Partial p53-dependence of anisomycin-induced apoptosis in PC12 cells.

The bacterial antibiotic anisomycin is known to induce apoptosis by activating several mitogen-activated protein kinases and by inhibiting protein synthesis. In this study, the influence of p53 protein on the apoptosis-inducing effect of anisomycin was investigated. The effect of protein synthesis-inhibiting concentration of anisomycin on apoptotic events was analyzed using Western blot, DNA fragmentation, and cell viability assays in wild-type PC12 and in mutant p53 protein expressing p143p53PC12 cells. Anisomycin stimulated the main apoptotic pathways in both cell lines, but p143p53PC12 cells showed lower sensitivity to the drug than their wild-type counterparts. Anisomycin caused the activation of the main stress kinases, phosphorylation of the p53 protein and the eukaryotic initiation factor eIF2α, proteolytic cleavage of protein kinase R, Bid, caspase-9 and -3. Furthermore, anisomycin treatment led to the activation of TRAIL and caspase-8, two proteins involved in the extrinsic apoptotic pathway. All these changes were stronger and more sustained in wtPC12 cells. In the presence of the dominant inhibitory p53 protein, p53- dependent genes involved in the regulation of apoptosis may be less transcribed and this can lead to the decrease of apoptotic processes in p143p53PC12 cells.

Immunohistochemical localization of cardio-active neuropeptides in the heart of a living fossil, Nautilus pompilius L. (Cephalopoda, Tetrabranchiata).

Neuropeptides play an important role in modulating the effects of neurotransmitters such as acetylcholine and noradrenaline in the heart and the vascular system of vertebrates and invertebrates. Various neuropeptides, including substance P (SP), vasoactive intestinal polypeptide (VIP) and FMRFamide, have been localized in the brain in cephalopods and the neurosecretory system of the vena cava. Previous studies involving cephalopods have mainly focussed on the modern, coleoid cephalopods, whereas little attention was paid to the living fossil Nautilus. In this study, the distributions of the peptides related to tachykinins (TKs) and the high affinity receptor for the best characterized TK substance P (tachykinin NK-1), VIP, as well as FMRFamide were investigated in the heart of Nautilus pompilius L. by immunohistochemistry. TK-like immunoreactivity (TK-LI) was seen associated to a sub-population of hemocytes, VIP-LI glial cells in larger nerves entering the heart, whereas FMRFamide immunoreactivity was distributed throughout the entire heart, including the semilunar atrioventricular valves. The pattern of FMRFamide immunoreactivity matched that of Bodian silver staining for nervous tissue. The NK-1-LI receptor was located on endothelial cells, which were also positive for endothelial nitric oxide synthase-LI (eNOS). The results indicate that neuropeptides may be involved in the regulation of the Nautilus heart via different mechanisms, (1) by direct interaction with myocardial receptors (FMRFamide), (2) by interacting with the nervus cardiacus (VIP-related peptides) and (3) indirectly by stimulating eNOS in the endothelium throughout the heart (TK-related peptides).

Hemocyanin re-uptake in the renal and branchial heart appendages of the coleoid cephalopod Sepia officinalis.

The renal and branchial heart appendages of Sepia officinalis L. were investigated in order to elucidate a possible involvement of their excretory epithelia in hemocyanin metabolism. Immunocytochemical findings and tracer experiments indicate that after passing the barrier of ultrafiltration the hemocyanin molecules are taken up by the epithelial cells of the renal and branchial heart appendages and are subsequently carried back to the circulatory system, suggesting a mechanism of hemocyanin recycling. Apart from a function in maintaining constant hemocyanin levels, the present study indicates that the renal and branchial heart appendages are also sites of temporary hemocyanin storage.

Tracer studies of food absorption in the digestive tract of Nautilus pompilius (Cephalopoda, Tetrabranchiata).

In Nautilus pompilius, tracer experiments with 14C-labelled food show that the midgut gland, caecum and crop are involved in absorption of nutrients. According to liquid scintillation and light- and electron-microscopic autoradiography, the midgut gland exhibits the highest activity, followed by the caecum and crop. The density of silver precipitates is highest in the terminal alveoli of the midgut gland. Precipitates are also seen in the main cells of the caecal epithelium. Few precipitates are found in the lamina epithelialis mucosae of the crop, indicating that, in addition to food storage, digestive processes begin in this organ. These results have been confirmed by injection of the protein ferritin into the buccal cavity. The largest amount of ferritin is seen in the dense bodies of the main cells of the midgut gland, whereas those of the main cells of the caecum and crop contain less ferritin.

Inhibitory cholinergic effects on the autonomously contractile bulbus cordis branchialis of the cephalopod Sepia officinalis L.

In vitro experiments were performed on a standardized preparation of the autonomously contractile bulbus cordis branchialis of the branchial heart of Sepia officinalis to investigate its cholinergic neuroregulation. Apart from acetylcholine, nicotine and carbachol (nicotinic agonists), the muscarinic agonists muscarine, arecoline, pilocarpine, and oxotremorine also exerted concentration-dependent negative inotropic effects on the preparations. As both the muscarinic antagonist quinuclidinylbenzilate and the nicotinic antagonist alpha-bungarotoxin blocked the ACh action there might be a special, possibly mixed muscarinic/nicotinic ACh-receptor system in the myocytes of the bulbus cordis branchialis, which is different from the cholinergic receptor in the central part of the branchial heart.

Monoamines and the isolated auricle of sepia officinalis: are there &bgr;-like receptors in the heart of a cephalopod?

Pharmacological examinations of isolated auricles from Sepia officinalis were carried out to analyze the putative role of the monoaminergic transmitter/receptor system in the control of auricle function. In conjunction with histofluorescence studies and HPLC analyses, evidence of a double excitatory serotonergic and noradrenergic innervation of the auricles was obtained. Serotonin-induced positive chronotropic and inotropic effects were blocked by mianserin (5-HT1 and 5-HT2) but not by cyproheptadine (5-HT2). It is assumed that the auricular serotonin (5-HT) receptor represents a 5-HT1-like subtype and is not identical to the ventricular 5-HT receptor. Noradrenaline, adrenaline and dopamine evoked mainly positive chronotropic reactions and less prominent positive inotropic reactions. The potency range (pD2 frequency: noradrenaline 6.65 >> adrenaline 5.69 > dopamine 5.34; pD2 amplitude: noradrenaline 6.09 (greater than or equal to) adrenaline 5.91 > dopamine 5.33) indicates out that noradrenaline might be the effective neurotransmitter in vivo. The &agr; -mimetics clonidine ( &agr; 2) and phenylephrine ( &agr; 1) induced positive chronotropic and inotropic effects, while the &bgr;-mimetics albuterol (&bgr;2>&bgr;1) and dobutamine (&bgr;1) revealed only positive inotropic reactions. The &bgr;-agonist isoprenaline mimicked the positive chronotropic effects of noradrenaline and induced the strongest positive inotropic effects of all the agonists tested. Urapidil ( &agr; 1) or phentolamine ( &agr; 1 and &agr; 2) blocked only the positive chronotropic effects of noradrenaline and isoprenaline. The positive inotropic effects of isoprenaline could be blocked by the adenylate cyclase inhibitors MDL-12,330A or SQ-22, 536, which had no effect on the chronotropic effects of isoprenaline. These results suggest that two catecholaminergic receptors are present in the auricles of Sepia officinalis: an &agr; -like adrenoreceptor mediating mainly chronotropic effects, and a &bgr;-like receptor which appears to mediate inotropic effects by activating the cyclic AMP pathway. These results suggest that the auricles exert a regulatory effect on ventricular performance.

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.

Cytological and enzyme-histochemical investigations on the digestive organs of Nautilus pompilius (Cephalopoda, Tetrabranchiata).

The foregut, stomach, caecum, midgut, and rectum of the digestive tract of Nautilus pompilius L.were investigated with ultrastructural and enzyme-cytological methods. Three different cell types were identified within the lamina epithelialis mucosae: main cells, goblet cells, and cells with secretory granules. The main cell type is the epithelial cell with microvilli, a basal nucleus surrounded by dictyosomes, rough endoplasmic reticulum, mitochondria, and electron-dense granules identified as lysosomes in the apical part of the cell. In the caecum this cell type contains endosymbiotic bacteria. The presence of endocytotic vesicles and the storage of lipids in the caecum indicate that this organ is involved in the process of absorption. In the caecum and the longitudinal groove of the rectum the main cells are, in addition, ciliated, facilitating the transport of food particles and faeces. Two types of goblet cells are found in all organs except in the stomach, forming a gliding path for food particles and protecting the epithelium. In the foregut and rectum, cells with electron-dense granules were recognized as the third type. The conspicuous secretory cells of the rectum represent a delimited rectal gland; its possible biological function is discussed. The tunica muscularis in all organs of the digestive tract consists of obliquely striated muscle cells innervated by axons containing transparent, osmiophilic and dense-cored vesicles. Positive reactions for acid and alkaline phosphatase, monoamine oxidase, beta-glucuronidase, and trypsin- and chymotrypsin-like enzymes are localized in the lamina epithelialis mucosae.

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.

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.

Localization of catecholamine-containing nerve fibres in the branchial heart and cardiac ganglion of the common cuttlefish Sepia officinalis L. (cephalopoda).

The innervation of the branchial heart of Sepia officinalis was examined using TEM and glyoxylic acid induced fluorescence. In the cardiac ganglion and in cardiac nerves bluish-green fluorophores were seen associated with perikarya and varicose nerve fibres. Microspectrofluorometric analysis provided clear evidence that monoaminergic neurons in the branchial heart contain only catecholamines. Considering pharmacological data, it is more than likely that 5-hydroxytryptamine (serotonin) is not present in this system.

Early development and myogenesis of the posterior anuran lymph hearts.

The early development of anuran posterior lymph hearts studied by light- and electron-microscopy in frog larval stages 25-29 (Gosner 1960) can be subdivided into three phases. In phase I, mesenchymal myogenic cells are found, each possessing a single 9 + 0 cilium and numerous filopode-like processes aggregated near the vena caudalis lateralis, forming up to three metameric organ anlagen arranged like a cuff around the preexisting lymphatics (stages 26/27). In phase II, cell proliferation starts at stage 28 within the lymph heart wall as does the formation of primarily polynuclear myofibres by fusion of several myoblasts. At this stage immature myofibres show a vast sarcoplasm, a poorly developed SR and only few myofibrils with not yet distinguishable A- and I-bands. In phase III, the afferent and efferent valves are formed at the onset of pulsation in stage 29. Contractile myofibres contain large glycogen fields and a considerable amount of myofibrils which frequently branch and show distinct Z-lines, A-, I-, H- and M-bands; 1-3 cilia were found lying within a channel-like cell invagination. The peculiarities of organogenesis and myofibre development are discussed.

Effects of biogenic amines and related agonists and antagonists on the isolated heart of the common cuttlefish Sepia officinalis L.

1. Effects of noradrenaline and the related compounds adrenaline, dopamine, octopamine, tyramine, clonidine and isoprenaline were studied in isolated heart preparations from the cuttlefish Sepia officinalis L. 2. All analogues produced a positive inotropic affect, with noradrenaline being the most potent substance. The chronotropic effects of the tested compounds differed widely. 3. The action of substances of the phenylethanolamine group were not antagonized by propranolol but were partly antagonized by phentolamine. 4. Serotonin and its analogues also produced cardio-excitation. These effects were blocked by cyproheptadine but not by methysergide. 5. These results indicate the presence of two different receptors in the Sepia myocardium: one type reacting with noradrenaline most effectively and a second type being stimulated by serotonin.

Cytology of the renal appendages of Nautilus (Cephalopoda, Tetrabranchiata).

The renal appendages of tetrabranchiate cephalopods, Nautilus pompilius (L.) and Nautilus macromphalus (G.B. Sow.), were studied using light- and electron-microscopical methods. The appendages, homologous to the renal appendages of dibranchiate cephalopods, possess in both species a folded transporting epithelium characterized by a secreting brush border and a high content of mitochondria and lysosome-like dense bodies as well as a basal labyrinth, and are separated from the blood lacunae by a multilayered lamina basalis. In the extracellular crypt-like infoldings of the epithelium, numerous concentric stratified concrements are found. The results strengthen the hypothesis that these concrements represent mineral-storage structures only found in calcium shell-supporting species within the class Cephalopoda.

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.