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

The roles of calcium in cell signaling consequent to chromatophorotropin action and as an activator of mechanochemical transport proteins responsible for pigment granule translocation were investigated in the red ovarian chromatosomes of the freshwater shrimp Macrobrachium olfersii. Chromatosomes were perfused with known concentrations of free Ca++ (10(-3) to 10(-9) M) prepared in Mg(++)-EGTA-buffered physiological saline after selectively permeabilizing with 25 microM calcium ionophore A23187 or with 10(-8) M red pigment concentrating hormone (RPCH). The degree of pigment aggregation and the translocation velocity of the leading edges of the pigment mass were recorded in individual chromatosomes during aggregation induced by RPCH or A23187 and dispersion induced by low Ca++. Aggregation is Ca++ dependent, showing a dual extracellular and intracellular requirement. After perfusion with reduced Ca++ (10(-4) to 10(-9) M), RPCH triggers partial aggregation (approximately 65%), although the maximum translocation velocities (approximately 16.5 microns/min) and velocity profiles are unaffected. After aggregation induced at or below 10(-5) M Ca++, spontaneous pigment dispersion ensues, suggesting a Ca++ requirement for RPCH coupling to its receptor, or a concentration-dependent, Ca(++)-induced Ca(++)-release mechanism. The Ca(++)-channel blockers Mn++ (5 mM) and verapamil (50 microM) have no effect on RPCH-triggered aggregation. An intracellular Ca++ requirement for aggregation was demonstrated in chromatosomes in which the Ca++ gradient across the cell membrane was dissipated with A23187. At free [Ca++] above 10(-3) M, aggregation is complete; at 10(-4) M, aggregation is partial, followed by spontaneous dispersion; below 10(-5) M Ca++, pigments do not aggregate but disperse slightly. Aggregation velocities diminish from 11.6 +/- 1.2 microns/min at 5.5 mM Ca++ to 7.4 +/- 1.3 microns/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 velocities increase from 0.6 +/- 0.2 to 3.1 +/- 0.5 microns/min. Half-maximum dispersion 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 intracellular 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-dependent properties of the granule translocation apparatus.