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Modulation of Na+/alanine cotransport in liver sinusoidal membrane vesicles by internal divalent cations
T W Simmons1, R H Moseley, J L Boyer
1Department of Biophysics, University of Rochester School of Medicine, NY 14642.
Abstract:
Rat liver basolateral plasma membrane (blLPM) vesicles resuspended in 5 mM Mg2(+)-, Ca2(+)-, Mn2(+)- or Co2(+)-containing media exhibited a markedly lower rate of Na(+)-stimulated L-alanine transport. Divalent cation inhibition of L-alanine uptake was dose dependent, and was observed only when the vesicles were pre-loaded with the divalent cations. The presence or absence of the metal ions in the extravesicular incubation media had no effect on L-alanine transport. Conversely, pretreatment of the vesicles with 0.2 mM of either EGTA or EDTA resulted in higher initial rates of L-alanine transport. This stimulation was overcome by addition of excess divalent cation to the vesicle suspension solution. Since these blLPM vesicles are primarily oriented right-side-out, the divalent cation inhibition of L-alanine transport appears to be a result of their interaction with cytosolic components of the cell membrane. Total Na+ flux as measured with 22Na+ was not affected by intravesicular 5 mM Mg2+ or Ca2+, indicating that the inhibition was not due to dissipation of the Na+ gradient. These observations suggest that intracellular divalent cations may serve to modulate L-alanine transport across the liver cell plasma membrane.
Insights
Intracellular divalent cations like magnesium and calcium inhibit sodium-stimulated L-alanine transport in rat liver cells. This suggests a regulatory role for these cations in amino acid uptake.
Area of Science:
- Cell Biology
- Biochemistry
- Membrane Transport
Background:
- The basolateral plasma membrane (blLPM) of liver cells plays a crucial role in nutrient transport.
- Sodium-coupled amino acid transporters are vital for cellular uptake of essential amino acids.
- The influence of intracellular divalent cations on these transport systems is not fully understood.
Purpose of the Study:
- To investigate the effect of intracellular divalent cations on sodium-stimulated L-alanine transport in rat liver basolateral plasma membrane vesicles.
- To determine if divalent cations modulate L-alanine uptake by interacting with intracellular components of the membrane.
Main Methods:
- Preparation of right-side-out rat liver blLPM vesicles.
- Measurement of Na(+)-stimulated L-alanine transport rates under varying intravesicular divalent cation concentrations (Mg2+, Ca2+, Mn2+, Co2+).
- Assessment of Na+ flux using 22Na+ to rule out effects on the Na+ gradient.
Main Results:
- Intravesicular divalent cations (Mg2+, Ca2+, Mn2+, Co2+) significantly inhibited Na(+)-stimulated L-alanine transport in a dose-dependent manner.
- Inhibition was observed only when cations were present inside the vesicles, not externally.
- EGTA/EDTA treatment, which chelates divalent cations, increased L-alanine transport rates, confirming cation-mediated inhibition.
- Na+ flux remained unaffected, indicating the inhibition was specific to L-alanine transport and not due to Na+ gradient dissipation.
Conclusions:
- Intracellular divalent cations, particularly Mg2+ and Ca2+, act as negative modulators of Na(+)-stimulated L-alanine transport across the liver blLPM.
- This suggests a physiological mechanism where intracellular cation levels can regulate amino acid uptake in hepatocytes.
- Further research is warranted to identify the specific cytosolic targets involved in this regulatory process.