Adenosine triphosphatase localization in the branchial heart of Sepia officinalis L. (Cephalopoda)

Sodium- and potassium-dependent adenosine triphosphatase (Na+--K+-ATPase) is demonstrated in the branchial heart of Sepia officinalis L. by biochemical, cytochemical and autoradiographical methods. The biochemical data indicate the presence of Na+--K+-ATPase, shown by potassium and magnesium dependency and inhibition by ouabain. Cytochemically and autoradiographically, the enzyme is localized in the sarcolemma of the muscle cells. The positive reaction of the transparent cells (type I cells) is due to activity of alkaline phosphatases. The dark cells (type II cells) react negatively. In addition to the Na+--K+-ATPase, a magnesium-activated adenosine triphosphatase (Mg2+-ATPase) and a bicarbonate-stimulated ATPase (HCO3(-)-ATPase) are localized in the mitochondria.     

Adenosine triphosphatase localization in the branchial heart appendage of Sepia officinalis L. (Cephalopoda)

Summary Sodium- and potassium-dependent adenosine triphosphatase (Na⁺–K⁺-ATPase) has been demonstrated in the branchial heart appendage (pericardial gland) of Sepia officinalis L. by biochemical, cytochemical and autoradiographical methods. The biochemical data indicate the presence of Na⁺–K⁺-ATPase, judging from the potassium dependency and, with some restrictions, the inhibition by ouabain. Cytochemically and autoradiographically, the enzyme could be localized on the cytoplasmic surfaces of the lateral plasma membranes and the basal membrane infoldings (basal labyrinth) of the folded epithelium of the branchial heart appendage. The pdocytes of the peripheral zone of the organ reacted negatively. In addition to the Na⁺–K⁺-ATPase, a magnesium-activated adenosine triphosphatase (Mg²⁺-ATPase) was demonstrated in the folded epithelium, localized mainly in the mitochondria but also at the brush border and in the apical intercellular space, whereas a bicarbonate-stimulated ATPase (HCO ₃ ^– -ATPase) was present only in the mitochondria.This study was supported by the Deutsche Forschungsgemeinschaft     

Serotonergic regulation of the central heart auricles of Sepia officinalis L. (Mollusca, Cephalopoda)

In pharmacological bioassays on isolated isotonically suspended auricles of Sepia officinalis, the regulatory action of the neurotransmitter serotonin (5-hydroxytryptamine, 5-HT) on these autonomous contractile compartments was demonstrated. 5-HT induced concentration-dependent positive effects on frequency and tone, whereas the concentrations/response curve for the amplitude showed a biphasic course. All applied antagonists inhibited mainly the effect of 5-HT on frequency and amplitude. The chronotropic effects of 5-HT were blocked mainly by the 5-HT(1,2) antagonist methiothepin (pA(2)=8.01), the 5-HT(1a) antagonist NAN-190 (pA(2)-) and in lesser extent by the 5-HT(1,2) antagonist mianserin (pA(2)=6.81). In the presence of each antagonist applied the 5-HT action on amplitude was transformed into a positive inotropic effect with the maximum under influence of NAN-190 and the 5-HT(2,1c)-antagonist ketanserin. The auricular tone was also influenced by the antagonists and in combination with methiothepin it turned into strong negative tonotropic effect. In addition to the pharmacological bioassays, the presence of 5-HT in nerve endings within the auricle wall was demonstrated by immunohistochemical and fluorescence microscopic findings. Altogether the findings presented here confirm that 5-HT evokes excitatory effects on the autonomous contractile auricle of S. officinalis and acts obviously over different receptors, whereby a 5-HT(1)- and a 5-HT(2)-like seem to be involved.     

Histochemical localization of cholinesterases and monoamines in the central heart of Sepia officinalis L. (Cephalopoda)

The central heart of the coleoid cephalopod, Sepia officinalis, was studied using acetylcholinesterase and fluorescence histochemistry. Using histo- and cytochemical reactions, acetylcholinesterase was localized in the axolemma and axoplasm of specific cardiac nerve fibres, as well as in the sarcolemma and within the sarcotubular system of the muscle cells. Butyrylcholinesterase exhibited a different distribution, being found only in the luminal trabecular muscle layer. Glyoxylic-acid-induced fluorescence indicated the presence of catecholamines (emission maximum, 470 nm) in cardiac nerve axons. These histochemical findings support the hypothesis that noradrenaline and/or dopamine and acetylcholine act antagonistically as natural transmitters. Fluorophores indicating the presence of serotonin were not observed. The present results are discussed in the light of previous pharmacological findings.     

Histochemical localization of cholinesterases and monoamines in the central heart of Sepia officinalis L. (Cephalopoda)

Summary The central heart of the coleoid cephalopod, Sepia officinalis, was studied using acetylcholinesterase and fluorescence histochemistry. Using histo- and cytochemical reactions, acetylcholinesterase was localized in the axolemma and axoplasm of specific cardiac nerve fibres, as well as in the sarcolemma and within the sarcotubular system of the muscle cells. Butyrylcholinesterase exhibited a different distribution, being found only in the luminal trabecular muscle layer. Glyoxylic-acid-induced fluorescence indicated the presence of catecholamines (emission maximum 470 nm) in cardiac nerve axons. These histochemical findings support the hypothesis that noradrenaline and/or dopamine and acetylcholine act antagonistically as natural transmitters. Fluorophores indicating the presence of serotonin were not observed. The present results are discussed in the light of previous pharmacological findings.     

Adenosine triphosphatase localization in the branchial heart appendage of Sepia officinalis L. (Cephalopoda).

Sodium- and potassium-dependent adenosine triphosphatase (Na+--K+-ATPase) has been demonstrated in the branchial heart appendage (pericardial gland) of Sepia officinalis L. by biochemical, cytochemical and autoradiographical methods. The biochemical data indicate the presence of Na+--K+-ATPase, judging from the potassium dependency and, with some restrictions, the inhibition by ouabain. Cytochemically and autoradiographically, the enzyme could be localized on the cytoplasmic surfaces of the lateral plasma membranes and the basal membrane infoldings (basal labyrinth) of the folded epithelium of the branchial heart appendage. The pdocytes of the peripheral zone of the organ reacted negatively. In addition to the Na+--K+-ATPase, a magnesium-activated adenosine triphosphatase (Mg2+-ATPase) was demonstrated in the folded epithelium, localized mainly in the mitochondria but also at the brush border and in the apical intercellular space, whereas a bicarbonate-stimulated ATPase (HCO-3-ATPase) was present only in the mitochondria.     

An antagonistic 5-HT receptor system in the auricles of the systemic heart complex of Sepia officinalis L. (Cephalopoda) shows 5-HT1 and 5-HT4 subtype properties

In pharmacological bioassays on isolated ring-shaped auricle preparations of Sepia officinalis, the action of the specific 5-hydroxytryptamine (5-HT) agonists 8-OH-DPAT (5-HT1a), CP-93129 (5-HT1b), TFMPP (5-HT1b) and RS-67333 (5-HT4) on these autonomously contractile compartments was studied. 8-OH-DPAT and CP-93129 induced mainly positive effects on frequency and tone on the isotonically suspended auricles. The positive effect of 8-OH-DPAT on frequency was blocked by the specific 5-HT1a antagonist NAN-190. The 5-HT1b agonist TFMPP caused similar effects on tone and a positive impact on the auricular amplitude. The highly specific 5-HT4 agonist RS-67333 induced an effect opposite to the action of 5-HT1 agonists inducing mainly negative effects on frequency, amplitude and tone, causing a diastolic standstill at a concentration of 10(-6) M. These negative effects were blocked by the adenylyl cyclase inhibitor SQ-22,536 in the absence of a diastolic standstill. The opposing action of 5-HT1 and 5-HT4 agonists on auricular contractile activity suggests that an antagonistic 5-HT-receptor system exists within the auricular myocardial cells of S. officinalis, probably consisting of 5-HT1- and 5-HT4-like subtypes. The results also suggest that adenylyl cyclase acts as the intracellular target enzyme of both signal transduction mechanisms.     

Cytobiological studies on hemocyanin metabolism in the branchial heart complex of the common cuttlefish Sepia officinalis (Cephalopoda, Dibranchiata)

The present study confirms previous investigations that demonstrated a high copper content in the branchial heart and its appendage, and that gave the first indication that this organ complex might be involved in hemocyanin metabolism in Sepia officinalis L. Immunocytochemical localization of hemocyanin molecules within the endocytotic lysosomal system of the ovoid cells and tracer experiments with ¹²⁵I-labeled Sepia hemocyanin suggest its endocytotic uptake. Energy-dispersive X-ray microanalysis and histochemical methods reveal a high copper content within the ovoid cells of the branchial heart. In view of the turnover of the respiratory pigment in the branchial heart of Sepia officinalis L., we believe that the ovoid cells are a site of hemocyanin catabolism.     

Zur Struktur und Entwicklung des Riesenfasersystems erster Ordnung von Sepia officinalis L. (Cephalopoda)

Summary The differentiation and course of the first order giant nerve fibres in the medial and posterior suboesophageal lobes of the brain of Sepia officinalis is examined in different developmental stages. In earlier embryonic stages one pair of first order giant cells differentiates on each side. Later, as a normal phenomenon, one cell of each pair degenerates.The two remaining giant fibres cross in the palliovisceral lobe. On either side of the intersection one branch of the contralateral and one branch of the ipsilateral axon are connected with the second order giant fibres. This structure, which differs from that found in Loligo, apparently mediates the functional bilaterality of the giant fibre system.

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Supported by grant NONR 2100 through the Anton and Reinhard Dohrn foundation.     

The localization of enzyme activities in the pancreatic appendages of Sepia officinalis L. (Cephalopoda).

In this study, enzyme activities of the pancreatic appendages of the ductus hepatoPancreas (the so-called "pancreas") in Sepia officinalis L. have been demonstrated by light and electron micicroscopical methods: Malate dehydrogenase, monoamine oxidase, acid phosphatase, beta-glucuronidase, adenosine triphosphatase and carbonic anhydrase were shown by the former, and monoamine oxidase, catalase, glutamic oxalacetic transaminase, choline esterase (non-specific), alkaline phosphatase, acid phosphatase and carbonic anhydrase by the latter technique. The correlation between enzyme activity and distribution, and the presumed function of the two pancreatic epithelia is discussed.     

Nitric oxide: a vasodilatatory mediator in the cephalic aorta of Sepia officinalis (L.) (Cephalopoda)

Evidence is presented that nitric oxide (NO) may regulate blood pressure in cephalopod molluscs. In vitro tests performed on the cephalic aorta of Sepia officinalis (L.) (Cephalopoda) showed that the NO releasers (glyceroltrinitrate, sodium nitroprusside, 3-morpholinylsydnoneimine chloride and KNO(2)) induced concentration-dependent vasodilatation of vessel segments (without the tunica adventitia/periadventitia) precontracted by dopamine. These vasodilatatory actions could be totally blocked by oxadiazolo[4,3-a] quinoxalin-1-one, an inhibitor of the NO-sensitive guanylyl cyclase, and partially mimicked by the cyclic guanosine monophosphate (cGMP) analogue 8-bromo cGMP and by the phosphodiesterase inhibitor, zaprinast. The NO-precursor, L-arginine, showed vasodilatatory effects only on segments of the aorta in which the layers containing nerves (tunica adventitia/periadventitia) had been left intact, suggesting that NO synthase may be located within peripheral nerves.     

Nitric oxide: a vasodilatatory mediator in the cephalic aorta of Sepia officinalis (L.) (Cephalopoda)

Evidence is presented that nitric oxide (NO) may regulate blood pressure in cephalopod molluscs. In vitro tests performed on the cephalic aorta of Sepia officinalis (L.) (Cephalopoda) showed that the NO releasers (glyceroltrinitrate, sodium nitroprusside, 3-morpholinylsydnoneimine chloride and KNO₂) induced concentration-dependent vasodilatation of vessel segments (without the tunica adventitia/periadventitia) precontracted by dopamine. These vasodilatatory actions could be totally blocked by oxadiazolo[4,3-a] quinoxalin-1-one, an inhibitor of the NO-sensitive guanylyl cyclase, and partially mimicked by the cyclic guanosine monophosphate (cGMP) analogue 8-bromo cGMP and by the phosphodiesterase inhibitor, zaprinast. The NO-precursor, l-arginine, showed vasodilatatory effects only on segments of the aorta in which the layers containing nerves (tunica adventitia/periadventitia) had been left intact, suggesting that NO synthase may be located within peripheral nerves. Accepted: 11 August 1998     

Cytomorphological aspects on the response of the branchial heart complex of Sepia officinalis L. (cephalopoda) to xenobiotics and bacterial infection

The ovoid cells of the branchial heart complex of Sepia officinalis L. were investigated with respect to their role in detoxification processes. The electron microscopical localization of in situ injected ferritin in the endocytotic-lysosomal system, and the fluorescence microscopical localization of protein-bound Evans blue in the ovoid cells of in vivo incubated animals, indicate that foreign materials are eliminated from the hemolymph by the branchial heart tissue. In addition to the non-circulating ovoid cells of the branchial heart, hemocytes in the circulating blood and in the wall of the branchial heart are also involved in the incorporation of allogeneic substances and bacteria, or their debris. Based on these observations, we propose that the ovoid cells, together with circulating and adhesive hemocytes in the branchial hearts, are an important component of a more comprehensive defence and detoxification system in dibranchiate cephalopods that prevents contamination of the whole organism by endocytotic removal of noxious substances from the hemolymph.     

Initial stages of food ingestion by Sepia officinalis (Mollusca: Cephalopoda)

The cuttlefish ingests much skeleton from the crustaceans and fish it preys upon. The skeletal pieces are relatively large and their dimensions bear a close relationship to the length of the buccal mass and diameter of the oesophagus. The structures of the buccal mass are instrumental in the breakdown of prey and orientation of long pieces of skeleton to ensure their entry into the oesophagus. Many pieces of skeletal material present in the stomach contents still have attached muscles, showing that there is little, or no, external digestion. Skeletal material may be important for long-term maintenance of young Sepia in captivity.     

Fine structure and function of the digestive cell of Sepia officinalis (Mollusca: Cephalopoda)

The post–embryonic development of the digestive gland has been studied by light and electron microscopy on animals reared in the laboratory. The gland reaches its "adult" structure around the end of the first month after hatching.
The infrastructural changes of the digestive gland have been followed during digestion at various ages (5,10,20,30 days and "adult"); ferritin was used for a tracer to study absorption.
These experiments have produced evidence of endocytosis intake of large proteins and further intracellular digestion inside the digestive cell. The residues of digestion are excreted in a "brown body" while the metabolites are used in new syntheses for the cell itself. These results demonstrate that "ancestral" digestive processes have been kept in at least one cephalopod.     

Food of the cuttlefish Sepia officinalis and S. elegans in the Ria de Vigo (NW Spain) (Mollusca: Cephalopoda)

A study of the diet of Sepia officinalis and S. elegans in the Ria de Vigo has shown that crustaceans are the most abundant prey in both species, followed by fish. Changes in the food composition of both species occur with growth. The type of prey eaten by the two sexes of these species is very similar. The possibility of trophic competition between juveniles of S. qficinalis and S. elegans is discussed.     

Integumental amino acid uptake in a carnivorous predator mollusc (Sepia officinalis, Cephalopoda)

The epithelial cells of the integument of body, arms and tentacles of Sepia officinalis present on their apical membrane a well-organised brush border and show the morphological and histochemical characteristics of a typical absorptive epithelium. The ability of the integument to absorb amino acids was investigated both in the arms incubated in vitro and in a purified preparation of brush border membrane vesicles (BBMV). Autoradiographic pictures of the integument after incubation of the arms in sea-water with or without sodium, showed that proline intake was Na+-dependent, whereas leucine intake appeared to be a largely cation-independent process. Time course experiments of labelled leucine, proline and lysine uptakes in BBMV evidenced that these amino acids are accumulated within the vesicles in the presence of an inwardly directed sodium gradient. The sodium-driven accumulation proves that cationic and neutral amino acids are taken up by the apical membrane of the epithelium of Sepia integument through a secondary active mechanism. For leucine, a 90% inhibition of the uptake was recorded in the presence of a large excess of the substrate. In agreement with the autoradiography results, an analysis of the cation specificity transport in BBMV showed that leucine uptake had a low cation specificity, whereas lysine and proline uptakes were Na+-dependent. An excess of lysine and proline, which share with alanine two different transport systems in the gill epithelium of marine bivalves, reduced eucine uptake. The possible role of the absorptive ability of the integument in a carnivorous mollusc is discussed.     

The cytoskeleton of chondrocytes of Sepia officinalis (Mollusca, Cephalopoda): an immunocytochemical study

Our previous electron microscope study showed that chondrocytes from cephalopod cartilage possess a highly developed cytoskeleton and numerous cytoplasmic processes that ramify extensively through the tissue. We have now carried out a light microscope immunocytochemical study of chondrocytes from the orbital cartilage of Sepia officinalis to obtain indications as to the nature of the cytoskeletal components. We found clear positivity to antibodies against mammalian tubulin, vimentin, GFAP, and actin, but not keratin. The simultaneous presence of several cytoskeletal components is consistent with the hypothesis that cephalopod chondrocytes have the characteristics of both chondrocytes and osteocytes of vertebrates, which endow the tissue as a whole with some of the properties of vertebrate bone. We confirm, therefore, the presence in molluscs of the ubiquitous cytoskeletal proteins of metazoan cells that have remained highly conserved throughout phylogenetic evolution.