Cellular signalling of PCH-induced pigment aggregation in the crustacean Macrobrachium potiuna erythrophores

The cellular system responsible for the transduction of the pigment-concentrating hormone (PCH) signal was investigated in erythrophores of the freshwater shrimp, Macrobrachium potiuna. Dose-response curves to the hormone were determined in the absence and in the presence of several drugs that affect sequential steps of the Ca²⁺-dependent signalling pathway. Additionally, the ability of forskolin to induce pigment dispersion was evaluated. Neomycin sulphate (10⁻⁴ and 10⁻³ mol · l⁻¹), trifluoperazine (10⁻⁵ and 10⁻⁴ mol · l⁻¹), 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (10⁻⁷ and 10⁻⁵ mol · l⁻¹) and okadaic acid (10⁻⁷ mol · l⁻¹) significantly (P<0.05) decreased the responses to PCH. However, okadaic acid at low concentration (10⁻⁹ mol · l⁻¹) and cyclosporin A (10⁻⁶ and 10⁻⁵ mol · l⁻¹) did not significantly (P>0.05) affect PCH activity. Forskolin (10⁻⁴ mol · l⁻¹) was able to half-maximally reverse the hormone-induced aggregation. Our results suggest that the pigment-concentrating hormone induces pigment aggregation through a Ca²⁺-dependent pathway with a posteriori phosphatase activation, probably the serine/threonine phosphatase 1. Accepted: 30 June 1997     

Cyclic GMP and cyclic GMP-dependent protein kinase in brown adipose tissue of developing rats

In order to ascertain the possible involvement of cyclic GMP in the physiological regulation of the function and development of brown fat of the rat, we have determined its tissue concentration in vivo under a variety of conditions. The steady-state concentration of cyclic GMP in interscapular brown adipose tissue of late foetus was about 80 pmol per g fresh weight. The concentration gradually declined during the first 2 weeks after birth to reach 40 pmol/g fresh weight and then remained constant into adulthood. The cyclic GMP content of brown fat was decreased by chemical sympathectomy and was increased after complete acclimatization of the animals to the cold. The activity of cyclic GMP-dependent protein kinase was also highest in tissue from newborn and cold-acclimatized rats.Both acute cold stress and injection of norepinephrine resulted in a significant but temporary increase in the concentration of cyclic GMP in brown fat, which was followed by a depression of the concentration below values in untreated animals. The concentration of cyclic AMP showed similar pattern of changes. Injection of phenylephrine was followed by a pronounced increase in the cyclic GMP content of brown fat, with little effect upon cyclic AMP. Injection of isoproterenol raised the content of cyclic AMP but not that of cyclic GMP. The ability of norepinephrine and phenylephrine to increase the concentration of cyclic GMP was abolished by pre-treatment of the animals with phenoxybenzamine, but not by pre-treatment with propranolol. Conversely, propranolol but not phenoxybenzamine abolished the effects of norepinephrine on the cyclic AMP content of the tissue.Thus we have established the responsiveness of the cyclic GMP content of brown fat to physiological and pharmacological stimuli and have evidence of the possible participation by cyclic GMP in the α-adrenergic stimulation and in the regulation of proliferative processes in the tissue.     

A point mutation in the pore region alters gating, Ca(2+) blockage, and permeation of olfactory cyclic nucleotide-gated channels

Upon stimulation by odorants, Ca(2+) and Na(+) enter the cilia of olfactory sensory neurons through channels directly gated by cAMP. Cyclic nucleotide-gated channels have been found in a variety of cells and extensively investigated in the past few years. Glutamate residues at position 363 of the alpha subunit of the bovine retinal rod channel have previously been shown to constitute a cation-binding site important for blockage by external divalent cations and to control single-channel properties. It has therefore been assumed, but not proven, that glutamate residues at the corresponding position of the other cyclic nucleotide-gated channels play a similar role. We studied the corresponding glutamate (E340) of the alpha subunit of the bovine olfactory channel to determine its role in channel gating and in permeation and blockage by Ca(2+) and Mg(2+). E340 was mutated into either an aspartate, glycine, glutamine, or asparagine residue and properties of mutant channels expressed in Xenopus laevis oocytes were measured in excised patches. By single-channel recordings, we demonstrated that the open probabilities in the presence of cGMP or cAMP were decreased by the mutations, with a larger decrease observed on gating by cAMP. Moreover, we observed that the mutant E340N presented two conductance levels. We found that both external Ca(2+) and Mg(2+) powerfully blocked the current in wild-type and E340D mutants, whereas their blockage efficacy was drastically reduced when the glutamate charge was neutralized. The inward current carried by external Ca(2+) relative to Na(+) was larger in the E340G mutant compared with wild-type channels. In conclusion, we have confirmed that the residue at position E340 of the bovine olfactory CNG channel is in the pore region, controls permeation and blockage by external Ca(2+) and Mg(2+), and affects channel gating by cAMP more than by cGMP.     

Formation of aerial hyphae and conidia under orthophosphate-limited conditions inNeurospora crassa: Role of repressible cyclic phosphodiesterase

A wild type strain ofNeurospora crassa produced aerial hyphae and luxuriant conidia in standing culture in low phosphate liquid media.nuc-1 andnuc-2, which have no ability to derepress repressible cyclic phosphodiesterase (cPDase) (3′; 5′-cyclic AMP 5′-nucleotidohydrolase, EC 3.1.4.17) and several other repressible enzymes, did not form them. Heterocaryon between them restored the abilities not only to produce aerial hyphae and conidia but also to produce cPDase. Revertants fromnuc-1 and a mutant in alkaline phosphatase,pho-2, produced aerial hyphae and conidia in low phosphate condition, whereas a mutant in cPDase,pho-3, produced only a limited amount of them. In media containing low levels of 2′, 3′-cAMP, the wild type, the revertants fromnuc-1, pho-2 andpho-3 produced aerial hyphae and conidia in abundance, whereas in media containing 3′, 5′-cAMP these strains produced no or only limited amounts of them. In low phosphate medianuc-1, nuc-2 andpho-3 showed higher levels of 3′, 5′-cAMP as compared with those strains which have the ability to derepress cPDase. The cPDase activities in crude mycelial extracts fromnuc-1 andpho-3 grown in low phosphate media were 5.6 and 17.5% of that ofpho-2 when assayed for 3′,5′-cAMP at an intracellular level of 2 μM.     

Inhibition of cyclic GMP formation and aggregation in Dictyostelium by the intracellular Ca2+ antagonist TMB-8

Aggregation in Dictyostelium discoideum was shown in previous studies employing EGTA to require Ca2+, but the intra- or extracellular site of action of this ion and its role in chemotaxis were not determined [1]. In this investigation we show that the intracellular Ca2+ immobilising agent TMB-8 does not affect binding of the signalling nucleotide, cAMP, to the cell surface receptors but abolishes the rapid accumulation of intracellular cGMP and subsequent chemotactic aggregation. We infer that movement of Ca2+ from membrane-bound stores is triggered by binding of cAMP to the cell-surface receptor and that this plays a primary role in stimulating cGMP formation and chemotaxis.     

Differential activation of cAMP- and cGMP-dependent protein kinases by cyclic purine and pyrimidine nucleotides

The cyclic purine nucleotides cAMP and cGMP are well-characterized second messengers and activators of PKA and PKG, respectively. In contrast, the functions of the cyclic pyrimidine nucleotides cCMP and cUMP are poorly understood. cCMP induces relaxation of smooth muscle via PKGI, and phosphodiesterases differentially hydrolyze cNMPs. Here, we report that cNMPs differentially activate PKA isoforms and PKGIα. The combination of cCMP with cAMP reduced the EC₅₀ of cAMP for PKA. PKGIα exhibited higher specificity for the cognate cNMP than PKA. Our data support a role of cCMP and cUMP as second messengers.     

Dual Role of Prostaglandins (PGE2) in Regulation of Channel Density and Open Probability of Epithelial Na+ Channels in Frog Skin (R. pipiens)

Prostaglandins are important in signaling pathways involved in modulating the rates of Na⁺ transport in a diverse group of tissues possessing apical membrane epithelial channels. PGE₂ is known to cause either stimulation, inhibition or transient stimulatory changes of Na⁺ transport. We have continued our studies of frog skins that are known to respond to forskolin and PGE₂ with large steady-state increases of transport and have used noninvasive methods of blocker-induced noise analysis of Na⁺ channels to determine their channel densities (N _T ) and open probabilities (P _o ). In the absence of exogenous hormones, baseline rates of Na⁺ transport are especially high in scraped skins (R. pipiens pipiens) studied in the fall of the year. Na⁺ transport was inhibited by indomethacin and by removal of the unstirred layers of the corium (isolated epithelia) alone suggesting that PGE₂ is responsible for the sustained and elevated rates of transport in scraped skins. Changes of transport caused by indomethacin, forskolin or PGE₂ were unquestionably mediated by considerably larger changes of N _T than compensatory changes of P _o . Since cAMP caused no change of P _o in tissues pretreated with indomethacin, PGE₂ appears in this tissue to serve a dual role, increasing the steady state N _T by way of cAMP and decreasing P _o by unknown mechanisms. Despite appreciable PGE₂-related decreases of P _o , the net stimulation of transport occurs by a considerably greater cAMP-mediated increase of N _T . Received: 28 February 1996/Revised: 22 August 1996     

Role of cyclic AMP in mitochondriogenesis in yeast

Cyclic AMP prevents the release of proteins from mitochondria (prelabelled with radioactive aminoacids) during glucose repression in yeast cells and chloramphenicol has no effect on this process. Using specific antisera, the release of the mitochondrially synthesised membrane factor of ATPase during repression and the blocking of this by cAMP has been demonstrated.     

A spring-matrix model for pigment translocation in the red ovarian chromatophores of the freshwater shrimp Macrobrachium olfersi (Crustacea, Decapoda)

A model for intracellular transport of pigment granules in the red ovarian chromatophores of the freshwater shrimp Macrobrachium olfersi is proposed on the basis of shifts in the equilibrium of resting forces acting on an elastic pigment matrix. The model describes a pigment-transport mechanism in which mechanochemical protein motors like kinesin and myosin alternately stretch and compress a structurally unified, elastic pigment matrix. Quantifiable properties of the spring-matrix obey Hooke's Law during the rapid phases of pigment aggregation and dispersion. The spring-like response of the pigment mass is estimated from previous kinetic experiments on pigment translocation induced by red pigment concentrating hormone, or by the calcium ionophore A23187. Both translocation effectors trigger an initial phase of rapid pigment aggregation, and their removal or washout after complete aggregation produces a phase of rapid pigment dispersion, followed by slow pigment translocation. The rapid-phase kinetics of pigment transport are in reasonable agreement with Hooke's Law, suggesting that such phases represent the release of kinetic energy, probably produced by the mechanochemical protein motors and stored in the form of matrix deformation during the slow phases of translocation. This semiquantitative model should aid in analyzing intracellular transport systems that incorporate an elastic component.     

A quench-flow kinetic investigation of calcium ion accumulation by isolated cardiac sarcoplasmic reticulum. Dependence of initial velocity on free calcium ion concentration and influence of preincubation with a protein kinase,MgATP, and cyclic AMP

Ca²⁺ accumulation at pH 6.8 by isolated rabbit heart microsomes derived chiefly from sarcoplasmic reticulum was investigated by a quench-flow technique. The reaction was terminated at preset times by addition to the reaction mixture of an equal volume of 10 to 50 mM ethyleneglycol-bis-(β-aminoethyl ether)-N,N′-tetraacetic acid buffered at pH 6.0. The initial velocity of Ca²⁺ accumulation by microsomal preparations exhibiting a steady state Ca²⁺ accumulation of 25.6 nmol Ca²⁺/mg increased from 3.67 to 33.4 nmol Ca²⁺/mg · s as the free Ca²⁺ concentration was raised from 0.2 to 18.9 μM. Preincubation of the cardiac microsomes with a partly purified soluble cardiac cyclic AMP-dependent protein kinase, MgATP, and cyclic AMP lead to a significant increase in the initial Ca²⁺ accumulation rate. The amounts of Ca²⁺ that were found to accumulate in the first 200 ms of the reaction are comparable to the quantities of the ion that according to literature data need to be removed from the myofilaments and the myoplasm for induction of relaxation of the myocardial fibers.     

Identification and quantification of carotenoid pigments during the embryonic development of the freshwater shrimp Macrobrachium olfersii (crustacea,decapoda)

The principal carotenoids present during the 16‐day embryonic development period and in the first post‐embryonic stage (zoea I) were identified and quantified in the diadromous, neotropical, freshwater shrimp Macrobrachium olfersii, employing differential extraction, absorption spectra analysis and thin layer chromatography. The characteristic crustacean pigments α‐ and β‐carotene and astaxanthin, and a mixture of four unidentified yellow pigments were found. Total carotenoids increase significantly at the mid‐point of embryogenesis (day 8), declining to a minimum in the recently hatched first zoea. This decrease appears to reflect the establishment of carotenoid catabolism, or of carotenoid use in protein and membrane formation and stabilization towards the end of embryogenesis, as well as the depletion of yolk protein reserves by the non‐feeding first zoea.     

Conserved role of cyclic nucleotides in the regulation of ecdysteroidogenesis by the crustacean molting gland

Molting processes in crustaceans are regulated by ecdysteroids produced in the molting gland (Y-organ), and molting is indirectly controlled by circulating factors that inhibit the production of these polyhydroxylated steroids. Two of these regulatory factors are the neuropeptides molt-inhibiting hormone (MIH) and crustacean hyperglycemic hormone (CHH). CHH appears to inhibit ecdysteroidogenesis in the Y-organ through the activation of a receptor guanylyl cyclase. The signaling pathway activated by MIH, however, remains a subject of controversy. It is clear that neuropeptides inhibit ecdysteroidogenesis by simultaneously suppressing ecdysteroid biosynthetic processes, protein synthesis, and uptake of high density lipoproteins. Data demonstrate that cAMP is the primary regulator of critical catabolic, anabolic, and transport processes, which ultimately support the capacity for ecdysteroid production by the Y-organ. While cAMP also regulates acute ecdysteroidogenesis to some extent, data indicate that cGMP is the primary signaling molecule responsible for acute inhibition by neuropeptides. It is clear that the regulatory roles filled by cAMP and cGMP are conserved among decapod crustaceans. It is unknown if these complementary second messengers are linked in a single signaling pathway or are components of independent pathways activated by different factors present in extracts of eyestalk ganglia.     

Cyclic adenosine 5′‐diphosphoribose (cADPR) cyclic guanosine 3′,5′‐monophosphate positively function in Ca2+ elevation in methyl jasmonate‐induced stomatal closure,cADPR is required for methyl jasmonate‐induced ROS accumulation NO production in guard cells

Methyl jasmonate (MeJA) signalling shares several signal components with abscisic acid (ABA) signalling in guard cells. Cyclic adenosine 5′‐diphosphoribose (cADPR) and cyclic guanosine 3′,5′‐monophosphate (cGMP) are second messengers in ABA‐induced stomatal closure. In order to clarify involvement of cADPR and cGMP in MeJA‐induced stomatal closure in Arabidopsis thaliana (Col‐0), we investigated effects of an inhibitor of cADPR synthesis, nicotinamide (NA), and an inhibitor of cGMP synthesis, LY83583 (LY, 6‐anilino‐5,8‐quinolinedione), on MeJA‐induced stomatal closure. Treatment with NA and LY inhibited MeJA‐induced stomatal closure. NA inhibited MeJA‐induced reactive oxygen species (ROS) accumulation and nitric oxide (NO) production in guard cells. NA and LY suppressed transient elevations elicited by MeJA in cytosolic free Ca²⁺ concentration ([Ca²⁺]_cyt) in guard cells. These results suggest that cADPR and cGMP positively function in [Ca²⁺]_cyt elevation in MeJA‐induced stomatal closure, are signalling components shared with ABA‐induced stomatal closure in Arabidopsis, and that cADPR is required for MeJA‐induced ROS accumulation and NO production in Arabidopsis guard cells.