Red Pigment-concentrating Hormone Research Articles

A high-resolution in vitro bioassay to identify neurons containing red pigment concentrating hormone

The release of red pigment concentrating hormone (RPCH) by single peptidergic neurons of the crayfish X organ/sinus gland system (XO-SG) was demonstrated using a novel in vitro bioassay in which XO neurons were co-cultured with tegumentary erythrophores. Local retraction of the pigmentary matrix within filipodia from erythrophores plated next to presumptive RPCH-containing neurons suggest spontaneous hormone release. Topical application of synthetic RPCH onto long filipodia also produced a local response. The time course of pigmentary matrix aggregation depended on the dose of synthetic RPCH. The effect of peptide on the cultured target cells was blocked by a polyclonal antiserum against RPCH. In co-culture conditions, the time course of pigmentary matrix aggregation was accelerated when presumptive RPCH-containing neurons were depolarized by intracellular current injection or by voltage-clamping to activate the Ca2+ current. The aggregation response evoked by these maneuvers was similar to that obtained with synthetic RPCH at a concentration of 1 fmol l-1. The immune serum was also used to identify a subset of 3-7 immunoreactive neurons localized in the external rim of the XO close to the medulla interna. Under culture conditions, this subset of neurons corresponded to the cells that induced the erythrophore response.

Sequential developmental acquisition of neuromodulatory inputs to a central pattern-generating network.

The activity of the adult stomatogastric ganglion (STG) depends on a large number of aminergic and peptidergic modulatory inputs. Our aim is to understand the role of these modulatory inputs in the development of the central pattern-generating networks of the STG. Therefore, we analyze the developmental and adult expressions of three neuropeptides in the stomatogastric nervous system of the lobsters Homarus americanus and Homarus gammarus by using wholemount immunocytochemistry and confocal microscopy. In adults, red pigment-concentrating hormone (RPCH)-like, proctolin-like, and a tachykinin-like immunoreactivity are present in axonal projections to the STG. At 50% of embryonic development (E50), all three peptides stain the commissural ganglia and brain, but only RPCH- and proctolin-like immunoreactivities stain axonal arbors in the STG. Tachykinin-like immunoreactivity is not apparent in the STG until larval stage II (LII). The RPCH-immunoreactive projection to the STG consists of two pairs of fibers. One pair stains for RPCH immunoreactivity at E50; the second RPCH-immunoreactive pair does not stain until about LII. One pair of the RPCH fibers double labels for tachykinin-like immunoreactivity. The adult complement of neuromodulatory inputs is not fully expressed until close to the developmental time at which major changes in the STG motor patterns occur, suggesting that neuromodulators play a role in the tuning of the central pattern generators during development.

Characterization of Chromatophorotropic Neuropeptides from the Kuruma PrawnPenaeus japonicus

Three chromatophorotropic neuropeptide hormones were purified from an aqueous extract of the sinus glands of the kuruma prawnPenaeus japonicusby two steps of reverse-phase HPLC and their amino acid sequences determined. One of them was found to show pigment concentrating activity and to have an amino acid sequence identical with that of the known red pigment concentrating hormone (RPCH), and therefore it was named Pej-RPCH. The other two peptides showed pigment dispersing hormone (PDH) activity and were named Pej-PDH-I and -II. They both consisted of 18 amino acid residues with a free amino-terminus and an amidated carboxyl-terminus, the sequences of Pej-PDH-I and -II being NSELINSLLGIPKVMTDAamide and NSELINSLLGLPKFMIDAamide, respectively. Three amino acid residues at positions 11, 14, and 16 differed between the two PDHs. Pej-PDH-II was about 5-, 7-, and 10-fold more potent than Pej-PDH-I for erythrophores, xanthophores, and melanophores, respectively. The major reason for the difference in potency between the two PDHs was attributed to differences in residues at position 16. In addition, they were found to be produced by a single individual. The order of sensitivity of the four types of chromatophores to Pej-RPCH and both PDHs was found to be erythrophores = xanthophores > melanophores > leukophores.

Kinetic characterization of pigment migration and the role of the cytoskeleton in granule translocation in the red chromatophores of the shrimpMacrobrachium olfersii (Crustacea, Decapoda)

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. J. Exp. Zool. 283:19–30, 1999. © 1999 Wiley-Liss, Inc.

Peptidergic Neurons in Barnacles: An Immunohistochemical Study Using Antisera Raised Against Crustacean Neuropeptides.

Antisera raised against neuropeptides from decapod crustaceans were used to investigate whether balanomorph barnacles produce peptides analogous to those identified in some decapods. The distribution and structure of immunoreactive neurons was examined in Balanus balanus, Balanus perforatus, and Chirona (Balanus) hameri by whole-mount immunohistochemistry. In these species, no immunoreactivity was observed to antisera against CHH (crustacean hyperglycemic hormone), MIH (molt-inhibiting hormone), or RPCH (red-pigment-concentrating hormone), but neurons immunoreactive for pigment-dispersing hormone (PDH) and crustacean cardioactive peptide (CCAP) were observed. In all three species, PDH immunoreactivity was primarily associated with a pair of large (30-50 {mu}m diam.) anterio-ventral perikarya in the ventral ganglion, projecting prominent axons along the great splanchnic nerves, which branched extensively in the segmental splanchnic nerves, directing several arborizing dendrites to the somatic extensor muscles. Occasionally, three pairs of anterio-dorsal perikarya were observed, which projected fine ipsilateral and contralateral axons along the great splanchnic nerves. A further 12 pairs of perikarya, apparently segmentally arranged, were observed in the thoracic ganglion. Several PDH-immunoreactive perikarya and associated branching plexus were observed in the supra-esophageal ganglion. CCAP immunoreactivity was mainly restricted to the ventral ganglion, where three pairs of perikarya (ca. 30-50 {mu}m diam.) projected contralateral descending axons to the cirri. Occasionally a single pair of immunoreactive neurons were observed in the supra-esophageal ganglia. Although the anatomy of the CCAP-immunoreactive neurons in the ventral ganglion of barnacles might be homologous to conserved neural architectures in higher crustaceans, the anatomy of the PDH-immunoreactive neurons seems unique, and the morphology of the two large neurons in the ventral ganglion suggests a neuromodulatory role for this peptide, possibly associated with somatic extension.

Organization of the stomatogastric neuropil of the crab, Cancer borealis, as revealed by modulator immunocytochemistry.

We used antibodies to a number of neuromodulatory substances, including serotonin, FLRF amide, red pigment-concentrating hormone, substance P, proctolin and cholecystokinin, to investigate the distribution of molecules similar to these substances in the stomatogastric ganglion of the crab, Cancer borealis. No immunoreactivity was seen in the region of the cell bodies that surrounds the neuropil and little was found in the core of the neuropil (where the primary neurites of the intrinsic neurons occupy most of the space). Instead, modulator immunolabel was densely packed in the more peripheral portion of the neuropil that surrounded the core. Within this peripheral neuropil, profiles appeared quite uniformly distributed. Double-labeling showed that there were limited differences in distribution between the labels examined in our study. The only immunolabeled structures that showed a distinct differential distribution within the stomatogastric neuropil were a population of >/=10 microm varicosities that arose from a pair of input fibers that we termed the large varicosity fibers. These varicosities were immunolabelled by antisera for three different peptides. Taken collectively, these data shows that there is a stereotyped distribution of modulator immunoreactivity within the crab stomatogastric neuropil. However, this segregation is more rudimentary than that reported for the intrinsic stomatogastric neurons.

Neurotransmitter interactions in the stomatogastric system of the spiny lobster: one peptide alters the response of a central pattern generator to a second peptide.

Two of the peptides found in the stomatogastric nervous system of the spiny lobster, Panulirus interruptus, interacted to modulate the activity of the cardiac sac motor pattern. In the isolated stomatogastric ganglion, red-pigment-concentrating hormone (RPCH), but not proctolin, activated the bursting activity in the inferior ventricular (IV) neurons that drives the cardiac sac pattern. The cardiac sac pattern normally ceased within 15 min after the end of RPCH superfusion. However, when proctolin was applied within a few minutes of that time, it was likewise able to induce cardiac sac activity. Similarly, proctolin applied together with subthreshold RPCH induced cardiac sac bursting. The amplitude of the excitatory postsynaptic potentials from the IV neurons to the cardiac sac dilator neuron CD2 (1 of the 2 major motor neurons in the cardiac sac system) was potentiated in the presence of both proctolin and RPCH. The potentiation in RPCH was much greater than in proctolin alone. However, the potentiation in proctolin after RPCH was equivalent to that recorded in RPCH alone. Although we do not yet understand the mechanisms for these interactions of the two modulators, this study provides an example of one factor that can determine the "state" of the system that is critical in determining the effect of a modulator that is "state dependent," and it provides evidence for yet another level of flexibility in the motor output of this system.

Roles of neurotransmitters in regulating reproductive hormone release and gonadal maturation in decapod crustaceans

Summary Experiments done in this laboratory showed 5-hydroxytryptamine (5-HT) stimulates gonadal maturation in male and female sand fiddler crabs, Uca pugilator, and red swamp crayfish, Procambarus clarkii. This action of 5-HT is indirect, 5-HT apparently stimulating release of the gonad-stimulating hormone (GSH) that is present in the brain and thoracic ganglia. For example, studies with ovarian explants showed 5-HT has no direct effect on the ovary. But, when ovarian explants were incubated with 5-HT and brain or thoracic ganglia, the incubation medium produced greater ovarian maturation than did the medium when ovarian explants were incubated with brain or thoracic ganglia alone, 5-HT presumably enhancing GSH release. In males 5-HT not only induces testicular maturation but also development of the androgenic glands. 5-HT in males, as in females, apparently triggers GSH release; but in males GSH in turn stimulates the androgenic glands which release the androgenic gland hormone, resulting in testicular maturation. In contrast, dopamine (DA) inhibits gonadal maturation in both sexes. Methionine enkephalin, like DA, slows ovarian maturation. Red pigment-concentrating hormone (RPCH), like 5-HT, stimulates ovarian maturation both in vivo and in vitro, apparently by stimulating GSH release. RPCH does not affect the ovary directly. Calcium appears to act here as a second messenger for RPCH. The implications of these findings for enhancing the culture of crustaceans is discussed.

Seasonal Rhythm of Red Pigment Concentrating Hormone in the Crayfish

The content of red pigment concentrating hormone (RPCH) in the eye-stalk of the crayfish Procambarus clarkii varies seasonally, with maximum values during the summer months and the lowest values in winter. The responsiveness of tegumentary chromatophores to synthetic RPCH varies concurrently. (Chronobiology International, 14(6), 639–645, 1997)

Hydrolysis of Insect Neuropeptides by an Angiotensin-Converting Enzyme From the Housefly, Musca domestica

Lamango, N. S., R. J. Nachman, T. K. Hayes, A. Strey and R. E. Isaac. Hydrolysis of insect neuropeptides by an angiotensin-converting enzyme from the housefly, Musca domestica. Peptides 18(1) 47–52, 1997.—The presence in insect tissues of peptides with structural similarities to angiotensin I and to bradykinin, the two best known substrates of mammalian angiotensin-converting enzyme, has not been reported. As part of our study to identify potential substrates for insect angiotensin-converting enzyme, we have investigated the susceptibility of a number of known insect peptide hormones and neurotransmitters to hydrolysis by Musca domestica angiotensin-converting enzyme. Insect peptides belonging to the red pigment-concentrating hormone, leucokinin, locust tachykinin, and depolarizing peptide families were hydrolyzed by housefly angiotensin-converting enzyme, whereas proctolin and crustacean cardioactive peptide were not substrates. Cus-DP II, LK I, LK II, and Lom-TK I were all cleaved at the penultimate C-terminal peptide bond to release a dipeptide amide as a major fragment with K m values of 94 ± 11, 634 ± 81, and 296 ± 35 μM for Cus-DP II, LK I, and Lom-TK I, respectively. The ability of insect angiotensin-converting enzyme to hydrolyze C-terminally amidated peptides in vitro might be of functional significance because the enzyme has been localized to neuropile regions of the insect brain and is present in the hemolymph of houseflies.

Modulation of crayfish retinal function by red pigment concentrating hormone

The role of the crustacean octapeptide red pigment concentrating hormone (RPCH) in the control of crayfish retinal activity was explored. RPCH injection into intact animals resulted, after a latency of 10­30 min, in a dose-dependent enhancement of electroretinogram (ERG) amplitude lasting 60­120 min. RPCH was able to potentiate ERG amplitude in both light-adapted and dark-adapted animals. Following light-adaptation, responsiveness to RPCH was five times higher than following dark-adaptation. In conjunction with ERG enhancement, in light-adapted animals, RPCH injection elicited a dose-dependent retraction of distal retinal pigment, but did not affect proximal retinal pigment position. The effects of RPCH were blocked by a polyclonal antibody raised against a tyrosinated form of RPCH (A-tyr-RPCH). The antibody was also capable of partially blocking the nocturnal phase of the circadian rhythm of ERG amplitude and the darkness-induced retraction of distal retinal pigment. These results suggest that RPCH acts both on the retinal photoreceptors and on the distal pigment cells, playing a physiological role as a mediator of the effects induced by darkness and by the nocturnal phase of the circadian rhythm.

A Highly Conserved Red Pigment-Concentrating Hormone Precursor in the Blue Crab Callinectes Sapidus

A cDNA library was established from the eyestalk ganglia of the blue crab Callinectes sapidus. One clone was isolated (644 bp excluding the poly(A) tail) which encodes the red pigment-concentrating hormone (RPCH)-precursor, consisting of the 25 amino acid residue signal peptide, the RPCH, and a 73 amino acid residue RPCH-precursor related peptide. This clone displays high sequence similarity with a clone isolated from an eyestalk cDNA library of the shore crab Carcinus maenas, in accordance with the close phylogenetic relationship between these species. Northern blot experiments indicated the presence of two different mRNA transcripts which hybridized with a specific RPCH-cDNA probe pointing to the possibility of multiple RPCH isoforms in the blue crab. Although crustacean RPCH and the insect adipokinetic hormones (AKH) are structurally related, their precursors show little similarity.

A neurotransmitter role for red-pigment-concentrating hormone in ovarian maturation in the red swamp crayfish Procambarus clarkii

The influence of red-pigment-concentrating hormone (RPCH) on ovarian maturation in the red swamp crayfish Procambarus clarkii was studied using both in vivo and in vitro techniques. In vivo, RPCH stimulated ovarian maturation. However, RPCH did not affect the ovary in vitro when only RPCH, muscle and ovarian explants were used. But when RPCH, thoracic ganglia, which are known to contain gonad-stimulating hormone-like (GSH-like) activity, and ovarian explants were incubated together, significant ovarian maturation ensued. The calcium ionophore A23187 mimicked RPCH both in vivo and in vitro. These results provide evidence to support the hypothesis that RPCH has a role as a neurotransmitter in Procambarus clarkii to stimulate GSH release, with calcium acting as a second messenger for RPCH.

Identification and expression of the Drosophila adipokinetic hormone gene

Drosophila adipokinetic hormone (DAXH) is an eight amino acid member of a large arthropod neuropeptide family. The gene encoding the peptide precursor has been identified and sequenced providing an inferred precursor structure of 79 amino acids including a 46 amino acid carboxy-terminal fragment of unknown function. In situ hybridization identifies sites of DAKH synthesis towards the base of the third larval instar ring gland. Like other RPCH (red pigment concentrating hormone)/AKH family peptides, DAKH can act as a cardioaccelerator at least in prepupae. Peptide levels measured in wildtype and mutant flies possessing one or three copies of the DAKH gene suggest that the amount of neuropeptide per fly is tightly regulated.

Neuronal pathways of classical crustacean neurohormones in the central nervous system of the woodlouse, Oniscus asellus (L.)

Neuropeptide-immunoreactive neurons have been mapped by immunocytochemistry in whole-mount preparations and sections of the central nervous system of Oniscus asellus . We tested rabbit antisera against decapod crustacean hyperglycemic hormone (CHH), moult inhibiting hormone (MIH ), pigment dispersing hormone (PDH) and red pigment concentrating hormone (RPCH). four CHH- and three PDH-immunoreactive neurons localized in the superior median protocerebrum of the brain constitute neurosecretory pathways to the neurohaemal sinus gland. No immunoreactive structures have been detected with an antiserum against MIH of Carcinus maenus . Another, newly identified neurosecretory pathway is formed by a group of RPCH-immunoreactive neurons in the mandibular ganglion. These neurons project to the neurohaemal lateral cephalic nerve plexus, further PDH- and RPCH-immunoreactive neurons and fibres occur in the brain and the ventral nerve cord (VNC). Two groups of PDH-immunoreactive neurons supply brain and optic lobe neuropils, the bases of the ommatidia, and probably give rise to descending fibres innervating all VNC-neuropils. Two groups and five individuals of RPCH-immunoreactive neurons that innervate several brain neuropils or occur as ascending neurons in the VNC have been reconstructed. The CHH-immunoreactive neurons, and distinct types of PDH- and RPCH-immunoreactive neurons obviously belong to classical hormone-producing neurosecretory pathways. At least the CHH-immunoreactive cells seem to be part of an isopod homologue of the decapod X-organ. The existence of other PDH- and RPCH-immunoreactive interneurons suggests additional functions of these peptides as neurotransmitters or neuromodulators, which is in agreement with similar observations in the decapod central nervous system.

Circadian rhythm of content of red pigment-concentrating hormone in the crayfish eyestalk

The content of red pigment-concentrating hormone (RPCH) was determined in the crayfish eyestalk at different times of day and various conditions of illumination. Two types of preparations were used: (a) eyestalks removed from otherwise intact animals; and (b) isolated eyestalks kept in organ culture. The RPCH content was determined under (a) 12:12 hr light:dark cycles, (b) continuous darkness, and (c) continuous illumination under 500 Ix. In all three conditions, RPCH content varied in a 24-hr rhythm. Under LL and DD, the period deviated from 24 hr both in intact animals and in isolated eyestalks. The rhythm amplitude ranged from 2 to 3 log units of RPCH content, depending on the experimental conditions, being highest in the intact animals under LD (1200 pg at night to 2 pg in daytime), and the lowest in the isolated eyestalks under DD (400 pg at night to 6 pg in daytime). These results suggest that the isolated eyestalk is capable of generating a self-sustaining circadian rhythm of RPCH secretion, with similar properties to those in the intact animal.

Circadian rhythm of content of red pigment-concentrating hormone in the crayfish eyestalk

The content of red pigment-concentrating hormone (RPCH) was determined in the crayfish eyestalk at different times of day and various conditions of illumination. Two types of preparations were used: (a) eyestalks removed from otherwise intact animals; and (b) isolated eyestalks kept in organ culture. The RPCH content was determined under (a) 12:12 hr light:dark cycles, (b) continuous darkness, and (c) continuous illumination under 500 Ix. In all three conditions, RPCH content varied in a 24-hr rhythm. Under LL and DD, the period deviated from 24 hr both in intact animals and in isolated eyestalks. The rhythm amplitude ranged from 2 to 3 log units of RPCH content, depending on the experimental conditions, being highest in the intact animals under LD (1200 pg at night to 2 pg in daytime), and the lowest in the isolated eyestalks under DD (400 pg at night to 6 pg in daytime). These results suggest that the isolated eyestalk is capable of generating a self-sustaining circadian rhythm of RPCH secretion, with similar properties to those in the intact animal.

Molecular Cloning of Crustacean Red Pigment Concentrating Hormone Precursor

A cDNA encoding the precursor of the red pigment concentrating hormone (RPCH) of the shore crab, Carcinus maenas, was isolated and sequenced. The predicted preprohormone sequence consists of a putative 25 amino acid signal peptide, the eight amino acid RPCH sequence followed by a Gly as amide donor, a dibasic processing site and a 74 residue peptide of unknown function, referred to as RPCH-precursor-related peptide (RPRP). In in situ hybridisation experiments, the RPCH gene transcript could be localised in two clusters of neurons in the medulla terminalis of the eyestalk.

The neuropeptide red pigment concentrating hormone affects rhythmic pattern generation at multiple sites.

1. The cardiac sac network, which controls the rhythmic contractions of the cardiac sac in the foregut of crustaceans, is distributed throughout the stomatogastric nervous system, including the oesophageal ganglion (OG), the commissural ganglia (CGs), and the stomatogastric ganglion (STG). A red pigment-concentrating hormone (RPCH)-like peptide is likewise widely distributed. 2. The effects that bath application of the neuropeptide RPCH to the different ganglia has on the cardiac sac pattern were studied. 3. RPCH applied to the STG, the OG, or the CGs elicited bursting activity in all the known components of the cardiac sac pattern, including the two motor neurons, cardiac sac dilators 1 and 2 (CD1 and CD2), and the inferior ventricular nerve (ivn) fibers. 4. A cardiac sac pattern was also elicited when RPCH was applied to either the STG, the OG, or the CGs after synapses in that ganglion had been blocked by low Ca2+ saline containing 20 mM Co2+. 5. These data suggest that the ivn fibers are sensitive to RPCH and respond to it by generating bursting activity at or near their terminals in all four ganglia. 6. Application of RPCH to either the STG or the OG also caused an increase in the amplitude of the postsynaptic potential (PSP) from the ivn fibers to both CD1 and CD2. The increase was largest in the ganglion to which the RPCH was applied. 7. Repeated stimulation of the ivn, mimicking the bursts that occur during cardiac sac activity, also caused an increase in PSP amplitude, and so facilitation resulting from activation of ivn bursting could account for a portion of the increased amplitude seen in RPCH.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparative Activities of the Chromatophorotropins RPCH, α-PDH, and β-PDH on Three Crustacean Species

We determined the potencies of the pigment-dispersing hormones, α-PDH and β-PDH, and the red pigment-concentrating hormone, RPCH, by the standard activity method in the marine isopod Ligia exotica, the fiddler crab Uca rapax, and the freshwater shrimp Macrobrachium acanthurus. In U. rapax, both PDHs exhibited the same dose-dependent activity, maximal dispersion being achieved at 3.0 pmol/animal; in L. exotica, α-PDH had a dose-related effect, with maximal pigment dispersion at 100 pmol/animal, and only a partial, dose-independent dispersion was elicited by β-PDH; in M. acanthurus, α-PDH caused a dose-dependent pigment dispersion, with maximal response at 3,000 pmol/animal, whereas β-PDH had no effect. There seems to be more similarity between L. exotica and Pandalus borealis PDH receptors than between L. exotica and U. rapax receptors, suggesting an evolutionary convergence of the dispersing chromatophorotropins and their receptor molecules. Concerning RPCH, no pigment aggregation was observed in melanoph...