Medizin - Open Access LMU - Teil 07/22 Archiv

Conditions are described for controlled plasma membrane permeabilization of rat pheochromocytoma cells (PC12) and cultured bovine adrenal chromaffin cells by Streptolysin O (SLO). The transmembrane pores created by SLO invoke rapid efflux of intracellular 86Rb+ and ATP, and also permit passive diffusion of proteins, including immunoglobulins, into the cells. SLO-permeabilized PC12 cells release [3H]dopamine in response to micromolar concentrations of free Ca2+. Permeabilized adrenal chromaffin cells present a similar exocytotic response to Ca2+ in the presence of Mg2+/ ATP. Permeabilized PC12 cells accumulate antibodies against synaptophysin and calmodulin, but neither antibody reduces the Ca2+-dependent secretory response. Reduced tetanus toxin, although ineffective when applied to intact chromaffin cells, inhibits Ca2+-induced exocytosis by both types of permeabilized cells studied. Omission of dithiothreitol, toxin inactivation by boiling, or preincubation with neutralizing antibodies abolishes the inhibitory effect. The data indicate that plasma membrane permeabilization by Streptolysin O is a useful tool to probe and define cellular components that are involved in the final steps of exocytosis.

We examined the expression of the neural cell adhesion molecule NCAM in a number of endocrine tissues of adult rat and in an endocrine tumor cell line. NCAM was found by immunoelectron microscopy to be present on the surface of all endocrine cells in the three lobes of the hypophysis, although staining was relatively less intense in the intermediate lobe, and in pancreatic islets. Pituicytes, hypophyseal glial cells, were also labeled for NCAM. A rat insulinoma cell line (RIN A2) also expressed NCAM as judged by immunocytochemistry. Analysis of NCAM antigenic determinants (Mr 180, 140, and 120 KD) revealed large variations in the relative proportions of NCAM polypeptides present in the different tissues. Although all tissues and cell lines expressed NCAM-140, NCAM-180 was not detected in the adenohypophysis, pancreas, or adrenal medulla, and NCAM-120 was found in none of the endocrine tissues or cell lines except at low levels in the neurohypophysis. The tumor cell line expressed significant levels of NCAM-180, which was most abundant in the neurohypophysis. These results show that NCAM expression appears to be a general property of endocrine cells, although the antigenic composition differs markedly from that in brain tissue. These data are discussed with regard to the embryological origins of the different endocrine tissues, and possible functional implications are suggested.

The inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ compartment of endocrine cells was studied with alpha-toxin- and digitonin-permeabilized rat insulinoma (RINA2) and rat pheochromocytoma (PC12) cells. The Ca2+ uptake was ATP-dependent, and submicromolar concentrations of IP3 specifically released the stored Ca2+. Half-maximal Ca2+ release was observed with 0.25-0.5 mumol of IP3/l, and the amount of Ca2+ released due to IP3 could be enhanced by additional loading of the Ca2+ compartment. Consecutive additions of the same concentration of IP3 for 1-2 h always released the same amount of Ca2+ without desensitization, providing an ideal basis to further characterize the IP3-induced Ca2+ release. Here we describe for the first time a reversible inhibitory effect of decavanadate on the IP3-induced Ca2+ release. Among the vanadium species tested (decavanadate, oligovanadate and monovanadate), only decavanadate was inhibitory, with a half-maximal effect at 5 mumol/l in both cell types. The effect of decavanadate could be overcome by increasing the amount of sequestered Ca2+ or added IP3. Decavanadate did not affect the ATP-driven Ca2+ uptake but oligovanadate was inhibitory on Ca2+ uptake. p-Hydroxymercuribenzoate (pHMB) at concentrations between 10 and 30 mumol/l also inhibited the Ca2+ release due to IP3. Thiol compounds such as dithiothreitol (DTT; 1 mmol/l) added before pHMB removed all its inhibitory effect on the IP3-induced Ca2+ release, whereas the inhibition caused by decavanadate was unaffected by DTT. Thus, the decavanadate-dependent inhibition functions by a distinctly different mechanism than pHMB and could serve as a specific tool to analyse various aspects of the IP3-induced Ca2+ release within endocrine cells.

The role of Mg2+ during the final steps of exocytosis was investigated using rat pheochromocytoma cells (PC12) permeabilized with bacterial pore forming toxins. Concentrations of free Mg2+ between 0.2 and 2 mM slightly lowered the basal but greatly enhanced the [3H]dopamine release elicited by 8 μM free Ca2+. Maximal effects were obtained at approximately 1 mM free Mg2+. At higher concentrations Mg2+ was less potent. Similar effects of Mg2+ were obtained in cells permeabilized either for small molecules (by α-toxin) or for large ones (by streptolysin O). It is concluded that millimolar concentrations of cytoplasmic Mg2+ play an important role in Ca2+ triggered exocytosis.

The heavy and light chains of botulinum A toxin were separated by anion exchange chromatography. Their intracellular actions were studied using bovine adrenal chromaffin cells permeabilized with streptolysin O. Purified light chain inhibited the Ca2+-stimulated [3H]noradrenaline release with a half-maximal effect at about 1.8 nM. The inhibition was incomplete. Heavy chain up to 28 nM was neither effective by itself nor did it enhance the inhibitory effect of light chain. It is concluded that the light chain of botulinum A toxin contains the functional domain responsible for the inhibition of exocytosis.

Cleavage of the disulfide bond linking the heavy and the light chains of tetanus toxin is necessary for its inhibitory action on exocytotic release ofcatecholamines from permeabi1ized chromaffin cells [(1989) FEBS Lett. 242, 245-248; (1989) J. Neurochern., in press]. The related botulinum A toxin also consists of a heavy and a light chain linked by a disulfide bond. The actions ofboth neurotoxins on exocytosis were presently compared using streptolysin O-permeabilized bovine adrenal chromaffin cells. Botulinum A toxin inhibited Ca2 +-stimulated catecholamine release from these cells. Addition of dithiothreitollowered the effective doses to values below 5 nM. Under the same conditions, the effective doses of tetanus toxin were decreased by a factor of five. This indicates that the interchain S-S bond of botulinum A toxin must also be split before the neurotoxin can exert its effect on exocytosis.

Tetanus toxin is one of the most poisonous substances known. It is first produced by Clostridium tetani as a single-chain protein [1,2]. Subsequent proteolytic processing leads to a pharmacologically more active [3], two-chain form consisting of disulfide-linked heavy (98 kDa) and light (53 kDa) chains. The individual chains alone produced by reduction are not neurotoxic [4]. Intoxication requires hours before transmitter release is blocked. This delay reflects the time required for the binding of the toxin to the plasma membrane and its subsequent internalization before the toxin can interact with its as yet unknown intracellular target (cf. [2]). Adrenal medullary chromaffin cells, closely related to neurones, lack the capacity to bind tetanus toxin [2] and therefore are insensitive to externally applied toxin [5,6]. Sensitivity may be introduced by pretreatment of such cells with a ganglioside mixture containing tri- and tetrasialogangliosides as putative receptors (Marxen, Fuhrmann and Bigalke, personal communication). When directly injected into these cells tetanus toxin inhibits Ca2+ -induced exocytosis as revealed by cell capacitance measurement [7]. Binding and internalization can be by-passed using permeabilized cell preparations. With these methods the cytoplasm becomes fully accessible to extrinsic substances including Ca2+, drugs and toxins. Pheochromocytoma (PC 12) or adrenal medullary chromaffin cells in culture, when permeabilized with pore-forming proteins (cf. [8]) still release catecholamines in response to micromolar concentrations of Ca2 + [9-13]. Cells can be made permeable to low molecular mass solutes with staphylococcal er-toxin [9-14] and to large proteins such as immunoglobulins with streptolysin 0 (SLO) [8,13,15]. We have recently looked for an interference of various forms of tetanus toxin or its chains with the Ca2+ -stimulated [3H]noradrenaline release from SLO-permeabilized chromaffin cells.

The pregnancy-specific glycoprotein (PSG) genes constitute a subgroup of the carcinoembryonic antigen (CEA) gene family. Here we report the cloning of four cDNAs coding for different members of the PSG family from a human fetal liver cDNA library. They are derived from three closely related genes (PSG1, PSG4 and PSG6). Two of the cDNA clones represent splice variants of PSG1 (PSG1a, PSG1d) differing in their C-terminal domain and 3′-untranslated regions. All encoded proteins show the same domain arrangement (N-RA1-RA2-RB2-C). Transcripts of the genes PSG1 and PSG4 could be detected in placenta by hybridization with gene-specific oligonucleotides. Expression of cDNA in a mouse and monkey cell line shows that the glycosylated PSG1a protein has a Mr of 65–66 kD and is released from the transfected cells. Sequence comparisons in the C-terminal domain and the 3′-untranslated regions of CEA/PSG-like genes suggests a complex splicing pattern to exist for various gene family members and a common evolutionary origin of these regions

Various rodent and primate DNAs exhibit a stronger intra- than interspecies cross-hybridization with probes derived from the N-terminal domain exons of human and rat carcinoembryonic antigen (CEA)-like genes. Southern analyses also reveal that the human and rat CEA gene families are of similar complexity. We counted at least 10 different genes per human haploid genome. In the rat, approximately seven to nine different N-terminal domain exons that presumably represent different genes appear to be present. We were able to assign the corresponding genomic restriction endonuclease fragments to already isolated CEA gene family members of both human and rat. Highly similar subgroups, as found within the human CEA gene family, seem to be absent from the rat genome. Hybridization with an intron probe from the human nonspecific cross-reacting antigen (NCA) gene and analysis of DNA sequence data indicate the conservation of noncoding regions among CEA-like genes within primates, implicating that whole gene units may have been duplicated. With the help of a computer program and by calculating the rate of synonymous substitutions, evolutionary trees have been derived. From this, we propose that an independent parallel evolution, leading to different CEA gene families, must have taken place in, at least, the primate and rodent orders.