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Mercurial perturbation of brush border membrane permeability in rabbit ileum
Abstract:
The sulfhydryl reagents Hg++ and p-chloromercuribenzene sulfonate (PCMBS) at millimolar concentrations reduced the mucosal entry of sugars and amino acids to 80-90% of control levels within several minutes. Based on 50% levels of inhibition, Hg++ proved to be 20 and 10 times as potent as PCMBS in blocking sugar and amino acid transport, respectively; both systems were equally sensitive to Hg++. Concomitant measurements of 203Hg-PCMBS demonstrated a progressive tissue uptake, which, unlike inhibition, did not saturate with increasing times of exposure, thus suggesting appreciable epithelial entry with prolonged exposures (less than 30 min at 1 mM). At similar dose levels, no significant change in mucosal Na+ entry was detected. Inhibition was not reversed by 30-min washes in cholinesalt solutions; however, 10-min exposures to dithiothreitol [10 mM] reversed Hg++ and PCMBS inhibition by 40 and 100%, respectively. Alanine and galactose influx kinetics measured at concentrations of 0-100 mM exhibited a linear or diffusional entry component in addition to the usual saturable component for both control and Hg++-treated ileum. The presence of a diffusional term in the flux equation resulted in two sets of parameters giving nearly equal fits to these measurements. It was shown that this ambiguity could be resolved by determining the change in diffusional entry with Hg++ treatment. A 20-min exposure to 0.5 mM Hg++ caused an increase from 0.050 and 0.045 to 0.064 and 0.070 cm/hr in the coefficient of diffusional entry for alanine and galactose, respectively. On the basis of this increase, it is argued that Hg++ causes a decrease in Jmax and little change in Km for both transport mechanisms. This analysis has a general bearing on kinetic measurements of transport in which passive fluxes are comparable to those mediated by specific pathways. The alanine results are consistent with bimolecular reactions between mercurial and two membrane inhibitory sites, each producing approximately 40% reduction in membrane translocation rate. The estimated reaction rate constants were 5.0 and 0.4 mM min.
Insights
Sulfhydryl reagents like mercury (Hg++) and PCMBS significantly inhibit sugar and amino acid transport in the ileum. Mercury is more potent, affecting both saturable and diffusional transport pathways.
Area of Science:
- Biochemistry
- Cell Biology
- Physiology
Background:
- The intestinal epithelium actively transports nutrients like sugars and amino acids.
- Sulfhydryl reagents are known to interact with proteins and can affect membrane transport.
- Understanding the mechanisms of nutrient transport inhibition is crucial for physiological and pharmacological research.
Purpose of the Study:
- To investigate the effects of sulfhydryl reagents, specifically Hg++ and p-chloromercuribenzene sulfonate (PCMBS), on mucosal nutrient entry in the ileum.
- To compare the potency and sensitivity of Hg++ and PCMBS in inhibiting sugar and amino acid transport.
- To elucidate the impact of these reagents on both saturable and diffusional transport components.
Main Methods:
- Inhibition studies using millimolar concentrations of Hg++ and PCMBS on mucosal entry of sugars and amino acids.
- Measurement of 203Hg-PCMBS tissue uptake to assess epithelial entry.
- Assessment of Na+ entry to rule out non-specific effects.
- Reversal studies using dithiothreitol to determine the reversibility of inhibition.
- Kinetic analysis of alanine and galactose influx in control and Hg++-treated ileum.
Main Results:
- Hg++ and PCMBS reduced sugar and amino acid entry by 80-90%.
- Hg++ was 20 times more potent than PCMBS for sugar transport and 10 times for amino acid transport.
- Hg++ and PCMBS increased the diffusional entry coefficient for alanine and galactose, suggesting an impact on passive transport.
- Hg++ treatment decreased Jmax and minimally affected Km for both transport mechanisms.
- Inhibition was partially or fully reversed by dithiothreitol, indicating a role for sulfhydryl groups.
Conclusions:
- Sulfhydryl reagents, particularly Hg++, significantly inhibit both carrier-mediated and diffusional nutrient transport across the ileal epithelium.
- Hg++ appears to decrease the maximal transport rate (Jmax) without significantly altering the affinity (Km) of the transport systems.
- The findings provide insights into the kinetic analysis of transport mechanisms when passive fluxes are comparable to carrier-mediated fluxes.