Mats Carlberg

Is nitric oxide (NO) produced by invertebrate neurones

NADPH-diaphorase (NADPHd) is known to be identical to nitric oxide (NO) synthase in the mammalian nervous system, and is therefore used as a marker of NO-producing neurones. Using the histochemical reaction for NADPHd, we searched for such neurones in a selection of invertebrates. Special emphasis was given to molluscs. No selective neuronal staining was found in representatives of coelenterates, turbellarians, nematodes and urochordates. In all annelids, arthropods and molluscs examined, with the exception of a chiton, specific neurones were selectively stained. The reaction was particularly strong in pulmonate molluscs where scattered positive neurones were found in various ganglia and clustered symmetrically in the paired buccal ganglia. Biochemical assay of NO synthase in osphradia of the gastropod mollusc Lymnaea stagnalis revealed a formation of citrullin that was inhibited by the specific NO synthase N omega-nitro-L-arginine (NO2Arg). Both histochemical and biochemical methods indicate that NO can be used as a signal molecule by specific neurones in advanced invertebrates.

NADPH-diaphorase histochemistry and nitric oxide synthase activity in deutocerebrum of the crayfish, Pacifastacus leniusculus (Crustacea, decapoda)

The activity of an nitric oxide synthase in the deutocerebrum of the crayfish Pacifastacus leniusculus was investigated with histochemical and biochemical methods. By using the NADPH-diaphorase histochemical reaction, known as a selective marker for NO synthase in mammals, it was possible to localize specific neuronal elements in the crayfish. Pronounced diaphorase-staining was observed in peripheral olfactory sensory cells and in the neuropil of the olfactory lobes. Less intense diaphorase-staining also occurred in other deutocerebral neuropils, such as the accessory lobes, the lateral antennular neuropil and in the deutocerebral commissure neuropil. The biochemical assay revealed a calcium/calmodulin-dependent formation of citrulline from L-arginine in brain homogenate. It was also possible to show that the selective NO synthase inhibitor L-NOARG decreased the formation of citrulline. These data indicate a role for NO as an intercellular messenger in the crayfish.

Assay of neuronal nitric oxide synthase by HPLC determination of citrulline

Biological membranes from different tissue sources were incubated for nitric oxide synthase (NOS) activity under standard conditions at 20 degrees C and compared with rat cerebellar cytosol. NOS activity was monitored as the formation of L-citrulline from L-arginine. Samples were purified on Amprep CBA cation-exchange minicolumns prior to derivatization with o-phthaldialdehyde (OPA) and HPLC analysis. The OPA derivatives of L-citrulline and L-arginine eluted well separated within 15 min during isocratic elution at room temperature. A linear relation between peak height and quantity of L-citrulline was seen down to the detection limit at 0.1 pmol L-citrulline. Formation of L-citrulline was measurable in rat cerebellar cytosol as well as in preparations not previously assayed for NOS activity, including rat colon, cat oesophagus and crayfish (Pacifastacus leniusculus) brain. The method provides a sensitive and non-radioactive method for assaying NOS activity in small tissue samples and in tissues with low to moderate levels of NOS activity.

Presence of serotonin in early chick embryos

With biochemical analysis and with autoradiography based on injection of 5-[3H]hydroxytryptophan, it was possible to demonstrate the presence of serotonin (5-hydroxytryptamine) in early chick embryos as early as the pre-streak stage. The biochemical analysis which covered the early developmental period (0.5-6 days of incubation) revealed an elevated concentration of serotonin at gastrulation; from then it stayed at a lower and fairly even level. Autoradiographs of embryos at the pre-streak stage, the primitive streak stage, the head fold stage and the 4-6 somites stage indicated the presence of serotonin in intracellular yolk granules and in cell nuclei. Moreover, the amine appeared associated with microfilaments and microtubules, particularly in developing neural cells. Notably the elevated concentration of serotonin at gastrulation, but also the intracellular distribution of the amine during early organogenesis, indicates a prominent role for it in cell-shape changes and morphogenesis in the early chick embryo.

Complex avoidance behaviour and its neurochemical regulation in the land snail Cepaea nemoralis

1. In hot plate experiments, the pulmonate land snail Cepaea nemoralis displays a biphasic passive/active avoidance behaviour composed of retraction and subsequent searching mediated by antagonistic muscular systems. The switch, between the behaviours, is under neuronal control. 2. Leu- and met-enkephalin, as well as FMRFamide-antiserum, attenuated the retraction response and potentiated the searching behaviour. Opposite effects were achieved by injection of antisera to the enkephalins. 3. Both retraction and searching behaviours were potentiated by 5-HT. Methysergide antagonized the effects of the enkephalins on the searching behaviour. 4. We conclude that endogenous opioids act antagonistic to FMRFamide in the neuronally controlled switch between passive and active avoidance behaviour.

NO-synthase: What can research on invertebrates add to what is already known?

The present study attempts to review presently known data regarding the distribution of nitric oxide (NO) synthase and the function of NO in invertebrate species. NO is synthesized from L-arginine by the enzyme NO-synthase, and activates guanylate cyclase which in turn leads to an increase in levels of cGMP in target cells. Major contributions to the knowledge of NO as a messenger molecule in invertebrates have been made by NADPH-diaphorase histochemistry and biochemical assays. These techniques suggest the presence of a L-arginine/NO pathway in a variety of tissues, thus implicating multiple roles for NO in invertebrates.

Localization of dopamine in the freshwater hydrozoanHydra attenuata

SummaryHigh performance liquid chromatography (HPLC), with electrochemical detection, is an analytical method sensitive enough to permit quantification of dopamine, dihydroxyphenylalanine (DOPA) and 5-S-cysteinyl DOPA in single or hemisected specimens ofHydra attenuata. Dopamine and 5-S-cysteinylDOPA appear to be the quantitatively predominant catechol compounds inH. attenuata, whereas DOPA is present in minor amounts. The presence of DOPA and 5-S-cysteinylDOPA, and the quantitative correlation between dopamine and these compounds in many specimens, suggests that dopamine inH. attenuata, as in higher animals, is formed through decarboxylation of DOPA. Contrary to the dopaminergic nerves in higher animals, DOPA inHydra seems to be oxidized and 5-S-cysteinyl DOPA is formed as a by-product. The oxidation of DOPA indicates that the hydroxylation of tyrosine into DOPA in the tissues ofH. attenuata is mediated by a tyrosinase rather than a tyrosine hydroxylase. Immunocytochemical methods demonstrate a highly variable distribution of dopamine in the tissues of different specimens ofH. attenuata. Dopamine immunoreactivity is confined to ectodermal tissue and can be found in several different cell types including nerve cells, battery cells, nematocytes, epithelial cells and interstitial undifferentiated cells. The large amounts of dopamine found in some specimens ofH. attenuata indicate some biological function, although its sporadic occurrence in neurites makes it less plausible as a generally utilized neurotransmitter in this animal.

Gland cells in the tentacles of the jellyfish Cyanea lamarcki reactive with an antibody against 5-hydroxytryptamine.

SummaryA lightand electron-microscopic immunocytochemical study on the localization of 5-hydroxytryptamine in Cyanea lamarcki revealed a reaction product in the epidermal gland cells surrounding the nematocyst clusters. The closely related scyphozoan medusa Cyanea capillata lacked a reaction product in the similar mucus-producing gland cells. Earlier conflicting views on the presence and localization of 5-hydroxytryptamine in coelenterates can thus be due to large species differences. It can be stated that 5-hydroxytryptamine is lacking in the venom of Cyanea.

Enzymatic 5-hydroxylation of l-dopa by a tyrosinase isolated from the sea anemone Metridium senile

A particulate tyrosinase has been extracted and purified from tentacles of the sea anemone Metridium senile. The purified enzyme had properties in common with both mushroom and vertebrate tyrosinase and catalyzed three different reactions: oxidation of catechols, hydroxylation of L-tyrosine with L-dopa as cofactor and 5-hydroxylation of L-dopa. 5-Hydroxylation of L-dopa by an animal tyrosinase has not been reported earlier. The reaction could be analyzed under reducing conditions when the much faster oxidation of L-dopa to dopaquinone was inhibited. The conditions required for the accumulation of L-dopa and 5-hydroxydopa observed in vivo in tentacles of Metridium are discussed.

Presence of 3,4-dihydroxyphenylalanine and 3,4,5-trihydroxyphenylalanine in a coelenterate nervous system: Possible tyrosinase-mediated accumulation

3,4-dihydroxyphenylalanine (DOPA), 3,4,5-trihydroxyphenylalanine (5-OH-DOPA), 5-S-cysteinylDOPA (5-SC.D) and 2-S-cysteinylDOPA (2-SC.D) in the tentacles of the sea anemone, Metridium senile, were studied by the combined use of differential centrifugation of tissue homogenates, ultracentrifugation on sucrose density gradients, HPLC and electron microscopy. DOPA, 5-OH-DOPA and o-diphenol:O(2) oxidoreductase (Tyrosinase) were enriched in fractions containing membranes and subcellular particles, and the cysteinylDOPAs in the cytosol fractions. Ultrastructurally studied fractions rich in DOPA and 5-OH-DOPA contained large numbers of highly osmium-reducing vesicles. Identical structures were localized in ectodermal nerves and epidermal sensory cells. The results suggest that previous findings of catecholderivatives in the tentacles of Metridium and other sea anemone species by histochemical methods, are explained by a tyrosinase-based accumulation of DOPA and 5-OH-DOPA in the ectodermal nerve net. These substances are confined in specific compartments (vesicles) in the neurons and sensory cells.