Investigation vesicle cycle in nerve formations in somatic muscle of the earthworm Lumbricus terrestris

Luminous spots with a diameter of 1-2 microm, which are clusters of "synaptic buds", were revealed in the muscular wall of the earthworm using endocytotic fluorescent dyes FM1-43, FM2-10 and FM4-64. Application of the membrane probe Dil that is capable of being subjected to anterograde axonal transport to abdominal ganglia of the nervous chain, and subsequent (in a day) staining of nerve formations by endocytotic dye FM4-64 showed complete imposition of the emission data of the dyes that fluoresce in different parts of the spectrum. Using fluorescent marker DiBAC4(3) showed an increased emission of neural elements with increasing concentration of K+ in the extracellular environment. Application of FM2-10 showed that the higher concentration of K+ in solution, and hence the depolarization of the nerve cells, the faster the upload of the dye, and vice versa, the process slowed down in the absence of K+ in the medium. The seizure and removal of FM2-10 were blocked in calcium-free solutions in the presence of Ca2+ buffers, BABTA or BABTA-AM, but only after a preliminary 40 min incubation. The processes of exo- and endocytosis occurred in the clusters of synaptic "buds" and were preserved in conditions of "rest". This vesicle cycle depends on membrane potential and concentration of K+ and Ca2+, and, it is very likely that the calcium sensor operates on the principle "all or nothing".     

Immunocytochemical studies on the central nervous system of the earthworm, Lumbricus terrestris

There are numerous aldehyde fuchsin (AF)-positive, neurosecretory cells of medium size (A cells) and a small number of large, AF-negative neurons (B cells) in the cortical layer of the cerebral ganglion. In the subesophageal ganglion, symmetrical groups of AF-positive cells lie ventrally. The peroxidase--antiperoxidase (PAP) method was used for the immunocytochemical study of substance P and ACTH in these ganglia. In addition, the presence of L-enkephalin and alpha endorphin could be confirmed. Using rabbit antibodies to substance P we found small immunoreactive neurons among negative A and B cells in the cerebral ganglion. The processes of these immunoreactive cells could be traced to the subcortical synaptic neuropil. With antibodies to ACTH, activity was visible in perikarya similar in size to A neurons. A part of the nerve terminals of the synaptic zone, some of the B neurons and further several nerve cells of the subesophageal ganglion reacted positively. Successive demonstration of substance P and ACTH on the same section showed that the two materials occurred in different cell types. Using antiopsin antibody in an indirect immunocytochemical test we observed strong reaction in numerous medium-sized perikarya and in nerve fibres of the synaptic zone of the cerebral ganglion, further in some neurons of the subesophageal and abdominal ganglia. In contrast to this result, the photoreceptor cells of the prostomium and cerebral ganglion were negative. Presumably, substance P is present in a perikaryon type hitherto unrecognized while ACTH and antiopsin reactions seem to be located first of all in A cells.     

Immunocytochemical demonstration of vertebrate neuropeptides in the earthworm Lumbricus terrestris (Annelida,Oligochaeta)

Summary The localisation and distribution of 10 vertebrate-derived neuropeptides in the earthworm, Lumbricus terrestris, have been determined by an indirect immunofluorescence technique. The peptides are pancreatic polypeptide (PP), peptide tyrosine tyrosine (PYY), neuropeptide Y (NPY), glucagon (C-terminal), vasoactive intestinal polypeptide (VIP), peptide histidine isoleucine (PHI), gastrinreleasing peptide (GRP), calcitonin gene-related peptide (CGRP), neurotensin (NT), and met-enkephalin. For 6 of the peptides — PYY, NPY, PHI, glucagon, GRP and CGRP — this is the first demonstration of their presence in any annelid, and NT has not previously been described in an oligochaete. Cell bodies and nerve fibres immunoreactive to the 10 peptides occur throughout the CNS. In the PNS, epidermal sensory cells displayed immunoreactivities to PP and PYY, and PP-, PYY-, NPY-, PHI- and GRP-like immunoreactivities occurred in nerve fibres supplying the main body muscles. Nerve fibres immunoreactive to PP and PYY are also associated with the innervation of the gut (pharynx, oesophageal glands, and mid and posterior regions of the intestine). No endocrine cells immunoreactive for any of the antisera tested could be identified in the gut epithelium, suggesting that dual location of peptides in the brain and gut epithelium is a phenomenon that occurred at a later stage in evolution. No immunoreactive elements were detected in any of the organs and ducts of the reproductive and excretory systems.     

Occurrence of ceramide-glycanase in the earthworm, Lumbricus terrestris

We have detected the presence of ceramide-glycanase in the earthworm, Lumbricus terrestris. We have also devised a simple method for the preparation of this enzyme from the earthworm. This enzyme cleaved the linkage between the ceramide and the glycan chain in LacCer, GbOse3Cer, GbOse4Cer, GbOse5Cer, GM3, GM2, GM1 and GD1a. By using tritium-labeled GM2 as substrate, the optimum pH of this enzyme was found to be between pH 4 and 4.5. In the earthworm, the ceramide-glycanase was mainly found in the muscle. The intestine was found to contain a very low level of this enzyme. Because of their easy availability, earthworms should become a convenient source for the preparation of ceramide-glycanase.     

Fine structure of photoreceptor cells in the earthworm,Lumbricus terrestris

Summary Photoreceptor cells in the epidermis and nerve branches of the prostomium and in the cerebral ganglion of Lumbricus terrestris were investigated with the electron microscope. The photoreceptor cell is similar to the visual cell of Hirudo by having a central intracellular cavity (phaosome) filled with microvilli. Besides microvilli, several sensory cilia can also be found in the phaosome but they are structurally independent of the microvilli. A gradual branching of the phaosome cavity into smaller cavities makes its sectional profile extremely labyrinthic. Flattened smooth-surfaced cisternae in stacks of 2 to 5 are frequently observed around the phaosome. Characteristic constituents of the cytoplasm are vesicles and vacuoles filled with a substance of varying density. The photoreceptor cell is covered by glial cells or by their processes which at many places deeply invaginate the cell surface (trophospongium).

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Zusammenfassung Die Feinstruktur der Photorezeptorzellen in der Epidermis, in kleineren Nervenästen und im Zerebralganglion von Lumbricus terrestris wurde untersucht. Das Vorhandensein eines zentralen, intrazellulären Lumens (Phaosom), das mit Mikrozotten gefüllt ist, erinnert in der Struktur der Photorezeptorzelle des Regenwurms an Lichtsinneszellen von Hirudo. Außer Mikrozotten findet man im Phaosom einige Zilien vom Typ 9×2+0; solche Zilien sind von den Mikrozotten strukturell unabhängig. Durch eine Aufzweigung des Phaosoms in kleinere Buchten, die tief in das umgebende Zytoplasma eindringen, erhält es ein labyrinthartiges Aussehen.Glatte Zisternen in Gruppen von 2 bis 5 wurden oft um das Phaosom im Zytoplasma beobachtet. Charakteristische Bestandteile der Zelle sind noch Vesikel und Vakuolen, die mit einer Substanz von wechselnder Elektronendichte gefüllt sind. Die Photorezeptorzellen werden von Gliazellen und Gliafortsätzen umgeben, die an vielen Stellen die Zelloberfläche tief einstülpen (Trophospongium).

     

Purification and characterization of a Ca2+-binding protein in Lumbricus terrestris.

A Ca2+-binding protein which is capable of activating mammalian Ca2+-activatable cyclic nucleotide phosphodiesterase has been purified from Lumbricus terrestris and characterized. This protein and the Ca2+-dependent protein modulator from bovine tissues have many similar properties. Both proteins have molecular weights of approximately 18,000, isoelectric points of about pH 4, similar and characteristic ultraviolet spectra, and similar amino acid compositions. Both proteins bind calcium ions with high affinity. However, the protein from Lumbricus terrestris binds 2 mol of calcium ions with equal affinity, Kdiss = 6 X 10(-6) M, whereas the Ca2+-dependent protein modulator from bovine tissues binds 4 mol of calcium ions with differing affinities. Although the Ca2+-binding protein of Lumbricus terrestris activates the Ca2+-activatable cyclic nucleotide phosphodiesterase from mammalian tissues, we have failed to detect the existence of a Ca2+-activatable phosphodiesterase activity in Lumbricus terrestris. The activation of phosphodiesterase by the Ca2+-binding protein from Lumbricus terrestris is inhibited by the recently discovered bovine brain modulator binding protein (Wang, J. H., and Desai, R. (1977) J. Biol. Chem. 252, 4175-4184). Since the modulator binding protein has been shown to associate with the mammalian protein modulator to result in phosphodiesterase inhibition, it can be concluded that the Lumbricus terrestris Ca2+-binding protein also associates with the bovine brain modulator binding protein. Attempts to demonstrate the existence of a similar modulator binding protein in Lumbricus terrestris have been unsuccessful.     

Ultrastructure of the nerve-muscle junction in the pharynx of the earthworm,Lumbricus terrestris

Summary InLumbricus terrestris the wall of the pharynx is built up from obliquely striated longitudinal and circular muscle layers. The occurrence of perikarya and nerve bundles showing green fluorescence suggests the presence of aminergic innervation in the pharynx. A significant number of chemical synapses were detected in the neuropil among axon terminals. The junctional gap is generally 100–300 nm wide in type I junctions which resemble the cholinergic motor endplates of vertebrate skeletal muscle. A narrow junctional gap of about 25 nm is characteristic of the close contacts in the type II neuromuscular junction. Agranular spherical vesicles, together with small and large dense-cored granules, fill in these axon terminals.     

Immunochemical evidence for met-enkephalin-like and leu-enkephalin-like peptides in tissues of the earthworm, Lumbricus terrestris

The presence of met- and leu-enkephalin-like immunoreactive materials in nerve, gut, seminal vesicle and body wall tissues of the earthworm Lumbricus terrestris has been demonstrated by means of radioimmunoassay technique. The greatest activity of met- and leu-enkephalin-like immunoreactivity in earthworm gut appears in regions of high digestive enzyme activity and gastrin-like immunoreactivity where it presumably plays a role in regulation of gut function. In all tissues studied the levels of met-enkephalin-like immunoreactivity were higher than that of leu-enkephalin-like immunoreactivity. Dual localization of met- and leu-enkephalin-like immunoreactivity in earthworm gut and nerve tissues follows the pattern observed of peptide hormones in vertebrates which are common to both endocrine and non-endocrine tissues.     

Histochemical localization of production and secretion of trypsin in the earthworm Lumbricus terrestris

Summary The origin of trypsin in the digestive tract of Lumbricus terrestris was localized by using the chromogenic tryptic substrate carbobenzyloxy-L-arginine--napthylamide HCl (CANA) coupled with the azo dye Fast Garnet GBC. The specificity of earthworm trypsin toward the naphthylamide substrate was confirmed by disc gel electrophoresis of homogenates of the digestive tract and of intestinal fluid. Eluted fractions were assayed for tryptic activity using the synthetic substrates benzoyl-DL-arginine-p-nitroanilide (BAPA) and p-toluenesulfonyl-L-arginine methyl ester (TAME). The peak of activity toward these substrates corresponded in electrophoretic mobility to the band of CANA activity and all activities were abolished in the presence of the tryptic inhibitor N-tosyl-L-lysyl chloromethane HCl (TLCK).In frozen sections of the pharynx, esophagus, crop, gizzard and intestine, tryptic (CANA) activity was localized consistently only in the ventral and lateral fold epithelium of the pharynx. The chromogenic reaction was completely inhibited by pre-incubation of frozen sections with TLCK.Tissues from three regions of the pharynx were fixed and studied in an attempt to correlate electron microscopic observations with the histochemical results. Whereas mucousproducing cells are generally distributed in pharyngeal epithelium, heterogeneous spherical granules were found only in the ventral and lateral fold epithelium. Observations concerning the spherical secretory granules closely paralleled those of the histochemical reaction product suggesting that the spherical secretory granules may contain the tryptic enzyme.The authors wish to thank Ruth D. Merz and Sandra K. Elson for their technical assistance. The investigation was supported in part by funds to the Instrumentation Laboratories and from the Faculty Research Committee of Miami University.     

Acute toxicity of cadmium and zinc in the earthworm (Lumbricus terrestris)

Studies are continuing to explore the use of the earthworm (Lumbricus terrestris) for the determination of the acute toxicity of metal compounds. Worms were injected intraperitoneally with cadmium and zinc chlorides, and also zinc chloride followed by cadmium chloride to see if zinc could protect against the toxicity of cadmium. The 48 h acute toxicity (LD₅₀) values were 22 and 23 mg/kg for Cd and Zn respectively and 30 mg/kg for Cd after the worms were pretreated with Zn. It appears that the earthworm can be a useful test subject for obtaining preliminary information on metal toxicity.     

Isolation,purification and partial characterization of chloragocytes from the earthworm species Lumbricus terrestris

Chloragocytes were isolated from the earthworm species Lumbricus terrestris. After mechanical dissociation and sedimentation through Percoll, a highly purified fraction of viable chloragocytes was obtained. The isolated chloragocytes accumulated the vital dye neutral red and reduced the tetrazolium dye MTT, thereby indicating cellular integrity. Time of flight flow cytometric analyses revealed a main population of large and highly granulated cells in the 30-33 m size range. Hydrolase measurements showed that -D-N-acetyl-glucosaminidase and acid phosphatase exhibited the highest activities (146.6 and 24.9 mU/mg of protein, respectively), possibly indicating a major role for these 2 hydrolases in the physiological function of chloragocytes. In contrast, other acid hydrolases such as -D-galactosidase and -D-glucuronidase had specific activities of respectively 26 and 182 times lower than the glucosaminidase. The specific activity of the membrane-bound alkaline phosphatase was comparable to that of its acid counterpart (18.9 vs. 24.9 mU/mg of protein, respectively) and this level of activity may show an important trans-membrane activity in chloragocytes. The cytoplasmic and mitochondrial enzyme isocitrate dehydrogenase had a level of activity comparable to that of the exclusively cytoplasmic enzyme lactate dehydrogenase (6.6 vs. 8.1 mIU/mg of protein, respectively). When L. terrestris chloragocyte homogenates were separated on Percoll, results showed that hydrolases and dehydrogenases were mainly associated with the lighter materials that remained above the Percoll layer. Nonetheless, the detection of significant proportions (15-25%) of the total recovered activity of acid phosphatase and-galactosidase in the enriched chloragosome fraction supports the notion that some chloragosomes may be 'lysosome-like' organelles.     

Functional interaction of syntaxin and SNAP-25 with voltage-sensitive L- and N-type Ca2+ channels.

We have used an electrophysiological assay to investigate the functional interaction of syntaxin 1A and SNAP-25 with the class C, L-type, and the class B, N-type, voltage-sensitive calcium channels. Co-expression of syntaxin 1A with the pore-forming subunits of the L- and N-type channels in Xenopus oocytes generates a dramatic inhibition of inward currents (>60%) and modifies the rate of inactivation (tau) and steady-state voltage dependence of inactivation. Syntaxin 1-267, which lacks the transmembrane region (TMR), and syntaxin 2 do not modify channel properties, suggesting that the syntaxin 1A interaction site resides predominantly in the TMR. Co-expression of SNAP-25 significantly modifies the gating properties of L- and N-type channels and displays modest inhibition of current amplitude. Syntaxin 1A and SNAP-25 combined restore the syntaxin-inhibited N-type inward current but not the reduced rate of inactivation. Hence, a distinct interaction of a putative syntaxin 1A-SNAP-25 complex with the channel is apparent, consistent with the formation of a synaptosomal SNAP receptors (SNAREs) complex. The in vivo functional reconstitution: (i) establishes the proximity of the SNAREs to calcium channels; (ii) provides new insight into a potential regulatory role for the two SNAREs in controlling calcium influx through N- and L-type channels; and (iii) may suggest a pivotal role for calcium channels in the secretion process.     

Crystallographic identification of Ca2+ and Sr2+ coordination sites in synaptotagmin I C2B domain

Synaptotagmin I has two tandem Ca(2+)-binding C(2) domains, which are essential for fast synchronous synaptic transmission in the central nervous system. We have solved four crystal structures of the C(2)B domain, one of them in the cation-free form at 1.50 A resolution, two in the Ca(2+)-bound form at 1.04 A (two bound Ca(2+) ions) and 1.65 A (three bound Ca(2+) ions) resolution and one in the Sr(2+)-bound form at 1.18 A (one bound Sr(2+) ion) resolution. The side chains of four highly conserved aspartic acids (D303, D309, D363, and D365) and two main chain oxygens (M302:O and Y364:O), together with water molecules, are in direct contact with two bound Ca(2+) ions (sites 1 and 2). At higher Ca(2+) concentrations, the side chain of N333 rotates and cooperates with D309 to generate a third Ca(2+) coordination site (site 3). Divalent cation binding sites 1 and 2 in the C(2)B domain were previously identified from NMR NOE patterns and titration studies, supplemented by site-directed mutation analysis. One difference between the crystal and NMR studies involves D371, which is not involved in coordination with any of the identified Ca(2+) sites in the crystal structures, while it is coordinated to Ca(2+) in site 2 in the NMR structure. In the presence of Sr(2+), which is also capable of triggering exocytosis, but with lower efficiency, only one cation binding site (site 1) was occupied in the crystallographic structure.