Ionic requirements for catecholamine aggregating actions on the teleost Poecilia reticulata melanophores.

Norepinephrine (NE) and phenylephrine (Phe) were employed to study the ionic requirements for alpha adrenoceptor activation in the teleost Poecilia reticulata melanophores. As expected the beta adrenoceptor blocker, propranolol, increased the sensitivity of the preparation to NE (5.8 times), and was therefore employed in all the experimental procedures. Neither cocaine (a neuronal uptake blocker) nor dexamethasone (an extraneuronal uptake blocker) enhanced the sensitivity of the preparation to NE, suggesting that these inactivating mechanisms would not play a role in P. reticulata pigmentary system. However, in the absence of calcium, the dose-response curve (DRC) to NE was displaced to the left about 3.5 times, whereas the DRC to Phe was not affected. These results indicate that a neuronal uptake is active, but was not demonstrated by the classical pharmacological tools, probably due to an assymmetric display of the nervous endings. The DRC to NE was rightward displaced (14.1 times) in the presence of the calcium channel blocker Verapamil, whereas the DRC to Phe was not affected. These data suggest that P. reticulata melanophores possess a mixed population of alpha 1 and alpha 2 adrenoceptors, the activation of the latter eliciting an extracellular calcium influx. In sodium-free saline, the DRC to NE was rightward shifted (6.6 times) and the response to Phe was impaired in such a way that the maximal response was not achieved. The DRC to both NE and Phe were rightward displaced (7.9 and 2.7 times respectively) in the presence of the sodium channel blocker tetrodotoxin (TTX) 10(-7)M.(ABSTRACT TRUNCATED AT 250 WORDS)     

alpha-MSH (melanocyte stimulating hormone) and MCH (melanin concentrating hormone) actions in Bufo ictericus ictericus melanophores

1. The darkening actions of MCH (melanin concentrating hormone), alpha-MSH and the synthetic analog [Nle4, D-Phe7]-alpha-MSH on the toad, Bufo ictericus ictericus, melanophores were studied regarding the role of calcium in the hormone receptor coupling, signal transduction and intracellular pigment translocation. 2. In the absence of external calcium, MCH and both melanotropins still elicit maximal skin darkening. 3. Verapamil, a calcium-channel blocker, completely abolishes the alpha-MSH-induced response and partially inhibits MCH-induced darkening, although the calcium carrier, ionophore A23187, was unable to promote any pigment translocation. 4. Since darkening responses promoted by cyclic nucleotides proceeded normally in the presence of verapamil and extracellular calcium was not necessary for melanotropin dispersing action, it is suggested that the blocking activity obtained with verapamil is probably due to an impairment of the Ca2+-dependent adenylate cyclase activity. 5. Reversal of melanotropin-induced darkening could be obtained with melatonin, in both normal and Ca2+-free Ringer, whereas MCH darkening is reversed by melatonin only in the absence of calcium. 6. The results seem to indicate that calcium is not required for hormone receptor binding and pigment migration, whereas it is specifically needed for signal transduction.     

Melanin concentrating hormone (MCH) effects on teleost (Chrysiptera cyanea) melanophores

The in vitro biological actions of synthetic chum salmon melanin concentrating hormone (MCH) on melanophores of the blue damselfish (a teleost), Chrysiptera cyanea, were studied. This cyclic heptadecapeptide stimulated melanosome (melanin granule) aggregation (centripetal migration) within melanophores at a threshold concentration of about 10(-10) M. The action of this putative hormone was not blocked by alpha- or beta-adrenoceptor antagonists. It was concluded that the effects of MCH were direct and were not mediated indirectly through the actions of adrenergic neurotransmitters released from nerve terminals. Further evidence for this view comes from the observation that, unlike the case of neurotransmitter release, melanosome aggregation in response to MCH proceeded in the absence of calcium. The possible role of MCH in the control of color change of teleost fishes is discussed.     

Differential structural requirements for the MSH and MCH activities of melanin concentrating hormone

H-Asp-Thr-Met-Arg-Cys-Met-Val-Gly-Arg-Val-Tyr-Arg-Pro-Cys-Trp-Glu-Val-OH , melanin concentrating hormone (MCH), exhibits both melanin granule concentrating and dispersing (MSH-like) activities. Fragment analogues of MCH were synthesized as described herein and the melanotropic activities of the peptides were determined. In the frog (Rana pipiens) and lizard (Anolis carolinensis) skin bioassays, the 5-17 and 5-14 fragments of MCH were inactive (at concentrations of 10(-5)M or less), whereas the 1-14 sequence exhibited minimal (about 10%) MSH-like activity compared to MCH, which, as reported previously, was about 600 times less active than alpha-MSH. In the teleost (fish) skin bioassay, the MCH5-17 analogue was equipotent to MCH, whereas the 1-14 analogue was 10-30 times and the cyclic N- and C- terminal truncated analogue, MCH5-14, was about 300 times less active than MCH. These results suggest that the N-terminal sequence is particularly critical to MSH-like activity in the tetrapod species studied, whereas other structural regions of MCH, particularly in the C-terminal, are more related to MCH activity in teleosts.     

Ionic requirements for melanophore stimulating hormone (MSH) action on melanophores

• 1.1. Calcium is required for melanophore stimulating hormone (MSH) action on melanophores of the lizard, Anolis carolinensis, the toad, Scaphiopus couchi, and the frog Rana berlandieri forrei.
• 2.2. Ca²⁺ is required for an initial mechanism of MSH action, but not for melanosome dispersion per se, since melanophores respond to catecholamines, prostaglandins, theophylline or dibutyryl cyclic AMP in the absence of this cation.
• 3.3. Lithium, choline, rubidium and cesium, in the lizard and the toad, will replace the sodium and potassium of the Ringer medium and permit melanosome dispersion, but only if Ca²⁺ is present. These monovalent cations are irreversibly inhibitory to MSH action on melanophores of R. berlandieri and R. pipiens.
• 4.4. These results demonstrate that the Ca²⁺ is the only cation specifically required for MSH action on melanophores.

     

Melanin concentrating hormone exhibits both MSH and MCH activities on individual melanophores

Asp-Thr-Met-Arg-Cys-Met-Val-Gly-Arg-Val-Tyr-Arg-Pro-Cys-Trp-Glu-Val (melanin concentrating hormone, MCH) and several fragment analogs (MCH1-14, MCH5-17, MCH5-14) were synthesized and their biological activities determined in a very sensitive fish skin bioassay. The potency ranking and minimum effective doses of the peptides were determined to be: MCH1-17 (10(-12)M) greater than less than MCH5-17 (10(-12)M) greater than MCH1-14 (10(-11)M) greater than MCH5-14 (2 X 10(-10)M). The melanosome aggregating activity of MCH could be completely reversed by a 100-fold higher concentration of pounds-MSH. MCH was self-antagonized in a dose-related manner by higher concentrations of the peptide as was the activity of the MCH1-14 fragment analog. The MCH activities of the MCH5-17 and MCH5-14 analogs were not compromised by even the highest concentrations of the peptides employed. The MSH-like activity of MCH appears to relate to the N-terminus of the peptide whereas MCH activity is more a function of the C-terminus of the hormone. Self-antagonism of MCH at high concentrations appears to relate to the N-terminal tetrapeptide, which is responsible for the intrinsic MSH-like activity of the hormone.     

Synthesis and biological actions of melanin concentrating hormone

A melanin (melanosome) concentrating hormone, MCH, was synthesized and the methodology for its synthesis is detailed. This heptadecapeptide, H-Asp-Thr-Met-Arg-Cys-Met-Val-Gly-Arg-Val-Tyr-Arg-Pro-Cys-Trp-Glu-Val-OH , stimulated melanosome concentration (centripetal aggregation) within melanophores of all species of teleost fishes studied. Melanosome aggregation in response to MCH was not blocked by Dibenamine as was the response to norepinephrine (NE), demonstrating that melanosome aggregating responses to MCH and NE are mediated through separate receptors. Melanosome aggregation in response to MCH was reversed by an equimolar concentration of alpha-melanocyte stimulating hormone (alpha-MSH). In contrast, MCH stimulated melanosome dispersion (centrifugal movement) within melanophores of a frog (Rana pipiens) and a lizard (Anolis carolinensis). Therefore, MCH exhibits both melanosome concentrating and dispersing actions depending upon the species studied.     

Melanin concentrating hormone. III. Melanin concentrating hormone (MCH): the message sequence

Melanin concentrating hormone (MCH) is a heptadecapeptide synthesized by the hypothalamus and secreted by the neurohypophysis of the teleost pituitary gland. MCH stimulates melanosome aggregation within teleost melanocytes but also exhibits MSH-like (melanosome dispersing) activity on tetrapod (frog and lizard) melanocytes. We have synthesized a number of MCH analogues to determine the essential features of the primary structure necessary to stimulate either melanosome aggregation or dispersion in fish or tetrapod melanocytes, respectively. An analysis of the potencies and actions of these analogues on vertebrate melanocytes is provided and demonstrates that the two activities have different structural requirements.     

Identification of melanin concentrating hormone (MCH) as the natural ligand for the orphan somatostatin-like receptor 1 (SLC-1).

To identify possible ligands of the orphan somatostatin-like receptor 1 (SLC-1), rat brain extracts were analyzed by using the functional expression system of Xenopus oocytes injected with cRNAs encoding SLC-1 and G protein-gated inwardly rectifying potassium channels (GIRK). A strong inward current was observed with crude rat brain extracts which upon further purification by cation exchange chromatography and high performance liquid chromatography (HPLC) yielded two peptides with a high agonist activity. Mass spectrometry and partial peptide sequencing revealed that one peptide is identical with the neuropeptide melanin concentrating hormone (MCH), the other represents a truncated version of MCH lacking the three N-terminal amino acid residues. Xenopus oocytes expressing the MCH receptor responded to nM concentrations of synthetic MCH not only by the activation of GIRK-mediated currents but also by the induction of Ca(2+) dependent chloride currents mediated by phospholipase C. This indicates that the MCH receptor can couple either to the G(i)- or G(q)-mediated signal transduction pathway, suggesting that MCH may serve for a number of distinct brain functions including food uptake behavior.     

Melanin concentrating hormone (MCH): synthesis and bioactivity studies of MCH fragment analogues

Nineteen analogues of melanin concentrating hormone (MCH) were synthesized and tested for their skin-lightening activities in the in vitro eel skin (Synbranchus marmoratus) bioassay. All the analogues synthesized were fragments of the native sequence: Asp-Thr-Met-Arg-Cys-Met-Val-Gly-Arg-Val-Tyr-Arg-Pro-Cys-Trp-Glu-Val with sequential elimination of substituents from both the carboxy- and amino-termini. All the analogues that contained tryptophan in position 15 were found to be full agonists and equipotent to MCH. In the absence of Trp15, full agonist activity was maintained but potency was reduced ten-fold or more. The minimal fragment analogue possessing equipotency to the parent peptide, MCH, was the MCH(5-15) sequence. These observations coupled with results from work reported previously by our laboratories suggest the importance of the Trp15 residue for interaction with the MCH receptor in this assay system.     

Ionic Requirements for Catecholamine Aggregating Actions on the Teleost Poecilia reticulata Melanophores

Norepinephrine (NE) and phenylephrine (Phe) were employed to study the ionic requirements for alpha adrenoceptor activation in the teleost Poecilia reticulata melanophores. As expected the beta adrenoceptor blocker, propranolol, increased the sensitivity of the preparation to NE (5.8 times), and was therefore employed in all the experimental procedures. Neither cocaine (a neuronal uptake blocker) nor dexamethasone (an extraneuronal uptake blocker) enhanced the sensitivity of the preparation to NE, suggesting that these inactivating mechanisms would not play a role in P. reticulata pigmentary system. However, in the absence of calcium, the dose-response curve (DRC) to NE was displaced to the left about 3.5 times, whereas the DRC to Phe was not affected. These results indicate that a neuronal uptake is active, but was not demonstrated by the classical pharmacological tools, probably due to an assymmetric display of the nervous endings. The DRC to NE was rightward displaced (14.1 times) in the presence of the calcium channel blocker Verapamil, whereas the DRC to Phe was not affected. These data suggest that P. reticulata melanophores possess a mixed population of alpha₁ and alpha₂ adrenoceptors, the activation of the latter eliciting an extracellular calcium influx. In sodium-free saline, the DRC to NE was rightward shifted (6.6 times) and the response to Phe was impaired in such a way that the maximal response was not achieved. The DRC to both NE and Phe were rightward displaced (7.9 and 2.7 times respectively) in the presence of the sodium channel blocker tetrodotoxin (TTX) 10^?7M. In potassium-free saline, the melanophore sensitivity to Phe was increased, whereas the responses to NE were not affected, suggesting a differential sensitivity of the two alpha adrenoceptor subtypes to the resulting membrane hyperpolarization. Based on the literature and on our data we propose that P. reticulata melanophores possess a mixed population of alpha₁ and alpha₂ receptors. The activation of both subtypes of alpha adrenoceptors elicits a Na⁺ ion influx through TTX-sensitive sodium channels. The stimulation of alpha₂ adrenoceptors also requires an extracellular calcium influx, through the opening of slow calcium channels.     

Bicyclo[3.1.0]hexyl urea melanin concentrating hormone (MCH) receptor-1 antagonists: impacting hERG liability via aryl modifications

Herein, we report the discovery of an effective strategy to modulate liabilities related to affinity of previously disclosed bicyclohexane MCHR-1 antagonists for the hERG channel. This paper describes one of several strategies incorporated to limit hERG binding via modifications of a terminal aryl group in an otherwise promising bicyclohexyl urea series.     

Gametic isolation in guppies (Poecilia reticulata)

Post-mating reproductive isolating mechanisms may be among the earliest reproductive barriers to emerge among incipient species. Trinidadian guppy, Poecilia reticulata, populations in the Caroni and Oropouche drainages in Northern Trinidad exhibit marked genetic divergence and provide an ideal system in which to search for these barriers. We inseminated virgin females with equal amounts of sperm from two males, a 'native' male from the female's own population and a 'foreign' male from the other drainage. Artificial insemination ensured that mating order and mate choice did not affect the outcome. Paternities were assigned to the resulting broods using microsatellite markers. As predicted, sperm from native males had precedence over foreign sperm. Moreover, this effect was symmetrical for both drainages. In contrast, we detected no native sperm precedence in controls, in which females received sperm from the same and another population within the same drainage. Our results show that gametic isolation can arise between geographically proximate, though genetically divergent, populations of a single species and highlight the potential role of this process in speciation.     

Melanin concentrating hormone (MCH): structure-function aspects of its melanocyte stimulating hormone-like (MSH-like) activity

Melanin concentrating hormone (MCH) is a heptadecapeptide, Asp-Thr-Met-Arg-Cys-Met-Val-Gly-Arg-Val-Tyr-Arg-Pro-Cys-Trp-Glu-Val, synthesized in the brain and secreted from the pars nervosa of teleost fish. This hormone stimulates melanosome (melanin granule) aggregation within integumental melanocytes of fishes but, in contrast, stimulates melanosome dispersion within tetrapod (frog and lizard) melanocytes. We determined the message sequence of the primary structure of MCH which is responsible for its MSH-like component of activity. Removal of the N-terminal amino acid results in an almost total loss of MSH-like activity. The C-terminal amino acid is also essential for full MSH-like activity since the analogue, MCH(1-16), is about 100 times less active than MCH. Therefore, the entire heptadecapeptide sequence of MCH appears to contribute to the MSH-like activity of MCH. Ring-contracted analogues (e.g., [Ala5, Cys10]MCH) of MCH are almost devoid of any melanosome aggregating (MCH-like) activity but generally possess considerable or as great an MSH-like activity as MCH. Racemization of MCH by heat-alkali treatment drastically reduces the MCH-like activity of MCH, but does not enhance the MSH-like activity of the hormone.     

Structure of the female gonoduct of the viviparous teleost Poecilia reticulata (Poeciliidae) during nongestation and gestation stages

Female teleosts do not have oviducts because Müllerian ducts do not develop. Instead, the caudal region of the ovary, the gonoduct, connects to the exterior. Because of the lack of oviducts in viviparous teleosts, the embryos develop in the ovary, as an intraovarian gestation, unique in vertebrates. This is the first study to address the histology of the gonoduct in a viviparous teleost. The gonoduct of Poecilia reticulata was analyzed during previtellogenesis, vitellogenesis, and gestation. The gonoduct lacks germinal cells. From deep to superficial, the wall has simple cuboidal or columnar epithelium, loose connective tissue, longitudinal layer of smooth muscle, and visceral peritoneum. Cells of the immune system occur in the lumen and in the mucosa. The gonoduct was divided in three regions: 1) cephalic, 2) middle, and 3) caudal. At the initial part of each region, thin mucosal folds extend into the lumen. The cephalic region forms a tubular structure with light and irregular folds. The middle region has a wider lumen and is more irregular due to ventral invaginations and irregular and short mucosal folds; beneath the epithelium there are melano‐macrophage centers. The caudal region is delimited from the middle region by folds; however, they are thinner than these of the other regions. Ventral invaginations form exocrine glands, and the smooth muscle is thicker than in the other regions. During gestation, cells of the immune system are abundant; melano‐macrophage centers become larger and the glands exhibit desquamated cells. These observations suggest roles of the gonoduct in reducing the diameter of the lumen; receiving sperm during vitellogenesis; producing secretions, more abundant during vitellogenesis; and in immunological activity throughout the reproductive cycle. The ciliated epithelium and the thick muscle of the caudal region may be involved during birth. J. Morphol. 275:247–257, 2014. © 2013 Wiley Periodicals, Inc.     

Aerial Jumping in the Trinidadian Guppy (Poecilia reticulata)

Many fishes are able to jump out of the water and launch themselves into the air. Such behavior has been connected with prey capture, migration and predator avoidance. We found that jumping behavior of the guppy Poecilia reticulata is not associated with any of the above. The fish jump spontaneously, without being triggered by overt sensory cues, is not migratory and does not attempt to capture aerial food items. Here, we use high speed video imaging to analyze the kinematics of the jumping behavior P. reticulata. Fish jump from a still position by slowly backing up while using its pectoral fins, followed by strong body trusts which lead to launching into the air several body lengths. The liftoff phase of the jump is fast and fish will continue with whole body thrusts and tail beats, even when out of the water. This behavior occurs when fish are in a group or in isolation. Geography has had substantial effects on guppy evolution, with waterfalls reducing gene flow and constraining dispersal. We suggest that jumping has evolved in guppies as a behavioral phenotype for dispersal.     

Female preference for blue in Japan blue guppies (Poecilia reticulata)

Guppies (Poecilia reticulata) are widely used as a model species in mate choice studies. Although native to South America, guppies have been introduced to natural water bodies in disparate regions of the globe. Here, for the first time, we examine guppies from one such introduced population in Japan where males have evolved a predominantly blue color pattern. Previous studies of wild-type guppies have shown blue to play a relatively minor role in the mate choice decisions of females compared to other traits, such as orange, and the importance of blue is not universally supported by all studies. The Japanese population therefore presents an ideal opportunity to re-examine the potential significance of blue as a mate choice cue in guppies. Mate choice experiments, in which female Japan blue guppies were given a choice between pairs of males that differed in their area of blue coloration but were matched for other traits, revealed that females prefer males with proportionately larger amounts of blue in their color patterns. We discuss possible factors, including sexual and ecological selection, which may have led to the evolution of unusually large areas of blue at the expense of other colors in Japan blue guppies. However, further studies are needed to distinguish between these scenarios.     

Observational Learning and Predator Inspection in Guppies (Poecilia reticulata)

Prior work has demonstrated that, following a predator inspection visit of their own, guppies prefer to associate with individuals who inspected a predator most closely. Based on this work, as well as studies of social learning in the context of mate choice, we predicted that male guppies that observed but did not participate in an inspection trial would subsequently choose to associate with the closer of two inspectors. Our experimental protocol consisted of three treatments: a control test in which an observer watched two fish consecutively, only one of which was exposed to a predator, a sequential test in which an observer watched two fish consecutively, both of which were exposed to the predator, and a social test in which an observer watched two fish inspect simultaneously. We found no preferences by the observer for either of the fish in any of the trials. Our results suggest that direct interaction is a critical component to the development of preferences in male guppies. We discuss our findings in light of game theoretical treatments of cooperation.