The calcium dependence of pigment translocation in freshwater shrimp red ovarian chromatophores

The roles of calcium in cell signaling consequent to chromatophorotropin ac tion and as an activator of mechanochemical transport proteins responsible for pigment granule translocation were investigated in the red ovarian chro matosomes of the freshwater shrimp Macrobrachium olfersii. Chromatosomes we re perfused with known concentrations of free Ca++ (10(-3) to 10(-9) M) pre pared in Mg++-EGTA-buffered physiological saline after selectively permeabi lizing with 25 mu M calcium ionophore A23187 or with 10(-8) M red pigment c oncentrating hormone (RPCH). The degree of pigment aggregation and the tran slocation velocity of the leading edges of the pigment mass were recorded i n individual chromatosomes during aggregation induced by RPCH or A23187 and dispersion induced by low Ca++. Aggregation is Ca++ dependent, showing a d ual extracellular and intracellular requirement. After perfusion with reduc ed Ca++ (10(-4) to 10(-9) M), RPCH triggers partial aggregation (approximat e to 65%), although the maximum translocation velocities (approximate to 16 .5 mu m/min) and velocity profiles are unaffected. After aggregation induce d at or below 10(-5) M Ca++, spontaneous pigment dispersion ensues, suggest ing a Ca++ requirement for RPCH coupling to its receptor, or a concentratio n-dependent, Ca++-induced Ca++-release mechanism. The Ca++-channel blockers Mn++ (5 mM) and verapamil (50 mu M) have no effect on RPCH-triggered aggre gation. An intracellular Ca++ requirement for aggregation was demonstrated in chromatosomes in which the Ca++ gradient across the cell membrane was di ssipated with A23187. At free [Ca++] above 10(-3) M, aggregation is complet e; at 10(-4) M, aggregation is partial, followed by spontaneous dispersion; below 10(-5) M Ca++, pigments do not aggregate but disperse slightly. Aggr egation velocities diminish from 11.6 +/- 1.2 mu m/min at 5.5 mM Ca++ to 7. 4 +/- 1.3 mu m/min at 10(-4) M Ca++. Half-maximum aggregation occurs at 3.2 X 10(-5) M Ca++ and half-maximum translocation velocity at 4.8 x 10(-5) M Ca++. Pigment redispersion after 5.5 mM Ca++-A23187-induced aggregation is initiated by reducing extracellular Ca++: slight dispersion begins at 10(-7 ) M, complete dispersion being attained at 10(-9) M Ca++. Dispersion veloci ties increase from 0.6 +/- 0.2 to 3.1 +/- 0.5 mu m/min. Half-maximum disper sion occurs at 7.6 X 10(-9) M Ca++ and half-maximum translocation velocity at 2.9 X 10(-9) M Ca++. These data reveal an extracellular and an intracell ular Ca++ requirement for RPCH action, and demonstrate that the centripetal or centrifugal direction of pigment movement, the translocation velocity, and the degree of pigment aggregation or dispersion attained are calcium-de pendent properties of the granule translocation apparatus.