Effects of High K⁺ Media on Leucine Incorporation into Aplysia Nervous Tissue

Citation

Ram, Jeffrey Lewis (1974) Effects of High K⁺ Media on Leucine Incorporation into Aplysia Nervous Tissue. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/qtf0-tz26. https://resolver.caltech.edu/CaltechTHESIS:11202021-011429389

Abstract

To study possible coupling between membrane polarization and protein synthesis, elevated external K⁺ levels were used to depolarize the cell membranes in isolated Aplysia californica parieto-visceral ganglia (PVG). The effect of this treatment on the incorporation of labeled leucine into proteins in the ganglion was analyzed on sodium dodecyl sulfate polyacrylamide gels. PVGs were preincubated 3 hours and then incubated 4 hours in either control medium (¹⁴C-leucine, 10 mM [ K⁺]) or experimental medium (³H-leucine, 10 + x mM [K⁺] with equimolar [Na⁺] reduction). These were homogenized together, separated into aqueous soluble and aqueous insoluble fractions, and run on gels.

In the aqueous soluble fraction of the PVG High [K⁺] (90-110 mM [K⁺]} caused relative increases in incorporation in distinct peaks at 50K (K = 1000 daltons) and 40K. The larger peak, at 50K, was studied further.

The relative increase at 50K occurred when ¹⁴c-leucine (instead of the usual ³H-leucine) was incorporated in High [K⁺]. The relative increase at 50K did not occur (1) when [K⁺] was raised to only 50 mM; (2) when [Na⁺] was reduced by 80 mM, and tris⁺ (HCl to neutralize) was substituted instead of K⁺; (3) in pleura-visceral connective (PVC) nerve; and (4) in the aqueous insoluble fraction of the PVG.

The effect of High [K⁺] on incorporation into the giant cell (R2) of the PVG was examined by first labeling the PVG in control medium, rinsing it, and then labeling it in experimental medium. High [K⁺] in the experimental medium caused a significant relative increase at 50K in whole PVGs, half PVGs, and R2s dissected from the PVG following incubation. The results in R2 were marred by great variability in the control patterns.

Autoradiography of identified cells (R2 and R15) dissected from PVGs labeled with ³H-leucine in normal [K⁺] showed that contaminating cells {mostly glia), which always adhere to such dissected cells, generally account for less than 20% of the total incorporated formalin-fixed label. This contamination is large enough so that a glial origin of the High [K⁺] effect on incorporation at 50K cannot be positively excluded. However, the presence of this effect in dissected R2s and its absence in PVC nerves, which contain axons, glia, and connective tissue, but no nerve cell bodies, lend support to the notion that the effect is neuronal in origin.

High [K⁺] caused a reduction of approximately SO% in total incorporation into both aqueous soluble and aqueous insoluble proteins of the PVG. Similar decreases of 35% were seen in dissected R2s. [Na⁺] reduction (by 80 mM, tris⁺ substitution) had no significant effect on total incorporation (measured only in the aqueous soluble fraction of the PVG). High [K⁺] caused a reduction of approximately 85% in total incorporation into PVC nerve. Autoradiography of the nerve showed that this reduction occurred in both the connective tissue sheath and the axonal-glial region. High [K⁺] caused no significant change in non-volatile TCA soluble label in either the ganglion or the nerve.

Other effects of High [K⁺] on the PVG: (1) a small (not large enough to have caused the relative increase at 50K) decrease in the relative amount of label in the aqueous soluble, TCA insoluble fraction compared to the aqueous insoluble fraction, and (2) a relative decrease in incorporation in higher molecular weight peptides compared to lower molecular weight peptides in both aqueous soluble and aqueous insoluble fractions.

These results suggest, but do not prove, that High [K⁺] caused an increase in the synthesis of a neuronal peptide of approximately 50,000 daltons molecular weight. The possibility that this peptide may be a tubulin subunit is briefly discussed.

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