na+. ANG II had no effect on Na(+)-dependent succinate transport. ANG I (10(-9) M) and ANG III (10(-10) M) also stimulated the Na(+)-HCO3- cotransporter, and captopril (10(-4) M) attenuated the ANG I stimulation by 68 +/- 3.5% (P < 0.01) but not that of ANG II and III. Saralasin (10(-11) to 10(-8) M) by itself behaved as an agonist, and its stimulation was additive to that by ANG II. The nonpeptide ANG II receptor antagonist, losartan potassium (10(-6) M), and the disulfide-reducing agent, dithiothreitol (10 mM), each by itself had no effect on the cotransporter but each markedly attenuated the ANG II effect (by 77 +/- 1.4%, P < 0.01 and 74 +/- 1.6%, P < 0.005, respectively) in accord with the view that the basolateral receptor belongs to subtype 1. These results identify physiological concentrations of ANG II as a potent, direct, and specific stimulator of the basolateral Na(+)-HCO3- cotransporter.

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