Effects of divalent cations and metabolic poisons on the circadian rhythm from theAplysia eye

Summary

• 1. The isolated eyeof Aplysia has a circadian rhythm of compound optic nerve potentials. An attempt to inhibit entrainment of this rhythm by blocking synaptic transmission led to the discovery that one such inhibitor, manganese, produced phase shifts in the rhythm. Treatments of manganese (10 mM) of 6 h duration at different phases of the rhythm resulted in a phase response curve composed of only delay phase shifts (Fig. 1).

• 2. Many biological effects of Mn⁺⁺ involve Ca⁺⁺ dependent processes and this appears to be the case here since the phase shifting effect of Mn⁺⁺ was antagonized by increasing external Ca⁺⁺ concentrations (Fig. 3).

• 3. Mn⁺⁺ does not appear to be producing phase shifts by disrupting the flux of Ca⁺⁺ across the plasma membrane since changing such fluxes by either raising extracellular Ca⁺⁺ (50 mM) or lowering extracellular Ca⁺⁺ with EGTA (Ca⁺⁺ <10⁻⁷ M) did not cause phase shifts in the rhythm.

• 4. A Ca⁺⁺/Mg⁺⁺ ionophore, A23187, was used to affect the fluxes of these ions across intracellular membranes. Treatments of A23187 (7.5×10⁻⁶ M) produced delay phase shifts in the rhythm whether or not a Ca⁺⁺ or Mg⁺⁺ concentration gradient existed across the plasma membrane (Figs. 4, 5). In addition, an all-delay response curve resulted when eyes were treated at different phases with A23187 plus EGTA (Fig. 6).

• 5. Our results, and the known effects of A23187, led to the hypothesis that phase shifting by A23187 resulted either from the transport of Ca⁺⁺ and Mg⁺⁺ out of mitochondria or from oxidative phosphorylation becoming uncoupled as a result of the reuptake of these ions.

• 6. DNP (0.2 mM) and NaCN (2 mM) which both inhibit oxidative phosphorylation and cause release of Ca⁺⁺ and Mg⁺⁺ from mitochondria, also produced phase response curves with only delays (Fig. 9).

• 7. Mn⁺⁺, A23187, DNP, and NaCN have a common effect on oxidative phosphorylation and on the ability of mitochondria to regulate Ca⁺⁺ and Mg⁺⁺. The correlation between the similar phase response curves of these treatments (Fig. 10) and their common effects on mitochondria suggests that they are all affecting the rhythm by blocking energy production and/or causing the release of mitochondrial Ca⁺⁺ and Mg⁺⁺. The manner in which changes in energy production or in the concentrations of Ca⁺⁺/Mg⁺⁺ might affect the rhythm is discussed.