No abstract

The kinetic characteristics of pigment aggregation and dispersion were examined in the perfused, red ovarian chromatophores of the freshwater palaemonid shrimp Macrobrachium olfersii. Aggregation, induced by either red pigment concentrating hormone (RPCH) or the Ca ++ ionophore A23187, takes 24-26 min to complete. The associated velocity profiles consist of three distinct phases: an immediate velocity peak of 13.6 ± 3.23 and 11.6 ± 1.15 µm/min, respectively, of 8-min duration; 10-min plateaus of 1.7 ± 0.20 and 2.2 ± 0.29 µm/min, respectively; and an interval in which velocity declines irregularly and ceases. Subsequent pigment dispersion, induced by a Ca ++ -free saline, is likewise triphasic, attaining peak velocities of 5.0 ± 0.67 and 6.8 ± 1.72 µm/ min, respectively; plateau velocities were 1.7 ± 0.28 and 1.4 µm/min, respectively. The chromatophores contain two distinct types of pigment granule, bundles of microtubules, and a well-developed smooth endoplasmic reticulum. The effects of substances that affect the dynamics of cytoskeletal components were also examined. Neither colchicine nor the dynein inhibitor vanadate affect aggregation or dispersion, although colchicine itself induces 20-60% aggregation. However, cytochalsin B partially inhibits aggregation and markedly affects dispersion, while butanedione monoxime (an inhibitor of myosin ATPase) also partially inhibits aggregation and induces pigment dispersion. While the nature of the rapid component of pigment aggregation is obscure, these data suggest that the slow component may result from the interaction of the pigment granules with an actin-myosin-like system. These findings are compared with the kinetic characteristics of organelle translocation seen in fish chromatophores and neurons in which transport velocities are conspicuously greater than those recorded here for shrimp chromatophores.

Pigment granule migration within crustacean chromatophores provides an excellent model with which to investigate cytoplasmic movements, given the antagonistic, neurosecretory peptide regulation of granule translocation, and the absence of innervation in these large, brightly colored cells. Red pigment-concentrating hormone (RPCH) induces pigment aggregation in shrimp chromatophores via an increase in intracellular Ca2+; however, how this increase is brought about is not known. To examine the putative Ca2+ movements leading to pigment translocation in red, ovarian chromatophores of the freshwater shrimp, Macrobrachium olfersii, this study manipulates intra- and extracellular Ca2+ employing ER Ca2+-ATPase inhibitors, ryanodine-sensitive, ER Ca2+ channel blockers, and EDTA/EGTA-buffered A23187/Ca2+-containing salines. Our findings reveal that during pigment aggregation, cytosolic Ca2+ apparently increases from an intracellular source, the abundant SER, loaded by the SERCA and released through ryanodine-sensitive receptor/channels, triggered by capacitative calcium influx and/or calcium-induced calcium release mechanisms. Aggregation also depends on external calcium, which may modulate RPCH/receptor coupling. Such calcium-regulated pigment movements form the basis of a complex system of chromatic adaptation, which confers selective advantages like camouflage and protection against ultra-violet radiation to this palaemonid shrimp.

The cell signaling cascades that mediate pigment movements in crustacean chromatophores are not yet well established, although Ca(2+) and cyclic nucleotide second messengers are involved. Here, we examine the participation of cyclic guanosine monophosphate (cGMP) in pigment aggregation triggered by red pigment concentrating hormone (RPCH) in the red ovarian chromatophores of freshwater shrimp. In Ca(2+)-containing (5.5 mmol l(-1)) saline, 10 micromol l(-1) dibutyryl cGMP alone produced complete pigment aggregation with the same time course ( approximately 20 min) and peak velocity ( approximately 17 microm/min) as 10(-8) mol l(-1) RPCH; however, in Ca(2+)-free saline (9 x 10(-11) mol l(-1) Ca(2+)), db-cGMP was without effect. The soluble guanylyl cyclase (GC-S) activators sodium nitroprusside (SNP, 0.5 micromol l(-1)) and 3-morpholinosydnonimine (SIN-1, 100 micromol l(-1)) induced moderate aggregation by themselves ( approximately 35%-40%) but did not affect RPCH-triggered aggregation. The GC-S inhibitors zinc protoporphyrin IX (ZnPP-XI, 30 micromol l(-1)) and 6-anilino-5,8-quinolinedione (LY83583, 10 micromol l(-1)) partially inhibited RPCH-triggered aggregation by approximately 35%. Escherichia coli heat-stable enterotoxin (STa, 1 micromol l(-1)), a membrane-receptor guanylyl cyclase stimulator, did not induce or affect RPCH-triggered aggregation. We propose that the binding of RPCH to an unknown membrane-receptor type activates a Ca(2+)-dependent signaling cascade coupled via cytosolic guanylyl cyclase and cGMP to protein kinase G-phosphorylated proteins that regulate aggregation-associated, cytoskeletal molecular motor activity. This is a further example of a cGMP signaling cascade mediating the effect of a crustacean X-organ neurosecretory peptide.

Pigment aggregation in shrimp chromatophores is triggered by red pigment concentrating hormone (RPCH), a neurosecretory peptide whose plasma membrane receptor may be a G-protein coupled receptor (GPCR). While RPCH binding activates the Ca /cGMP signaling cascades, a role for cyclic AMP (cAMP) in pigment aggregation is obscure, as are the steps governing Ca release from the smooth endoplasmic reticulum (SER). A role for the antagonistic neuropeptide, pigment dispersing homone (α-PDH) is also unclear. In red, ovarian chromatophores from the freshwater shrimp Macrobrachium olfersi, we show that a G-protein antagonist (AntPG) strongly inhibits RPCH-triggered pigment aggregation, suggesting that RPCH binds to a GPCR, activating an inhibitory G-protein. Decreasing cAMP levels may cue pigment aggregation, since cytosolic cAMP titers, when augmented by cholera toxin, forskolin or vinpocentine, completely or partially impair pigment aggregation. Triggering opposing Ca /cGMP and cAMP cascades by simultaneous perfusion with lipid-soluble cyclic nucleotide analogs induces a "tug-of-war" response, pigments aggregating in some chromatosomes with unpredictable, oscillatory movements in others. Inhibition of cAMP-dependent protein kinase accelerates aggregation and reduces dispersion velocities, suggesting a role in phosphorylation events, possibly regulating SER Ca release and pigment aggregation. The second messengers IP and cADPR do not stimulate SER Ca release. α-PDH does not sustain pigment dispersion, suggesting that pigment translocation in caridean chromatophores may be regulated solely by RPCH, since PDH is not required. We propose a working hypothesis to further unravel key steps in the mechanisms of pigment translocation within crustacean chromatophores that have remained obscure for nearly a century.