The dependence of thermopreferendum in Helix pomatia L. on air temperature

Behavioural tests with 192 specimen of the roman garden snail Helix pomatia L. were performed in order to clarify whether the thermopreferendum of this pulmonate is influenced not only by the temperature of the substratum but also by air temperature. For this purpose, the snails were exposed to a horizontal temperature gradient of the substratum at different air temperatures between 10 and 30 degrees C. The results show that the selection of substratum temperature depends on the air temperature: the lower the air temperature, the lower is the temperature of the substratum preferred by the snails. Obviously, the animals tend to keep the difference between air temperature and temperature of the substratum as small as possible. This behaviour might be a mechanism to reduce the temperature gradient in the snail's body and to adjust the metabolic rate to cardiac output.     

The effects of aestivation on the catalytic and regulatory properties of pyruvate kinase from Helix aspersa.

1. Pyruvate kinase was partially purified from the foot, mantle, and digestive gland of active and aestivating snails. 2. At pH 7.0 the apparent Km values for phosphoenolpyruvate (PEP) were 0.064 mmol/l for the enzyme from foot and 0.071 mmol/l for the enzyme from mantle; those for ADP were 0.35 mmol/l for the foot enzyme and 0.33 mmol/l for the mantle enzyme. 3. Both enzymes were inhibited by alanine, and this could be reversed by fructose 1,6-bisphosphate (FBP), although FBP alone was a weak activator. 4. Decreasing the pH to 6.5 markedly increased the inhibition by alanine and reduced the response to FBP. 5. The enzymes from these tissues of aestivating snails showed a small decrease in their affinity for PEP and a small increase in the effectiveness of alanine as an inhibitor. 6. These changes are indicative of a down-regulation of this enzyme which is consistent with the observations in other species during metabolic depression. 7. In contrast the enzyme from the digestive gland of active animals showed sigmoidal saturation kinetics for PEP with a S0.5 of 1.2 mmol/l, but had a markedly higher affinity for PEP, S0.5 = 0.20 mmol/l during aestivation. This may be indicative of other metabolic changes occurring in the digestive gland.     

Pre- and postsynaptic effects of eugenol and related compounds on Helix pomatia L. neurons

We showed how eugenol blocks the synaptic transmission and gave a possible interpretation how it inhibits the excitation-contraction coupling that several authors described previously. Eugenol acts both in the pre- and postsynaptic side of the neurons. It blocks the Ca2+-currents, decreases the membrane potential of the neurons, increases the inward resistance and decreases the GABA, ACh and glutamate evoked excitatory responses in submillimolar concentration.     

The tubular endoplasmic reticulum in the amoebocytes of the shell-regenerating snail,Helix pomatia L.

Summary The tubular endoplasmic reticulum has been studied in the amoebocytes which are present in the connective tissue of the hepatopancreas of the snail, Helix pomatia. The reticulum is similar to that previously described within the glandular cells of the hepatopancreas. Two distinct components are recognizable in the reticulum—central main tubules approximately 100 m in diameter and connecting tubules about 20 m in width. The profile of this tubular network in cross-sections appears as a very regular, apparently crystalline array. The tubules are intimately associated with dense granular material, dense bodies and with mitochondria. The possible function of the tubular endoplasmic reticulum is discussed.This investigation was supported by grants from the Swedish Natural Science Research Council, which are gratefully acknowledged. I am indebted to Miss G. Drugge for her technical assistance.     

Ultrastructural study of the shell-repair membrane in the snail,Helix pomatia L.

Summary The shell-repair membrane of the snail, Helix pomatia, has been studied with the transmission electron microscope (TEM). The ultrastructure of the repair membrane, in the initial stages of calcification, revealed the presence of a fibrillar protein, proteoglycan granules, osmiophilic vesicles, and cytoplasmic dense bodies of different size and structure. The involvement of the cell constituents in the formation of calcifying centra and initial crystal formation is discussed. The amoebocytes present within the repair membrane appeared to be involved in three different functions: (1) phagocytosis, (2) release of granules, vesicles and dense bodies, and (3) secretion of a fibrillar protein. The possible lytic function of the amoebocytes is mentioned. The common features in the mineralizing process of the shell-repair membrane of the snail and the epiphyseal cartilage of the mammals were noted.This investigation was supported by a grant from the Swedish Natural Science Research Council, which is gratefully acknowledged. I am indebted to Miss Ch. Stensjö and Mrs. E. Hellmén for their technical assistance     

Effects of alpha-ketoisovalerate on bovine heart pyruvate dehydrogenase complex and pyruvate dehydrogenase kinase

The regulatory effects of alpha-ketoisovalerate on purified bovine heart pyruvate dehydrogenase complex and endogenous pyruvate dehydrogenase kinase were investigated. Incubation of pyruvate dehydrogenase complex with 0.125 to 10 mM alpha-ketoisovalerate caused an initial lag in enzymatic activity, followed by a more linear but inhibited rate of NADH production. Incubation with 0.0125 or 0.05 mM alpha-ketoisovalerate caused pyruvate dehydrogenase inhibition, but did not cause the initial lag in pyruvate dehydrogenase activity. Gel electrophoresis and fluorography demonstrated the incorporation of acyl groups from alpha-keto[2-14C]isovalerate into the dihydrolipoyl transacetylase component of the enzyme complex. Acylation was prevented by pyruvate and by arsenite plus NADH. Endogenous pyruvate dehydrogenase kinase activity was stimulated specifically by K+, in contrast to previous reports, and kinase stimulation by K+ correlated with pyruvate dehydrogenase inactivation. Maximum kinase activity in the presence of K+ was inhibited 62% by 0.1 mM thiamin pyrophosphate, but was inhibited only 27% in the presence of 0.1 mM thiamin pyrophosphate and 0.1 mM alpha-ketoisovalerate. Pyruvate did not affect kinase inhibition by thiamin pyrophosphate at either 0.05 or 2 mM. The present study demonstrates that alpha-ketoisovalerate acylates heart pyruvate dehydrogenase complex and suggests that acylation prevents thiamin pyrophosphate-mediated kinase inhibition.     

Nerve cells and synaptic connections in the intestinal nerve of the snail,Helix pomatia L.

Summary The ultrastructure of nerve cells and the finestructural organization of synaptic contacts have been investigated in the intestinal nerve in the snail Helix pomatia. Three types of nerve cells, occurring singly or in groups, can be distinguished on the basis of the ultrastructure of their perikaryon and content of granules. The peripheral output of these nerve cells has been verified by retrograde CoCl₂ and NiCl₂ staining. Both axosomatic and axo-axonic specialized synaptic contacts occur in the intestinal nerve. Presynaptic elements of these synaptic contacts contain 100–120 nm granular vesicles or 120–200 nm neurosecretory-like granules. Following intracellular horseradish peroxidase (HRP) labelling of identified central neurons responsible for peripheral regulatory processes, several labelled axons running toward the periphery can be followed throughout the branches of the intestinal nerve. These labelled axon processes (either primary axon or small collaterals) form specialized synaptic contacts, inside the intestinal nerve, and are always in a postsynaptic position. The occurrence of peripheral axo-somatic and axo-axonic synapses provides a morphological basis for integrative processes taking place in the intestinal nerve (peripheral nervous system) of Helix pomatia.     

The pathway mediating insulin's effects on pyruvate dehydrogenase bypasses the insulin receptor tyrosine kinase

The effect of insulin on pyruvate dehydrogenase activity was examined in two different cell types that over expressed either normal or defective human insulin receptors, RAT 1 embryonic fibroblasts and Chinese hamster ovary (CHO) cells. Insulin stimulated pyruvate dehydrogenase activity in cells that expressed normal insulin receptors (RAT 1 HIRc, and CHO-WT and CHO-T cells), or receptors in which lysine 1018 in the ATP-binding site of the tyrosine kinase domain was exchanged for alanine (RAT 1 A/K1018 and CHO-mut cells). For both rat and hamster cell lines, the insulin dose-response curves from cells that expressed the mutant receptors were identical to those from the appropriate controls that over expressed the normal insulin receptors. Insulin failed to stimulate pyruvate dehydrogenase activity in CHO-delta cells, which expressed a mutant human insulin receptor that was truncated by 112 amino acids at the carboxyl terminal of the beta chain. Control studies verified that all the cells used in this study exhibited the expected phenotypes with respect to the number of insulin receptors which they expressed, insulin-stimulated tyrosine kinase activity, and the biological consequences of inactivating the insulin receptor tyrosine kinase. These findings show that the insulin receptor tyrosine kinase does not play an obligatory role in the insulin signaling pathway that stimulates pyruvate dehydrogenase activity.     

Insulin reverses effects of starvation on the activity of pyruvate dehydrogenase kinase in cultured hepatocytes.

In tissue culture of hepatocytes, insulin (0.1-1 munits/ml for 4 h) reversed completely the effects of starvation of rats to decrease the activity of pyruvate dehydrogenase (PDH) complex and to increase the activities of PDH kinase and PDH kinase activator protein. It had no effect in hepatocytes from fed rats. Significant effects of insulin were detected with 0.01 munit/ml after 4 h, and in 1-2 h with 1 munit/ml.     

Cryoenzymologic studies on arginine kinase: solvent, temperature and pH effects on the overall reaction.

The overall reaction catalyzed by the phosphotransferase arginine kinase was studied at normal and subzero temperatures. Ethylene glycol was used as the antifreeze and its effects on the Km values of substances, kcat and pH profiles were investigated in detail. a) The Km values for the substrate (2 mM for ATP and 0.6 mM for arginine) were little affected by the solvent composition or temperature of the reaction mixture. b) At concentration of ethylene glycol higher than 40% there was a sharp drop of enzyme activity. c) Ethylene glycol induces a large shift in the enzymic pK D) At -5 degrees C in 40% of solvent there was a break in the Arrhenius plot suggesting a change of the rate-limiting step. The relevance of these results to the reaction pathway of arginine kinase is discussed. In addition, controlled perturbations induced by cosolvent and temperature appear as useful tools for further kinetic investigations.     

The fine structural organization of sensory nerve endings in the lip of Helix pomatia L.

The fine structural and cytochemical characteristics of sensory nerve cells have been studied in the lip of Helix pomatia. A ruthenium red positive cuticular layer was found on the surface of the sensory epithelium. Among the undifferentiated epithelial cells two types of sensory dendrites were observed, namely ciliated and non-ciliated ones. A large amount of smooth surfaced endoplasmic reticulum, microtubes and ribosomes were present in the neuroplasm of the sensory dendrites. However, rough surfaced endoplasmic reticulum, mitochondria, and electrodense bundles of long filaments were characteristic in the simple epithelial cells. The cell bodies of the sensory dendrites lie subepithelially among the muscle cells and they generally contain empty or dense core vesicles.     

Acid phosphatase in the contents of the oesophagus and stomach of the snail,Helix pomatia L.

• 1.1. In the contents of the oesophagus and stomach, one form of acid phosphatase is found. Its electrophoretic mobility is identical to that of the multiple form 3 of acid phosphatase from the hepatopancreas.
• 2.2. The enzyme is not stimulated by divalent cations. It is inhibited by molybdate, Cu²⁺, Hg²⁺. F⁻ and tartrate L+.
• 3.3. The optimum pH of the enzyme is 4.5. The K_m for paranitrophenylphosphate as substrate amounts to 0.25 mM. The enzyme is stable at a temperature of up to 55°C.

     

The ultrastructure of the sensory cells in the chemoreceptor of the ommatophore of Helix pomatia L.

Most of the sensory cells found in the chemoreceptor of the ommatophore of Helix pomatia are typical bipolar cells. The chemoreceptor is deveded by a furrow into two parts; within the ventral subdivision the layer of sensory cell bodiesis thicker than in the dorsal part. According to the differentiations of the apical surface of the dendrites, it is possible to distinguish six different classes: a) dendrites with one cilium and 75 nm thick cytofila (sometimes dendrites of identical appearance posses more than one cilium); b)dendrites with several cilial and 150 nm thick cytofila; c) dendrites with several cilia, 50 nm thick cytofila, and long, striated rootlets; d) dendrites with several cilia bur without cytofila; e) dendrites with 130 nm thick cytofila but without cilia; and f) dendrites with 65 nm thick cytofila but without cilia; dendrites of this class are the only ones with a cytoplasm more electron dense than that of the surrounding supporting cells. All these dendrites are connected to the surrounding supporting cells by terminal bars, each consisting of zonula adhaerens, aonula intermedia and zonula septata. The perikarya of the sensory cells measure approximately 15 mum by 8 mum and enclose 10 mum by 6 mum large nuclei. Axons, originating from these perikarya, extend to the branches of the digital ganglion. In the distal part of this gangloin the axons come into synaptic contact with interneurons, but in our electron micrography it was not possible to coordinate processes and synapses with the corresponding neurons.     

Granular cells in the connective tissue of Helix pomatia L. (Gastropoda,Pulmonata)

Summary Granular cells (cells crowded with colourless granules staining with paraldehyde fuchsin according to Gomori-Gabe and not containing calcium) are independent cells in the connective tissue of Helix pomatia. Histochemical data suggest that the granules are rich in sulfhydryl-containing proteins, but lack biogenic monoamines. Electron microscopic investigations confirm the supposed secretory activity of the granular cells. Secretory proteins are presumed to be synthetized in the endoplasmic reticulum and condensed in the Golgi apparatus giving rise to the granules. Extrusion occurs by exocytosis.Electrophoresis of homogenates, prepared from tissues containing numerous granular cells, results in the separation and identification of a secretory protein from the granular cells. An electrophoretically homologous protein is recognized in the hemolymph, but in very small quantities.Our findings and the work of others suggest the involvement of granular cells in neuroendocrine events.The author is indebted to Prof. Dr. D. Kuhlmann for suggesting the problem and for his valuable criticism during the investigation. I would like to thank Mr. J.N. Howell, who helped with the English.Part of this work has been supported by the Stiftung Volkswagenwerk and by the Deutsche Forschungsgemeinschaft