Signaling Events During Cyclic Guanosine Monophosphate-Regulated Pigment Aggregation in Freshwater Shrimp Chromatophores

Abstract

Crustacean color change results partly from granule aggregation induced by red pigment concentrating hormone (RPCH). In shrimp chromatophores, both the cyclic GMP (3', 5'-guanosine monophosphate) and Ca(2+) cascades mediate pigment aggregation. However, the signaling elements upstream and downstream from cGMP synthesis by GC-S (cytosolic guanylyl cyclase) remain obscure. We investigate post-RPCH binding events in perfused red ovarian chromatophores to disclose the steps modulating cGMP concentration, which regulates granule translocation. The inhibition of calcium/calmodulin complex (Ca(2+)/CaM) by N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide (W7) induces spontaneous aggregation but inhibits RPCH-triggered aggregation, suggesting a role in pigment aggregation and dispersion. Nitric oxide synthase inhibition by Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME) strongly diminishes RPCH-induced aggregation; protein kinase G inhibition (by rp-cGMPs-triethylamine) reduces RPCH-triggered aggregation and provokes spontaneous dispersion, disclosing NO/PKG participation in aggregation signaling. Myosin light chain phosphatase inhibition (by cantharidin) accelerates RPCH-triggered aggregation, whereas Rho-associated protein kinase inhibition (by Y-27632, H-11522) reduces RPCH-induced aggregation and accelerates dispersion. MLCP (myosin light chain kinase) and ROCK (Rho-associated protein kinase) may antagonistically regulate myosin light chain (MLC) dephosphorylation/phosphorylation during pigment dispersion/aggregation. We propose the following general hypothesis for the cGMP/Ca(2+) cascades that regulate pigment aggregation in crustacean chromatophores: RPCH binding increases Ca(2+)(int), activating the Ca(2+)/CaM complex, releasing NOS-produced nitric oxide, and causing GC-S to synthesize cGMP that activates PKG, which phosphorylates an MLC activation site. Myosin motor activity is initiated by phosphorylation of an MLC regulatory site by ROCK activity and terminated by MLCP-mediated dephosphorylation. Qualitative comparison reveals that this signaling pathway is conserved in vertebrate and invertebrate chromatophores alike.

Diffusible gas transmitter signaling in the copepod crustacean Calanus finmarchicus: Identification of the biosynthetic enzymes of nitric oxide (NO), carbon monoxide (CO) and hydrogen sulfide (H2S) using a de novo assembled transcriptome

“…Aonuma et al, 2000; Aonuma and Newland, 2001, 2002; Christie et al, 2003; Lee et al, 2000; Mahadevan et al, 2004; Scholz et al, 2001, 2002; Schuppe and Newland, 2004; Stein et al, 2005). In addition, there is growing evidence for the involvement of NO in the control of bioluminescence (Krönström et al, 2007), color change (Filgueira et al, 2010; Milograna et al, 2012; Vargas et al, 2008), immune response ( e.g. Fu et al, 2010; Jiang et al, 2006; Labbé et al, 2009; Rodríguez-Ramos et al, 2011; Yeh et al, 2006), molting/metamorphosis (Kim et al, 2004; Lee et al, 2007; McDonald et al, 2010; Zhang et al, 2012), and neurogenesis (Benton et al, 2007).…”

Pigment Translocation in Caridean Shrimp Chromatophores: Receptor Type, Signal Transduction, Second Messengers, and Cross Talk Among Multiple Signaling Cascades

“…VNP, an inhibitor of Ca 2+ /calmodulin‐dependent phosphodiesterase, partially inhibited RPCH‐induced pigment aggregation in M. olfersi chromatosomes. Formation of the Ca 2+ /calmodulin complex, an integral part of the aggregation cascade (Milograna et al., ), activates PDE1 that mainly hydrolyses cAMP (Oshima, ). Thus, inhibition of RPCH‐triggered aggregation results from diminished cAMP hydrolysis by PDE1 and elevated endogenous cAMP titers.…”

“…This Ca 2+ ‐binding effector stimulates nitric oxide synthase (NOS) ⑧ to synthesize nitric oxide (NO) that activates cytosolic guanylyl cyclase ⑨ to convert GTP into cGMP. This second messenger stimulates protein kinase G ⑩ that coordinates activity of the molecular motors dynein and nonmuscle myosin II ⑪ by regulating other downstream kinases and phosphatases (i.e., Rho‐associated protein kinase and myosin light chain phosphatase [Milograna et al., , ]), ultimately culminating in pigment aggregation ⑫.…”

“…Shrimps and crabs can readily change their color or tone, signaling overtly or camouflaging themselves by blending into the chromatic features of their substrates (Stevens and Merilaita, ) or exhibiting circadian rhythms of pigment movement (Thurman, ). Such chromomotor responses result from the translocation of colored pigment granules through the cytoplasm within their mono‐ or polychromatic chromatophores (McNamara, ; Bauer, ), the pigment‐bearing cells that render crustaceans an excellent model system in which to investigate the intracellular signaling cascades subjacent to color change (Milograna et al., ). Chromatophores are highly asymmetrical cells, consisting of a single cell body containing the main pigment granule mass, from which ramify a few fine cell extensions through which the granules migrate (McNamara and Taylor, ).…”

“…In caridean shrimps, RPCH binding causes an increase in both intracellular Ca 2+ (Milograna et al, 2010) and cyclic guanosine monophosphate (cGMP) levels (Ribeiro and McNamara, 2009;Milograna et al, 2012). Ca 2+ is first released from the smooth endoplasmic reticulum (SER) (Ribeiro and McNamara, 2007); an influx of extracellular Ca 2+ then follows down its gradient from the hemolymph through voltage-regulated Ca 2+ channels (Milograna et al, 2010).…”