Sea Pansy Renilla Koellikeri Research Articles

Retinoic acid and nitric oxide promote cell proliferation and differentially induce neuronal differentiation in vitro in the cnidarian Renilla koellikeri

Retinoic acid (RA) and nitric oxide (NO) are known to promote neuronal development in both vertebrates and invertebrates. Retinoic acid receptors appear to be present in cnidarians and NO plays various physiological roles in several cnidarians, but there is as yet no evidence that these agents have a role in neural development in this basal metazoan phylum. We used primary cultures of cells from the sea pansy Renilla koellikeri to investigate the involvement of these signaling molecules in cnidarian cell differentiation. We found that 9-cis RA induce cell proliferation in dose- and time-dependent manners in dishes coated with polylysine from the onset of culture. Cells in cultures exposed to RA in dishes devoid of polylysine were observed to differentiate into epithelium-associated cells, including sensory cells, without net gain in cell density. NO donors also induce cell proliferation in polylysine-coated dishes, but induce neuronal differentiation and neurite outgrowth in uncoated dishes. No other cell type undergoes differentiation in the presence of NO. These observations suggest that in the sea pansy (1) cell adhesion promotes proliferation without morphogenesis and this proliferation is modulated positively by 9-cis RA and NO, (2) 9-cis RA and NO differentially induce neuronal differentiation in nonadherent cells while repressing proliferation, and (3) the involvement of RA and NO in neuronal differentiation appeared early during the evolutionary emergence of nervous systems.

Immunohistochemical localization of a retinoic acid-like receptor in nerve cells of two colonial anthozoans (Cnidaria)

Retinoic acid is known to induce vertebrate stem cells to differentiate into a variety of cell types, including neurons. Although retinoic acid was reported to affect morphogenetic pattern specification in the hydrozoan Hydractinia (Müller, W.A., 1984. Retinoids and pattern formation in a hydroid. J. Embryol. Exp. Morph. 81, 253–271) and a retinoid RXR receptor was cloned in the jellyfish Tripedalia (Kostrouch, Z., Kostrouchova, M., Love, W., Jannini, E., Piatigorsky, J., Rall, J.E., 1998. Retinoic acid X receptor in the diploblast, Tripedalia cystophora. Proc. Natl. Acad. Sci. U.S.A. 95, 13442–13447), the cellular targets of retinoids were not investigated. We used Western immunoblotting and immunohistochemistry to investigate the presence and cellular distribution of a RXR-like receptor in the sea pansy Renilla koellikeri and in the staghorn coral Acropora millepora (Cnidaria, Anthozoa). Western blots revealed a 64 kDa protein from a sea pansy extract in a band that co-migrated with a RXR protein from the rat brain. Using antibodies raised against an epitope of human alpha RXR, we visualized putative ectodermal sensory cells in the polyp column of the adult sea pansy. Immunoreactivity was absent in staghorn coral larvae but present in the polyp column of adult colonies in the form of clusters of neuron-like cells in the basiectoderm near the ectoderm-mesoglea interface. These observations suggest that a RXR-like receptor is involved in epithelial nerve cell specification in adult anthozoans and that this role is conserved throughout evolution.

Nitric oxide modulates peristaltic muscle activity associated with fluid circulation in the sea pansyRenilla koellikeri

Nitric oxide (NO) is a well-known regulator of vascular activities in vertebrates and it has also been implicated as a vasodilatatory agent in a cephalopod. In the sea pansy Renilla koellikeri, an octocorallian representative of the most basal animals with a nervous system, we investigated the role of NO in peristalsis, an activity that moves body fluids through the coelenteron (gastrovascular cavity) of the polyps across the colony. NO donors increased the amplitude of peristaltic contractions and increased tonic contractions in relaxed preparations, but caused a relaxation of basal tension in contracted preparations. The NO synthase (NOS) inhibitors L-NAME (N(omega)-nitro-L-arginine methyl ester) and 7-nitroindazole reduced the amplitude of peristaltic contractions and lowered basal tension. In contrast, aminoguanidine, a specific inhibitor of inducible NOS, increased the amplitude but reduced the rate of peristalsis. Zaprinast, a cGMP-specific phosphodiesterase inhibitor, decreased the amplitude of peristaltic contractions, a decrease that was amplified by dibutyryl cGMP. In contrast, the inhibitor of soluble guanylyl cyclase ODQ (1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one) enhanced peristalsis. Putative NOS-containing neurons, revealed by NADPH-diaphorase activity and citrulline immunohistochemistry, were observed in the basiectoderm at the base of the autozooid polyp tentacles and in a nerve-net around the oral disc. Their neurites ran up the tentacles and down to the polyp body wall, crossing from the ectoderm through the mesoglea and into the endoderm musculature where musculo-epithelial cells were also reactive. These data suggest that two distinct nitrergic pathways, one of which is mediated by cGMP, regulate peristalsis and muscle tone in the sea pansy and that these pathways may involve NOS-containing ectodermal neurons and musculo-epithelial cells.

Identification of a novel aminergic-like G protein-coupled receptor in the cnidarian Renilla koellikeri

Biogenic amines exert various physiological effects in cnidarians, but the receptors involved in these responses are not known. We have cloned a novel G protein-coupled receptor cDNA from an anthozoan, the sea pansy Renilla koellikeri, that shows homology to mammalian catecholamine receptors and, to a lesser extent, to peptidergic receptors. This putative receptor, named Ren2, has a DRC pattern that replaces the well-conserved DRY motif on the cytoplasmic side of the transmembrane III and lacks the cysteine residues usually found in the second extracellular loop and C-terminus tail. Both the second extracellular loop and the N-terminal tail were seen to be short (six and three amino acids, respectively). Northern blot analysis suggests that the receptor gene codes for two transcripts. Localization of these transcripts by in situ hybridization demonstrated abundant expression in the epithelium of the pharyngeal wall, the oral disk and tentacles as well as in the endodermal epithelium lining the gastrovascular cavities.

Spawning and gamete follicle rupture in the cnidarian Renilla koellikeri: effects of putative neurohormones

The neuroendocrine control of spawning (release of intact gamete follicles) and of the ensuing exfoliation (freeing of gametes by follicle epithelium rupture) was investigated in colonies of the sea pansy Renilla koellikeri, an octocorallian of the sea pen family. Polyps of male colonies produce substantially more sperm follicles than female colonies do egg follicles, and significantly more sperm follicles are expelled than egg follicles during the summer spawning season. Spawning is accompanied by strong peristaltic contractions across the colony. Serotonin, a positive modulator of peristalsis in the sea pansy, induced spawning of either sperm or egg follicles, increasing both the proportion of spawning colonies and the number of expelled gamete follicles per colony in a dose-dependent manner. The serotonin antagonist 1-(1)naphthylpiperazine greatly reduced both spontaneous and serotonin-induced spawning. Antho-RFamide, a neuropeptide found in ciliated neurons within follicle epithelia, induced the exfoliation of the follicle epithelium from spawned follicles. Exposure of follicles to light enhanced the potency of Antho-RFamide. The actin-binding toxin phalloidin substantially reduced the incidence of Antho-RFamide-induced exfoliation and phalloidin-FITC staining was localized in the muscle feet of follicle epithelial cells. These results provide the first experimental evidence of neuroendocrine functions involved in cnidarian spawning.

Annual variations and sex-related differences of estradiol-17β levels in the anthozoan Renilla koellikeri

In the sea pansy Renilla koellikeri, estradiol-17β (E 2) levels exhibited an annual pattern of secretion that correlated with the reproductive cycle, and displayed sex-specific and tissue-specific differences. The E 2 levels were low during the non-reproductive period extending from autumn to winter. A first rise of E 2 concentrations occurred in March when gonad maturation resumed, as indicated by an increase in lipid storage. This suggests that E 2 may influence the beginning of gonad maturation in the sea pansy. Estradiol-17β returned to basal levels in April and May when lipids rose sharply. A second, more significant surge of E 2 levels occurred in June when spawning was initiated and it was more marked in female than in male colonies. This suggests that E 2 may participate in synchronising of oocyte maturation around spawning time to optimise the probability of fertilisation. The higher E 2 levels in somatic tissues (peduncle and polyps) than in eggs during the March peak revealed a non-reproductive origin for E 2 and the need for transport of E 2 through the gastrovascular cavity to reach reproductive tissues. Further analyses are required to determine the relative contribution of E 2 to reproductive events and its pathway of synthesis in this colonial anthozoan.

Tyrosine hydroxylase and dopamine-beta-hydroxylase immunoreactivities in the cnidarian Renilla koellikeri.

Western Blot and immunohistochemical studies were conducted in the sea pansy Renilla koellikeri, a representative of the earliest multicellular animals with a nervous system, using various antibodies raised against enzymes of the catecholamine biosynthetic pathway. Western blots of sea pansy extracts revealed a protein band that co-migrated with dopamine-beta-hydroxylase (DBH) from mouse adrenal glands. Similar experiments with antisera against tyrosine hydroxylase (TH) revealed several immunoreactive protein bands, all of larger molecular weight than mammalian tyrosine hydroxylase. DBH-like and, to a lesser extent, TH-like and phenylethanolamine N-methyltransferase-like immunoreactivities were detected in ectodermal sensory neurons and associated subectodermal neurites, in neurons of the mesogleal nerve-net and associated amoebocytes, and in some endodermal neurons. While it is still not clear whether the detected TH-immunoreactive proteins represent some form of TH, the presence in sea pansies of a DBH-like protein is in agreement with previously detected norepinephrine-like immunoreactivity in the same species. The widespread distribution of these immunoreactivities in various sea pansy neurons suggests important roles for catecholamines in nerve net activity.

Variations of lipid and fatty acid contents during the reproductive cycle of the anthozoan Renilla koellikeri.

Lipid and fatty acid contents were monitored in the sea pansy Renilla koellikeri during its reproductive cycle. The fluctuations of lipids and fatty acids were closely related to sexual development and gonads represented the principal site of lipid storage in the colony. Between April and May, fatty acids and lipids greatly accumulated just prior to spawning. The subsequent decrease of lipid and fatty acid contents in June probably resulted from loss due to spawning. Fatty acid composition was assessed for the whole animal and was compared between male and female gonads, and between gonads and somatic tissues. Arachidonic acid (AA) was the principal fatty acid in the whole colony followed by palmitic acid and eicosapentaenoic acid (EPA). The examination of fatty acid variations in gonads showed that EPA and AA were highly incorporated in eggs during the month preceding spawning, whereas in the same period, EPA was the only fatty acid to accumulate in spermatophores. The differences of fatty acid levels observed in gonads reflected sex differences observed in colonies. The seasonal variations of the omega6/omega3 ratio of the colonies were largely influenced by EPA and AA, and appeared as a reliable indicator of the sexual maturity of the sea pansy.

Monoamine release by neurons of a primitive nervous system: an amperometric study

We measured monoamine release from dissociated neurons of the sea pansy Renilla koellikeri, a representative of the most evolutionarily ancient animals with nervous systems, by real-time monitoring of exocytosis using the amperometric method with carbon-fiber microelectrodes. Depolarization-induced, as well as spontaneously active, neurons exhibited calcium-dependent exocytotic events at both the soma and the terminal bulb of neuritic processes. All spontaneously active neurons exhibited a bursting activity pattern in which amplitudes of exocytotic events appeared to be distributed in a quantal-like fashion. Fast Fourier transform analysis of bursting activity in 20 such neurons revealed burst harmonics with a major frequency of 8 Hz and a dominant rate of 95 Hz for individual exocytotic events within bursts. The results suggest that exocytotic transmitter release is as ancient as neurons and that endogenously bursting neurons in the sea pansy are as complex as those of higher animals. In addition, the observation that both soma and neuritic terminals of the same neuron can release transmitter suggests that local release sites in these cnidarian neurons are not critical for nerve net function.

Evidence for Gonadotropin-Releasing Hormone-like Peptides in a Cnidarian Nervous System

There is increasing evidence that peptides of the gonadotropin-releasing hormone (GnRH) family, long considered a vertebrate preserve, are also present in invertebrate (molluscan) nervous systems. The possibility was examined that GnRHs are present and bioactive in cnidarians, considered to be representatives of the most primitive animals possessing a nervous system. Immunoreactive GnRH was detected in endodermal neurons of two anthozoans, the sea pansy Renilla koellikeri and the sea anemone Nematostella vectensis. In the sea pansy, immunoreactivity was detected throughout the autozooid polyps, including gamete-producing endoderm. High-performance liquid chromatography and radioimmunoassays of extracts from whole sea pansy colonies yielded two elution peaks exhibiting GnRH immunoreactivity with antisera raised against shark or mammalian GnRH. Vertebrate GnRHs as well as the two sea pansy GnRH-like factors inhibited the amplitude and frequency of peristaltic contractions in the sea pansy, and these actions were blocked by the LHRH analog [D-pGlu1,D-Phe2,D-Trp3,6]-LHRH. These results suggest that the GnRH family of neuropeptides is more widespread in metazoans than previously thought. Although our physiological data are preliminary, they point to a role for GnRHs as inhibitory modulators of neuromuscular transmission in the sea pansy.

Distribution of serotonin uptake and binding sites in the cnidarian Renilla koellikeri: an autoradiographic study

Using [3H]-serotonin ([3H]-5-HT) as radiolabel and autoradiography, we have mapped the distribution of 5-HT uptake and binding sites in the sea pansy Renilla koellikeri in order to identify potential cellular pathways of 5-HT inactivation and to identify cellular substrates for the previously characterized 5-HT receptor involved in the modulation of peristaltic behavior. Uptake measured in fresh polyp tissues occurred via two processes: a high affinity (uptake1), clomipramine-sensitive process with a Km of 0.45 μM, and another of lower affinity (uptake2) with a Km of 11.6 μM. Autoradiograms of high affinity uptake sites revealed a diffuse distribution of label with higher density in the ectoderm and endoderm, and lower density in the mesoglea. No subsets of cells, including serotonergic neurons, appeared to retain label preferentially, thus suggesting that removal of 5-HT and its chemical conversion is a general property of sea pansy tissues. Under incubation conditions identical to those used in a previous radiobinding analysis, autoradiograms of binding sites were generated on sections from lightly fixed and cryosectioned polyps. In contrast to uptake sites, binding sites appeared as aggregations of label around neurons of the subectodermal, mesogleal and endodermal nerve nets. In the endoderm where the myoepithelia subtend peristalsis, myoepithelial cells appeared unlabeled, suggesting that 5-HT exerts its modulatory effect on peristalsis principally via neurons. Taken together, these results indicate that 5-HT is released as a neurohormone in the sea pansy and that it may act as a broad-range neuromodulator.

CHARACTERIZATION OF A SEROTONIN RECEPTOR IN THE CNIDARIAN RENILLA KOELLIKERI: A RADIOBINDING ANALYSIS

Serotonin (5-HT) binding sites in membrane preparations from the sea pansy Renilla koellikeri were identified using [3H]5-HT as radioligand and unlabelled 5-HT as displacer of specific binding. Saturation and kinetic studies revealed a mixed population of [3H]5-HT binding sites which as a whole displayed slow association kinetics, saturability, negligible dissociation and low affinity. The dopaminergic antagonist trifluoperazine specifically displaced the non-dissociated binding sites, allowing the characterization of a homogenous population of saturable sites exhibiting faster association and full dissociation. Scatchard analysis of this population revealed a KD of 23–34 nM and binding site density (Bmax) of 8.5–20.6 pmol/mg protein depending on body region. The pharmacological profile of the dissociable [3H]5-HT binding sites could not be categorized with that of any of the existing vertebrate and invertebrate 5-HT receptor subtypes in the current nomenclature. Especially noteworthy was the absolute inability of LSD, a ligand of choice for all hitherto identified invertebrate 5-HT receptors, to compete for the binding sites in sea pansy membrane preparations. It is proposed that these binding sites represent an evolutionary forerunner of the primordial 5-HT receptor that diversified as multiple subtypes in higher invertebrates and vertebrates. © 1997 Elsevier Science Ltd

Neuronal localization and evoked release of norepinephrine in the cnidarian Renilla koellikeri

AbstractNeuronal release of norepinephrine (NE) in the sea pansy Renilla koellikeri was investigated by searching for evidence 1) of neuronal localization of endogenous NE and 2) of synaptic‐like release of exogenously supplied NE in sea pansy tissues. Measurements of endogenous NE by high‐performance liquid chromatography with electrochemical detection corresponded with the visualization of specific NE immunoreactivity in six individual colonies thus sampled. Peroxidase‐antiperoxidase immunohistochemistry with antisera against NE‐aldehyde‐protein conjugates revealed that cellular NE immunostaining was predominantly associated with neurons and amoebocytes constituting the mesogleal nerve net. Transient increases of tritium outflow above background levels were evoked by field electrical stimulation of colonial (rachis) tissues pre‐loaded with 1 μM [3H]NE, under conditions that elicited nerve net‐mediated bioluminescence. This evoked release was largely reduced or abolished in a reversible manner by substituting high‐magnesium or calcium‐free seawater to normal seawater perfusate. Evoked release was significantly enhanced by exogenous norepinephrine (50 μM) and reduced in half or less by the α‐adrenergic blockers phentolamine and yohimbine (10 μM), thus providing support for the existence of an autoregulatory mechanism associated with release. These results suggest that neuronal, synaptic‐like release of NE occurs in the nerve‐net of the sea pansy, a representative of the phylum (Cnidaria) in which the first nervous systems of multicellular organisms are believed to have originated. © 1995 Wiley‐Liss, Inc.

Evidence for biosynthesis and catabolism of monoamines in the sea pansy Renilla koellikeri (CNIDARIA)

The biosynthesis of catecholamines and indoleamines was investigated in the sea pansy Renilla koellikeri by radiochemical screening of tissue samples exposed in vivo to labelled amino acid precursors and analysed by high-performance liquid chromatography coupled with electrochemical detection. Incubation of sea pansy tissues in [ 3H]tyrosine resulted in substantial accumulation of radioactivity recovered in chromatograms coeluting with tyrosine and 4-hydroxy-3-methoxy mandelic acid and, to a lesser extent, with 3,4-dihydroxy-phenylalanine, dopamine, norepinephrine, epinephrine, normetanephrine and 3,4-dihydroxyphenyl acetic acid. The catecholamine synthesis inhibitor α-methyl- p-tyrosine effectively reduced several of these [ 3H]tyrosine by-products formed as well as endogenous stores of these amines. Incubations in [ 3H]tryptophan resulted in large amounts of radioactivity associated with liquid chromatographic peaks coeluting with tryptophan and 5-hydroxytryptophan and lesser amounts with 5-hydroxytryptamine, N-acetyl-5-hydroxytryptamine and 5-hydroxy-3-indole acetic acid. The indoleamine synthesis inhibitor p-chlorophenylalanine reduced the amounts of products formed and depleted stores of the endogenous indoleamines. Enzyme activities which appear to involve tyrosine hydroxylase (EC 1. 12. 16. 2), tryptophan hydroxylase (EC 1. 14. 16. 4) and phenylethanolamine N-methyltransferase (EC 2. 1. 1. 28) were also detected in rachidial tissues by HPLC analysis of reaction products (hydroxylases) and by a radioenzymatic assay (methyltransferase). The sea pansy being a representative of the earliest invertebrates possessing a nervous system, these results support the hypothesis that vertebrate-like enzymatic pathways for the biosynthesis and degradation of monoamine neurotransmitters were conserved throughout evolution.

Distribution of ?2-like adrenergic receptors in the cnidarian Renilla koellikeri as revealed by autoradiography and in situ hybridization

Autoradiography and in situ hybridization were used to examine the histological distribution of the previously characterized beta 2-like adrenergic receptors involved in the bioluminescent activity of the sea pansy Renilla koellikeri. The use of [3H]-(+/-)CGP12177 as radioligand revealed autoradiographic labelling of the refringent granule-filled endoderm at the base of autozooid tentacles and autozooid columns, and in the corresponding endoderm of siphonozooid polyps, all areas where photocytes are concentrated. The presence of excess (10 microM) unlabelled (+/-)CGP12177 or atenolol in the incubation mixture substantially reduced total [3H]-(+/-)CGP12177 labelling. Under low stringency hybridization washing, human beta 2-adrenoceptor oligonucleotide probe signals were detected in granular cells located in those areas of polyp endoderm that were labelled by [3H]-(+/-)CGP12177. These cells were previously shown to be distinct from, but in close proximity to photocytes. No other cell or tissue type was labelled in polyps or throughout colonial tissues. The results suggest that a conserved form of beta 2-adrenergic receptors is present and synthesized in a unique type of endodermal cell indirectly involved in sea pansy bioluminescence control.

Modulation of rhythmic contractions by melatonin via cyclic GMP in the coelenterate Renilla koellikeri

1. The action of melatonin was investigated in a colonial anthozoan, the sea pansy Renilla koellikeri, in which rhythmic contractions underlying peristalsis were previously shown to be potentiated by serotonin (5-HT) and cAMP analogues (Anctil 1989). 2. Melatonin (2–1000 μmol·1-1) consistently and reversibly depressed the amplitude of rhythmic contractions, the latter all but vanishing at the most effective concentration (10 μmol·1-1). In addition, the frequency of rhythmic contractions incurred a 25% reduction in some preparations exposed to melatonin (10–20 μmol·1-1). Vaseline gap experiments showed that this response was restricted to tissues directly exposed to melatonin. 3. The potentiating effect of 5-HT on rhythmic contractions was eliminated in a reversible manner in the presence of melatonin. The latter also caused a 50% reduction of the amplitude of contractions elicited by field electrical stimulation. 4. Dibutyryl or 8-bromo cGMP, as well as the phosphodiesterase inhibitor IBMX, mimicked the melatonin-induced response and the effects of melatonin on the 5-HT potentiation or contractile response to electrical stimulation. cGMP levels, measured by radio-immunoassay (RIA) in rachidial tissues, increased 3-fold above basal levels in the presence of melatonin (0.01 mmol·1-1) and 2-fold with IBMX (1 mmol·1-1). In contrast, RIA measurements of cAMP levels showed the latter to fall to 50 and 40% of control values in the presence of melatonin (0.01 mmol·1-1) and IBMX (0.1 mmol·1-1), respectively. These results and other findings suggest that a cGMP-mediated melatonergic mechanism participates in an antagonistic fashion with 5-HT in the modulation of rhythmic contractions and peristalsis of the sea pansy.