Metal ion specificity in anaesthetic induced increase in the rate of monensin and nigericin mediated H+/M+ exchange across phospholipid vesicular membranes.

From a study of the decay of the pH difference across vesicular membranes (delta pH) it has been possible to show that H+ and alkali metal ion (M+) concentration gradients across bilayer membranes (which are responsible for driving important biochemical processes) can be selectively perturbed by anaesthetics such as chloroform and benzyl alcohol by combining them with a suitable exchange ionophore. On adding the anaesthetic to the membrane in an environment containing metal ions M+ = K+, the rate of delta pH decay by H+/M+ exchange increases by a larger factor or by a smaller factor (when compared to that in a membrane environment with M+ = Na+) depending on whether the exchange ionophore chosen is monensin or nigericin. A rational explanation of this "metal ion specificity" can be given using the exchange ionophore mediated ion transport scheme in which the equilibrations at the "interfaces" are fast compared to the "translocation equilibration" between the species in the two layers of the membrane. The following three factors are responsible for the observed "specificity": On adding the anaesthetic (i) translocation rate constants increase, (ii) the concentrations of the M+ bound ionophores increase at the expense of H+ bound ionophores. (iii) Under our experimental conditions the rate determining species are the complexes monensin-K (Mon-K) and nigericin-H (Nig-H) for M+ = K+ whereas they are monensin-H (Mon-H) and nigericin-Na (Nig-Na) for M+ = Na+. Possible anaesthetic induced membrane perturbations contributing to the above mentioned changes in the membrane are (A), the loosening of the membrane structure and (B), an associated increase in the membrane hydration (and membrane dielectric constant). An analysis of the consequent changes in the various transport step shows the following: (a), The anaesthetic induced changes in the translocation rates of electrically charged species are not relevant in the explanation of the observed changes in the delta pH decay rates. (b), Changes in the rates of fast equilibria at the interface contribute to changes in KH and KM. (c), A suggestion made in the literature, that a significant interaction between the dipole moment of the monensin-K complex and the membrane slows down its translocation, is not valid. (d), The ability to explain rationally all the delta pH decay data confirms the validity of the transport scheme used. In our experiments delta pH across the vesicular membrane was created by pH jump coming from a temperature jump.

Swelling of the vesicle is prerequisite for PTH secretion

Unlike most secretory cells, high extra cellular calcium inhibits rather than stimulates hormonal secretion in several cells such as parathyroid cells, Juxtaglomerular cells and osteoclast. To gain further insight into the common but unique stimulus-secretion coupling mechanism in these cells, bovine parathyroid slices were incubated in various conditions of Krebs-Ringer (KR) solution containing essential amino acids. Parathyroid cells showed the inverse dependency of secretion on extra cellular calcium concentration as we expected. Ammonium acetate overcame the inhibitory effect of 2.5 mM of calcium and the maximum effect was as much as the five times of the basal value, while there was a little additive effect under 0 mM CaCl2. PTH secretion was biphasic according to the change of extra cellular osmolarity and the lowest response was observed at 300 mOsm/l. In Na-rich KR solution, high concentration of nigericin (> 10(-4)M) completely overcame the inhibitory effect of 2.5 mM CaCl2 and the maximum stimulatory effect was 8 times greater whereas it was only 2 times greater without CaCl2. In K-rich KR solution that abolished the K-gradient between the extra cellular solution and the cytoplasm, the rate of PTH secretion increased, and furthermore the addition of nigericin increased the rate of secretion significantly. The results above suggested that the osmotic swelling of the secretory vesicle in parathyroid cells might promote exocytosis as in Juxtaglomerular cells. We propose that the swelling of the vesicle is also prerequisite for secretion in several cells inhibited paradoxically by Ca++, whatever the signal transduction pathway for swelling of the secretory granules induced by the lowering of Ca++ in cytoplasm are.