Localization of nitric oxide synthase-like immunoreactivity in the developing nervous system of the snail Ilyanassa obsoleta

Production of nitric oxide (NO), an evolutionarily conserved, intercellular signaling molecule, appears to be required for the maintenance of the larval state in the gastropod mollusc Ilyanassa obsoleta. Pharmacological inactivation of endogenous nitric oxide synthase (NOS), the enzyme that generates NO, can trigger metamorphosis in physiologically competent larvae of this species. Neuropils in the brains of these competent larvae display histochemical reactivity for NADPH diaphorase (NADPHd), an indication of neuronal NOS activity. The intensity of NADPHd staining is greatest in the neuropil of the apical ganglion (AG), a region of the brain that contains the apical sensory organ and that innervates the bilobed ciliated velum, the larval swimming and feeding organ. Once metamorphosis is initiated, the intensity of NADPHd staining in the AG and presumably, concomitant NO production, decline. The AG is finally lost by the end of larval metamorphosis, some 4 days after induction. To determine if the neurons of the AG are a source of larval NO, we conducted immunocytochemical studies on larval Ilyanassa with commercially available antibodies to mammalian neuronal NOS. We localized NOS-like immunoreactivity (NOS-IR) to 3 populations of cells in competent larvae: somata of the AG and putative sensory neurons in the edge of the mantle and foot. Immunocytochemistry on pre-competent larvae demonstrated that numbers of NOS-IR cells in the AG increase throughout the planktonic larval stage.     

Nitric oxide inhibits metamorphosis in larvae of Crepidula fornicata, the slippershell snail

This paper concerns the role of nitric oxide (NO) in controlling metamorphosis in the marine gastropod Crepidula fornicata. Metamorphosis was stimulated by the nitric oxide synthase (NOS) inhibitors AGH (aminoguanidine hemisulfate) and SMIS (S-methylisothiourea sulfate) at concentrations of about 100-1000 micromol l(-1) and 50-200 micromol l(-1), respectively. Metamorphosis was not, however, induced by the NOS inhibitor l-NAME (l-N(G)-nitroarginine methyl ester) at even the highest concentration tested, 500 micromol l(-1). Moreover, pre-incubation with l-NAME at 20 and 80 micromol l(-1) did not increase the sensitivity of competent larvae to excess K(+), a potent inducer of metamorphosis in this species; we suggest that either l-NAME is ineffective in suppressing NO production in larvae of C. fornicata, or that it works only on the constitutive isoform of the enzyme. In contrast, metamorphosis was potentiated by the guanylate cyclase inhibitor ODQ (1H-[1,2,4]oxadiazolo[4,3, -a]quinoxalin-1-one) in response to a natural metamorphic inducer derived from conspecific adults. Because NO typically stimulates cGMP production through the activation of soluble guanylate cyclase, this result supports the hypothesis that NO acts as an endogenous inhibitor of metamorphosis in C. fornicata. The expression of NOS, shown by immunohistochemical techniques, was detected in the apical ganglion of young larvae but not in older larvae, further supporting the hypothesis that metamorphosis in C. fornicata is made possible by declines in the endogenous concentration of NO during development.     

Timing is everything: the effects of putative dopamine antagonists on metamorphosis vary with larval age and experimental duration in the prosobranch gastropod Crepidula fornicata

The signal transduction pathway through which excess potassium ion stimulates the larvae of many marine invertebrates to metamorphose is incompletely understood. Recent evidence suggests that dopamine plays important roles in the metamorphic pathway of Crepidula fornicata. Therefore, we asked whether blocking dopamine receptors might prevent excess potassium ion from stimulating metamorphosis in this species. Surprisingly, the effects of the three putative dopamine antagonists tested (all at 10 microM) varied with exposure duration and the age of competent larvae. Chlorpromazine, a nonspecific dopamine antagonist known to have a number of other pharmacological effects, blocked the inductive action of excess potassium ion during the initial 5-8-h exposure periods in most assays, particularly for younger or smaller competent larvae. However, chlorpromazine in the absence of excess potassium ion also stimulated metamorphosis, particularly over the next 18 h, and worked faster on older competent larvae than on younger competent larvae. The specific D(1) antagonist R(+)-Sch-23309 had similar effects, blocking potassium-stimulated metamorphosis in short-term exposures and stimulating metamorphosis in longer exposures, particularly for older competent larvae. Although the specific D(2) antagonist spiperone (SPIP) blocked the inductive effects of excess potassium ion in only 1 of 6 assays during the first 6 h of exposure, it blocked metamorphosis in 2 of the assays during 24-h exposures. Our results indicate that dopamine receptors are involved in the pathway through which excess potassium ion stimulates metamorphosis in C. fornicata. In addition, the largely latent inductive effects of chlorpromazine, an inhibitor of nitric oxide synthase, suggest that endogenous nitric oxide may play a natural role in inhibiting metamorphosis in this species. Overall, our results would then suggest that exposing larvae of C. fornicata to excess K(+) leads to a shutdown of nitric oxide synthesis via a dopaminergic pathway, a pathway that can be blocked by some dopamine antagonists. Alternatively, chlorpromazine might eventually be stimulating metamorphosis by elevating endogenous cyclic nucleotide (e.g., cAMP) concentrations, again acting downstream from the steps acted on directly by excess K(+).     

Nitric Oxide Regulates Shikonin Formation in Suspension-Cultured Onosma paniculatum Cells

Endogenously occurring nitric oxide (NO) is involved in theregulation of shikonin formation in Onosma paniculatum cells.NO generated after cells were inoculated into shikonin productionmedium reached the highest level after 2 d of culture, whichwas 16 times that at the beginning of the experiment, and maintaineda high level for 6 d. A nitric oxide synthase (NOS) inhibitor,N-nitro-L-arginine (L-NNA), and a nitrate reductase (NR) inhibitor,sodium azide (SoA), consistent with their inhibition of NO biosynthesis,decreased shikonin formation significantly. This reduction couldbe alleviated or even abolished by exogenous NO supplied bysodium nitroprusside (SNP), suggesting that the inhibition ofNO biosynthesis resulted in decreased shikonin formation. However,when endogenous NO biosynthesis was up-regulated by the elicitorfrom Rhizoctonia cerealis, shikonin production was enhancedfurther, showing a dependence on the elicitor-induced NO burst.Real-time PCR analysis showed that NO could significantly up-regulatethe expression of PAL, PGT and HMGR, which encode key enzymesinvolved in shikonin biosynthesis. These results demonstratedthat NO plays a critical role in shikonin formation in O. paniculatumcells.     

Induction of metamorphosis in the marine gastropod Ilyanassa obsoleta: 5HT, NO and programmed cell death

The central nervous system (CNS) of a metamorphically competent larva of the caenogastropod Ilyanassa obsoleta contains a medial, unpaired apical ganglion (AG) of approximately 25 neurons that lies above the commissure connecting the paired cerebral ganglia. The AG, also known as the cephalic or apical sensory organ (ASO), contains numerous sensory neurons and innervates the ciliated velar lobes, the larval swimming and feeding structures. Before metamorphosis, the AG contains 5 serotonergic neurons and exogenous serotonin can induce metamorphosis in competent larvae. The AG appears to be a purely larval structure as it disappears within 3 days of metamorphic induction. In competent larvae, most neurons of the AG display nitric oxide synthase (NOS)-like immunoreactivity and inhibition of NOS activity can induce larval metamorphose. Because nitric oxide (NO) can prevent cells from undergoing apoptosis, a form of programmed cell death (PCD), we hypothesize that inhibition of NOS activity triggers the loss of the AG at the beginning of the metamorphic process. Within 24 hours of metamorphic induction, cellular changes that are typical of the early stages of PCD are visible in histological sections and results of a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay in metamorphosing larvae show AG nuclei containing fragmented DNA, supporting our hypothesis.     

Mitochondrial calcium uptake stimulates nitric oxide production in mitochondria of bovine vascular endothelial cells

Although nitric oxide (NO) is a known modulator of cell respiration in vascular endothelium, the presence of a mitochondria-specific nitric oxide synthase (mtNOS) in these cells is still a controversial issue. We have used laser scanning confocal microscopy in combination with the NO-sensitive fluorescent dye DAF-2 to monitor changes in NO production by mitochondria of calf vascular endothelial (CPAE) cells. Cells were loaded with the membrane-permeant NO-sensitive dye 4,5-diaminofluorescein (DAF-2) diacetate and subsequently permeabilized with digitonin to remove cytosolic DAF-2 to allow measurements of NO production in mitochondria ([NO]_mt). Stimulation of mitochondrial Ca²⁺ uptake by exposure to different cytoplasmic Ca²⁺ concentrations (1, 2, and 5 µM) resulted in a dose-dependent increase of NO production by mitochondria. This increase of [NO]_mt was sensitive to the NOS antagonist L-N⁵-(1-iminoethyl)ornithine and the calmodulin antagonist calmidazolium (R-24571), demonstrating the endogenous origin of NO synthesis and its calmodulin dependence. Collapsing the mitochondrial membrane potential with the protonophore FCCP or blocking the mitochondrial Ca²⁺ uniporter with ruthenium red, as well as blocking the respiratory chain with antimycin A in combination with oligomycin, inhibited mitochondrial NO production. Addition of the NO donor spermine NONOate caused a profound increase in DAF-2 fluorescence that was not affected by either of these treatments. The mitochondrial origin of the DAF-2 signals was confirmed by colocalization with the mitochondrial marker MitoTracker Red and by the observation that disruption of caveolae (where cytoplasmic NOS is localized) formation with methyl--cyclodextrin did not prevent the increase of DAF-2 fluorescence. The activation of mitochondrial calcium uptake stimulates mtNOS phosphorylation (at Ser-1177) which was prevented by FCCP. The data demonstrate that stimulation of mitochondrial Ca²⁺ uptake activates NO production in mitochondria of CPAE cells. This indicates the presence of a mitochondria-specific NOS that can provide a fast local modulatory effect of NO on cell respiration, membrane potential, and apoptosis. nitric oxide; nitric oxide synthase; calcium; endothelium; mitochondria     

Nitrergic relaxation in rat gastric fundus: influence of mechanism of induced tone and possible role of sarcoplasmic/endoplasmic reticulum Ca2+ ATPase

The aim of this study was to investigate the influence of the mechanism of induced tone and the role of sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA) in nitrergic relaxation of rat gastric fundus. Prostaglandin F(2alpha) (PGF(2alpha)), thapsigargin (TSG) and cyclopiazonic acid (CPA) were used in concentrations that induced a similar contraction (20 g force/g tissue). Nifedipine (3 x 10(-7) M) completely relaxed PGF(2alpha)-contracted tissues and relaxed tissues contracted by TSG and CPA by 20 +/- 6% and 56 +/- 12% respectively; contraction induced by the three contractile agents was fully reversed by a general Ca2+ entry blocker 1-[2-(4-methoxyphenyl)-2-[3-(4-metoxyphenyl)propoxy]ethyl-1H-imidazole HCl (SKF 96365; 10(-5) M). In the presence of nifedipine (3 x 10(-7) M) or verapamil (10(-5) M), PGF(2alpha) and CPA-induced contractions were still approximately 50% relaxed by SKF 96365. This suggests that contractions induced by PGF(2alpha) are related to Ca2+ entry through L-type voltage-operated Ca2+ channels and that contractions by TSG are mainly related to Ca2+ entry through store-operated Ca2+ channels. Relaxant responses to exogenous nitric oxide (NO), to endogenous NO released by electrical field stimulation, and to vasoactive intestinal polypeptide (VIP) were studied in tissues contracted by TSG and CPA and compared to responses in tissues contracted by PGF(2alpha). Responses to exogenous and endogenous NO were greatly attenuated in TSG-contracted tissues, but not in CPA-contracted tissues. When contraction was induced by CPA in the presence of nifedipine or verapamil, relaxations to exogenous and endogenous NO were also significantly reduced. Relaxation induced by VIP was reduced in tissues contracted by either TSG or CPA in the presence of nifedipine or verapamil. These results suggest that the ability of the nitrergic neurotransmitter to induce relaxation of rat gastric fundus is influenced by the mechanism used to induce tone and are indicative for a role for SERCA in nitrergic relaxation. However, activation of SERCA appears to not be unique for nitrergic relaxation, but might also be used by VIP, a co-transmitter of NO in this tissue.     

Analysis of nitric oxide-cyclic guanosine monophosphate signaling during metamorphosis of the nudibranch Phestilla sibogae Bergh (Gastropoda: Opisthobranchia)

SUMMARY The gas nitric oxide (NO), and in some cases its downstream second messenger, cyclic guanosine monophosphate (cGMP) function in different taxa to regulate the timing of life-history transitions. Increased taxonomic sampling is required to foster conclusions about the evolution and function of NO/cGMP signaling during life-history transitions. We report on the function and localization of NO and cGMP signaling during metamorphosis of the nudibranch Phestilla sibogae . Pharmacological manipulation of NO or cGMP production in larvae modulated responses to a natural settlement cue from the coral Porites compressa in a manner that suggest inhibitory function for NO/cGMP signaling. However, these treatments were not sufficient to induce metamorphosis in the absence of cue, a result unique to this animal. We show that induction of metamorphosis in response to the settlement cue is associated with a reduction in NO production. We documented the expression of putative NO synthase (NOS) and the production of cGMP during larval development and observed no larval cells in which NOS and cGMP were both detected. The production of cGMP in a bilaterally symmetrical group of cells fated to occupy the distal tip of rhinophores is correlated with competence to respond to the coral settlement cue. These results suggest that endogenous NO and cGMP are involved in modulating responses of P. sibogae to a natural settlement cue. We discuss these results with respect to habitat selection and larval ecology.     

Mechanical Stress Elicits Nitric Oxide Formation and DNA Fragmentation in Arabidopsis thaliana

The effect of mechanical stress (centrifugation) on the inductionof nitric oxide (NO) formation and DNA fragmentation was investigatedin leaf cells of Arabidopsis thaliana. Centrifuged and non-centrifugedleaves from wild-type and nitrate reductase (NR)nia1, nia2 doublemutant, defective in the assimilation of nitrate, were labelledwith 4,5-diaminofluorescein diacetate (DAF-2 DA) to visualizein vivo NO production. After these treatments, DNA fragmentationwas detected by the terminal deoxynucleotidyl transferase-mediateddUTP nick end in situ labelling (TUNEL) method. Exposure toan NO-releasing compound, sodium nitroprusside (SNP) mimickedthe cell response to centrifugation (20 g). The involvementof endogenous NO as a signal in mechanical stress and in DNAfragmentation was confirmed by inhibition of NO production usinga nitric oxide synthase (NOS) inhibitor viz. N^G-monomethyl-L -arginine (L -NMMA). These results indicate that NOS-likeactivity was present in A. thaliana leaves and was increasedby mechanical stress. The effect of leaf-wounding on nitricoxide production was identical to that of centrifugation. Experimentswith A. thaliana NR mutant also showed that NO bursts were inducedby mechanical and wounding stresses and that NO was not a by-productof NR activity. A positive and significant correlation betweenNO production and DNA fragmentation was recorded for both centrifugedand non-centrifuged cells. Our results suggest that factorsother than NO contribute to DNA damage and cell death, and furthermore,that an inducible form of NOS is present in A. thaliana. Copyright2001 Annals of Botany Company Arabidopsis thaliana, cell death, DNA fragmentation, NO, plant stress, wounding     

Exploring the regulatory role of nitric oxide (NO) and the NO-p38MAPK/cGMP pathway in larval settlement of the bryozoan Bugula neritina

The bryozoan Bugula neritina is a cosmopolitan marine fouling species that causes major fouling problems in sub-tropical waters. Settlement of B. neritina larvae can be triggered without an obvious external cue. Here, the negative regulatory role of nitric oxide (NO) during larval settlement of B. neritina was demonstrated to be mediated by cyclic guanosine monophosphate (cGMP). Although the regulatory role of the NO-p38 MAPK signaling axis in larval settlement was not evident, inhibition of nitric oxide synthase (NOS) led to the deactivation of p38 MAPK. Exclusive localization of NO and NO signaling components in sensory-related organs of the larvae is consistent with its signal transduction function in metamorphosis. Overall, this study provides new insights into the regulatory roles of the NO-p38MAPK/cGMP pathway in B. neritina settlement.     

Myosin Va plays a key role in nitrergic neurotransmission by transporting nNOSα to enteric varicosity membrane

Nitrergic neurotransmission at the smooth muscle neuromuscular junctions requires nitric oxide (NO) release that is dependent on the transport and docking of neuronal NO synthase (nNOS) α to the membrane of nerve terminals. However, the mechanism of translocation of nNOSα in actin-rich varicosities is unknown. We report here that the processive motor protein myosin Va is necessary for nitrergic neurotransmission. In wild-type mice, nNOSα-stained enteric varicosities colocalized with myosin Va and its tail constituent light chain 8 (LC8). In situ proximity ligation assay showed close association among nNOSα, myosin Va, and LC8. nNOSα was associated with varicosity membrane. Varicosities showed nitric oxide production upon stimulation with KCl. Intracellular microelectrode studies showed nitrergic IJP and smooth muscle hyperpolarizing responses to NO donor diethylenetriamine-NO (DNO). In contrast, enteric varicosities from myosin Va-deficient DBA (for dilute, brown, non-agouti) mice showed near absence of myosin Va but normal nNOSα and LC8. Membrane-bound nNOSα was not detectable, and the varicosities showed reduced NO production. Intracellular recordings in DBA mice showed reduced nitrergic IJPs but normal hyperpolarizing response to DNO. The nitrergic slow IJP was 9.1 ± 0.7 mV in the wild-type controls and 3.4 ± 0.3 mV in the DBA mice (P < 0.0001). Deficiency of myosin Va resulted in loss of nitrergic neuromuscular neurotransmission despite normal presence of nNOSα in the varicosities. These studies reveal the critical importance of myosin Va in nitrergic neurotransmission by facilitating transport of nNOSα to the varicosity membrane.     

L-NAME pre-treatment partially inhibits the agmatine-evoked depression of the electrically induced twitch contraction of isolated rat vas deferens

The effect of the putative endogenous ligand for alpha(2)-adrenoceptors and imidazoline receptors agmatine was studied in sympathetic neurotransmission in the rat epididymal vas deferens. Tissues were obtained from N(varpi)-nitro-l-arginine methyl ester (l-NAME)-treated or normal animals and were contracted by electrical stimulation or by exogenous adenosine 5'-triphosphate (ATP). In the electrically stimulated epididymal end, agmatine produced an inhibitory effect on twitch contraction that was partially reversed in l-NAME-treated animals, whereas the inhibition produced by clonidine was not affected by l-NAME treatment. The nitric oxide (NO)-donor S-nitroso-N-acetyl-penicillamine (SNAP) also inhibited twitch contraction. Neither agmatine nor SNAP interfered with the responses induced by exogenous ATP in the epididymal end. Removal of the epithelium of the preparation did not modify the agmatine response. We conclude that a nitrergic pathway activated by agmatine plays a role in its inhibitory effect in rat vas deferens, but it remains to be investigated whether it results from a direct action on the enzyme NO-synthase or a receptor-mediated mechanism.     

GABA is an inhibitory neurotransmitter in the neural circuit regulating metamorphosis in a marine snail

The marine mud snail, Tritia (=Ilyanassa) obsoleta, displays a biphasic life cycle. During the initial phase of early development, embryos hatch from benthic egg capsules to become weakly swimming veliger larvae. In the second phase, adult T. obsoleta are facultative carnivores and major agents of community disturbance. Metamorphosis is the irreversible developmental event that links these two life history stages. When physiologically competent, larvae can respond to appropriate environmental cues by settling onto their mudflat habitat and transforming themselves into miniature adult snails. Two neurotransmitters—serotonin and nitric oxide—have opposing effects on the metamorphic process in this species. In multiple other species of gastropod and bivalve molluscs, a third neurotransmitter, the classically inhibitory compound γ‐aminobutyric acid (GABA), can induce settlement or metamorphosis upon external application to competent larvae. In this situation, GABA is presumed to mimic the action of ligands from the juvenile environment that bind to larval chemosensory receptors and activate the metamorphic pathway. Results of our experiments contradict this commonly reported action of GABA on molluscan larvae. External application of GABA to competent larvae of T. obsoleta elicited no response, but instead attenuated the action of serotonin (5‐HT), a metamorphic inducer. Our investigations into the responses of larval T. obsoleta to multiple GABAergic reagents support our hypothesis that GABA functions internally as a neurotransmitter in the pathway that controls the initiation of metamorphosis. Our results also suggest that GABA acts directly on or downstream from serotonergic neurons to regulate the metamorphosis‐inducing effects of this neurotransmitter. © 2018 Wiley Periodicals, Inc. Develop Neurobiol 78: 736–753, 2018     

Nitric oxide, stomatal closure, and abiotic stress

Various data indicate that nitric oxide (NO) is an endogenoussignal in plants that mediates responses to several stimuli.Experimental evidence in support of such signalling roles forNO has been obtained via the application of NO, usually in theform of NO donors, via the measurement of endogenous NO, andthrough the manipulation of endogenous NO content by chemicaland genetic means. Stomatal closure, initiated by abscisic acid(ABA), is effected through a complex symphony of intracellularsignalling in which NO appears to be one component. ExogenousNO induces stomatal closure, ABA triggers NO generation, removalof NO by scavengers inhibits stomatal closure in response toABA, and ABA-induced stomatal closure is reduced in mutantsthat are impaired in NO generation. The data indicate that ABA-inducedguard cell NO generation requires both nitric oxide synthase-likeactivity and, in Arabidopsis, the NIA1 isoform of nitrate reductase(NR). NO stimulates mitogen-activated protein kinase (MAPK)activity and cGMP production. Both these NO-stimulated eventsare required for ABA-induced stomatal closure. ABA also stimulatesthe generation of H₂O₂ in guard cells, and pharmacological andgenetic data demonstrate that NO accumulation in these cellsis dependent on such production. Recent data have extended thismodel to maize mesophyll cells where the induction of antioxidantdefences by water stress and ABA required the generation ofH₂O₂ and NO and the activation of a MAPK. Published data suggestthat drought and salinity induce NO generation which activatescellular processes that afford some protection against the oxidativestress associated with these conditions. Exogenous NO can alsoprotect cells against oxidative stress. Thus, the data suggestan emerging model of stress responses in which ABA has severalameliorative functions. These include the rapid induction ofstomatal closure to reduce transpirational water loss and theactivation of antioxidant defences to combat oxidative stress.These are two processes that both involve NO as a key signallingintermediate. Key words: Abscisic acid, antioxidants, guard cells, hydrogen peroxide, nitric oxide, oxidative stress, stomata, water stress Received 19 June 2007; Revised 21 September 2007 Accepted 5 November 2007     

Efficiency of NO donors in substituting impaired endogenous NO production: a functional and morphological study

Two exogenous NO donors were used to act as substitutes for impaired endogenous nitric oxide (NO) production due to inhibition of NO synthase in rats. Six weeks' lasting inhibition of NO synthase by NG-nitro-L-arginine methyl ester (L-NAME) induced stabilized hypertension. Simultaneously administered isosorbide-5-mononitrate did not prevent the development of hypertension. Molsidomine, administered concomitantly with L-NAME, significantly attenuated the BP increase. However, BP was still found to be moderately increased compared to the initial values. Remarkable alterations in the geometry of the aorta, carotid and coronary artery found in NO-deficient hypertension were prevented in rats administered L-NAME plus molsidomine at the same time. In spite of 6 weeks' lasting inhibition of NOS, the NOS activators acetylcholine and bradykinin induced BP decrease; the maximum hypotensive value did not differ from the values recorded in the controls or in animals treated with L-NAME plus molsidomine. Notably enough, the hypotension was similar to that found in rats administered L-NAME alone for six weeks. After NO synthase inhibition, Isosorbide-5-mononitrate does not substitute and molsidomine substitute only partially the impaired endogenous NO production.     

Interspecific variation in metamorphic competence in marine invertebrates: the significance for comparative investigations into the timing of metamorphosis

Metamorphosis in marine invertebrate larvae is a dynamic, environmentallydependent process that integrates ontogeny with habitat selection.The capacity of many marine invertebrate larvae to survive andmaintain metamorphic competence in the absence of environmentalcues has been hypothesized to be an adaptive convergence (Hadfieldand others 2001). A survey of the literature reveals that asingle generalized hypothesis about metamorphic competence asan adaptive convergence is not sufficient to account for interspecificvariation in this character. In an attempt to capture this variation,we discuss the "desperate larva hypothesis" and propose twoadditional hypotheses called the "variable retention hypothesis"and the "death before dishonor hypothesis." To validate theseadditional hypotheses we collected data on taxa from the publishedliterature and performed a contingency analysis to detect correlationsbetween spontaneous metamorphosis, habitat specificity and/orlarval life-history mode, three characters relevant to environmentallyinduced settlement and metamorphosis. In order to account forphylogenetic bias in these correlations, we also constructeda phylogeny of these taxa and again performed a character-correlationanalysis. Both these tests suggest that juvenile habitat specificityis correlated to the capacity of individuals to retain the competentlarval state in the absence of substrate cues and thereforevalidate the existence of more than one hypothesis about metamorphiccompetence. We provide new data from the sea urchin Lytechinuspictus that suggest that nitric oxide (NO) and thyroxine hormonesignaling interact to determine the probability of settlementin response to a settlement cue. Similarly, we provide evidencethat thyroxine signaling in the sand dollar Dendraster excentricusincreases spontaneous metamorphosis in the absence of cues fromadult conspecifics in a manner that is independent of larvalage.     

Reactive oxygen species and anti-oxidant defenses in tail of tadpoles, Xenopus laevis

Tail regression in tadpoles is one of the most spectacular events in anuran metamorphosis. Reactive oxygen species and oxidative stress play an important role during this process. Presently, the cell- and tissue-specific localization of antioxidant enzymes such as superoxide dismutase (SOD) and catalase as well as neuronal and inducible nitric oxide synthase isoforms (nNOS and iNOS) responsible for production of nitric oxide (NO) were carried out during different stages of metamorphosis in tail of tadpole Xenopus laevis. NO also has profound effect on the mitochondrial function having its own nitric oxide NOS enzyme. Hence, in situ staining for NO and mitochondria also was investigated. The distribution of nNOS and iNOS was found to be stage specific, and the gene expression of nNOS was up-regulated by thyroxin treatment. In situ staining for NO and mitochondria shows co-localization, suggesting mitochondria being one of the sources of NO. SOD and catalase showed significant co-localization during earlier stages of metamorphosis, but before the tail regression begins, there was a significant decrease in activity as well as co-localization suggesting increased ROS accumulation. These findings are discussed in terms of putative functional importance of ROS and cytoplasmic as well as mitochondrial derived NO in programmed cell death in tail tissue.     

Induction of metamorphosis decreases nitric oxide synthase gene expression in larvae of the marine mollusc Ilyanassa obsoleta (say)

Many marine organisms spend the early part of their lives as larvae suspended in the water column before metamorphosing into benthic reproductive adults. Metamorphosis does not occur until a larva has become competent to respond to appropriate stimuli and after a suitable habitat for the young juvenile has been encountered. The gaseous neurotransmitter nitric oxide is thought to be important in the regulation of metamorphosis by holding the organism in the larval state. We have investigated expression of the neuronal nitric oxide synthase (nNOS) gene in larval and metamorphosing individuals of the marine mud snail Ilyanassa obsoleta. Our results indicate that nNOS is expressed at constant levels throughout larval development. In contrast, expression of nNOS decreases markedly during the first 24 h of metamorphosis. Our observations support previous findings that demonstrate that nitric oxide is present in larvae though competence. The decrease in nNOS gene expression that occurs during metamorphosis corresponds with a previously described reduction in nNOS activity.     

Regulation of metamorphosis in ascidians involves NO/cGMP signaling and HSP90

Treatment of larvae of the ascidians Boltenia villosa (Family: Pyuridae) and Cnemidocarpa finmarkiensis (Family: Styelidae) with drugs that inhibit the function of the molecular chaperone HSP90 increased the frequency of tail resorption, the primary morphogenetic event of metamorphosis. If treatment was initiated at hatching, metamorphic events subsequent to tail resorption failed to occur, indicating an ongoing role for HSP90 during morphogenesis. Removal of tails from heads of mature, but not newly hatched larvae, induced metamorphosis of the head. Decapitation experiments indicate that the capacity of tails to shorten in response to inhibition of HSP90 function requires communication with heads. To identify candidate proteins with which HSP90 may interact to regulate metamorphosis, we noted that in mammalian cells, nitric oxide synthase (NOS) interacts with HSP90 and its activity is sensitive to drugs that inhibit HSP90 function. In addition, nitric oxide (NO) signaling in the marine snail Ilyanassa obsoleta is an important regulator of metamorphosis. Inhibition of NOS activity in these ascidian larvae with L-NAME increased the frequency of metamorphosis, consistent with a putative interaction of NOS and HSP90. NOS is present in tail muscle cells, implicating them as targets for the drug treatments, consistent with the decapitation experiments. Inhibition of soluble guanylyl cyclase, the most common effector of NO signaling, also increased the frequency of metamorphosis. In contrast to treatment with anti-HSP90 drugs, metamorphosis induced with L-NAME or ODQ was complete. The results presented suggest that an HSP90-dependent, NO-based regulatory mechanism localized in tails represses ascidian metamorphosis. We discuss these results in relation to the induction of ascidian metamorphosis by several unrelated agents.