Tetanus and botulinum A toxins inhibit stimulated F-actin rearrangement in chromaffin cells.

Tetanus and botulinum A neurotoxins block exocytosis of noradrenaline in chromaffin cells. During exocytosis vesicles fuse with the plasma membrane. This requires an intact cytoskeleton. The cytoskeleton can be displayed by rhodamine-labelled phalloidin binding to F-actin. When chromaffin cells are stimulated with carbachol, F-actin forms clusters close to the plasma membrane. On withdrawal of the secretagogue the stimulated cells revert to their original appearance. The conversions, like exocytosis, depend on the presence of Ca2+, indicating the association of exocytosis with F-actin arrangement. When exocytosis is blocked in chromaffin cells by tetanus or botulinum A neurotoxins, F-actin fails to cluster during nicotinic stimulation. Thus, the toxins act somewhere between the rise in intracellular free Ca2+ and the rearrangement of F-actin.

Tetanus toxin: inhibitory action in chromaffin cells is initiated by specified types of gangliosides and promoted in low ionic strength solution.

Adrenal medullary chromaffin cells preloaded with [3H]noradrenaline release the hormone by exocytosis upon stimulation with carbachol. This exocytosis is not influenced by tetanus toxin (Tetx) which can block the release of transmitters from nerve endings. Preincubation of chromaffin cells with gangliosides followed by toxin exposure in low ionic strength solution results in a decrease of stimulated catecholamine release. The inhibition of exocytosis is most evident when cells are pretreated with the ganglioside GD lb. Cells treated with GTlb are 10 times less sensitive, those treated with GDla and GMl even 30 times less sensitive to Tetx. Thus GDlb appears to be the principal carrier for Tetx in chromaffin cells whereas the other gangliosides only play a minor role.

Distinct targets for tetanus and botulinum A neurotoxins within the signal transducing pathway in chromaffin cells.

Tetanus and botulinum A neurotoxins inhibited exocytosis evoked by various secretagogues in intact and permeabilized chromaffin cells. The block of exocytosis in intact chromaffin cells due to botulinum A neurotoxin could partially be overcome by enhancing nicotine- and veratridine-induced stimulation, whereas the block due to tetanus toxin persisted under the same conditions. The receptor-mediated restoration of 3H-noradrenaline release was specific for nicotinic stimulation, because exocytosis did not occur during muscarinic stimulation. Depolarization of intact chromaffin cells with increasing concentration of K+ failed to restore exocytosis that had been blocked by either toxin. When chromaffin cells, treated with tetanus or botulinum A neurotoxins, were exposed to the Ca2(+)-ionophore A 23187 or permeabilized by staphylococcal alpha-toxin, Ca2(+)-stimulated exocytosis was also inhibited. The inhibition was unaffected by increasing concentrations of free Ca2+. Activation of proteinkinase C and of G-proteins by phorbolester and GMPPNHP, respectively, increased Ca2(+)-induced exocytosis in control cells as well as in cells treated with tetanus and botulinum A neurotoxins. The block, however, could not be relieved by these manipulations, and it could not be relieved by activating the cGMP or cAMP pathways with analoga of cyclic nucleotides, phosphodiesterases inhibitors, and forskolin either. It is concluded that nicotine and veratridine trigger a mechanism within the sequence of events leading to exocytosis that is located beyond the increase in intracellular Ca2(+)-concentration. This pathway may not be affected by botulinum A neurotoxin. The target of tetanus toxin is probably located even closer to the fusion process, i.e. beyond the step upon which botulinum A neurotoxin acts.

The translocation of botulinum A neurotoxin by chromaffin cells is promoted in low ionic strength solution and is insensitive to trypsin.

Botulinum A neurotoxin (BoNtx) produced a partial inhibition of carbachol induced 3H-noradrenaline (3H-NA) release from bovine adrenal chromaffin cells in monolayer culture. Each of the polysialogangliosides GD1a, GT1b and GD1b enhanced the block of exocytosis when they were applied prior to the toxin exposure. The monosialoganglioside GM1 was not effective. Chromaffin cells treated with neuraminidase lost their sensitivity to BoNtx. Application of gangliosides to these cells, however, restored their susceptibility to the toxin. Treatment of the cells with trypsin did not affect the BoNtx-blockade of 3H-NA-release. The potency of botulinum A toxin was increased in a solution of low ionic strength in which sodium chloride was replaced by sucrose. In agreement with the potency of botulinum A neurotoxin in blocking exocytosis under the various conditions, binding of 125I-botulinum A neurotoxin to chromaffin cells was enhanced in low ionic strength solution and by pretreatment of the cells with gangliosides. The binding was decreased by digestion of gangliosides with neuraminidase. It is concluded that botulinum A neurotoxin binds exclusively to polysialogangliosides which subsequently serve as carriers for the toxin. The low ionic strength may increase some physico-chemical interaction of the toxin with the polysialogangliosides.

Gangliosides mediate inhibitory effects of tetanus and botulinum A neurotoxins on exocytosis in chromaffin cells.

Bovine chromaffin cells in monolayer culture were preloaded with 3H-NA (noradrenaline) and subsequently stimulated with carbachol. Botulinum A neurotoxin partially inhibited the evoked release of 3H-NA and the basal efflux of the hormone. The inhibition of evoked release did not exceed 40%, although the cells were exposed to 10 micrograms/ml of toxin for 6 days. The inhibitory effect of botulinum A neurotoxin was neutralized by its antibodies. In contrast to botulinum A neurotoxin, tetanus toxin at even higher concentrations did not influence evoked release. This difference in sensitivity could be explained by the ganglioside pattern of chromaffin cells. Ganglioside GD1a, a putative receptor for botulinum A neurotoxin, could be identified in lipophilic extracts, whereas the tetanus toxin binding gangliosides GT1b and GD1b could not be detected by means of thin-layer chromatography. Treatment of the cells with neuraminidase abolished both, GD1a and the inhibitory effect of botulinum A neurotoxin. Incubation of chromaffin cells with a mixture of gangliosides (21% GM1, 44% GD1a, 15% GD1b, 20% GT1b) not only increased the efficacy of botulinum A neurotoxin but also made the cells sensitive towards tetanus toxin. The concentration-response curve of botulinum A neurotoxin was shifted to the left about five-fold and the maximum inhibition of evoked release was increased up to 60%, even though the cells were exposed to the toxin for 3 days only. In contrast, the maximum inhibition that could be achieved by tetanus toxin was 40%. The results indicate that polysialogangliosides are important for the intracellular accumulation of these clostridial neurotoxins.

Tetanus antitoxin binds to intracellular tetanus toxin in permeabilized chromaffin cells without restoring Ca2(+)-induced exocytosis.

Tetanus toxin blocks Ca2(+)-evoked catecholamine release from permeabilized bovine adrenal chromaffin cells preloaded with gangliosides. Tetanus toxin preincubated with its specific antibodies F(ab')2 is without any effect on exocytosis. Specific antitetanus F(ab')2 presented to chromaffin cells which are pretreated with tetanus toxin and permeabilized by digitonin cannot restore exocytosis. Under the same conditions, however, 125I-labeled F(ab')2 accumulates in chromaffin cells. The accumulation depends on the presence and concentration of tetanus toxin and can be prevented by an excess of unlabeled F(ab')2. Once tetanus toxin has initiated block of exocytosis, it cannot be neutralized by binding to its specific antibody.

Urodilatin inhibits sodium reabsorption in the isolated perfused rat kidney.

The effect of human urodilatin (hURO) on kidney function has been examined in stable recirculating isolated perfused rat kidney. The concentration-dependent effects of urodilatin (URO) on isolated perfused rat kidney were analyzed at different perfusion pressures. The experiments show that at perfusion pressures of 100 or 130 mm Hg, 100 nmol/l hURO cause a significant increase of urine flow and a significant decrease of the fractional sodium reabsorption from 95.2 (SD 0.9, n = 5) to 85.6% (SD 0.9, n = 5) and 86.8 (SD 1.6, n = 5) to 77.6% (SD 3.2, n = 5), respectively. In contrast, at a perfusion pressure of 80 mm Hg sodium excretion was not increased by URO. The glomerular filtration rate was unchanged. Furthermore, the renal effects of hURO were compared with rat urodilatin (rURO) and the circulating form of rat cardiodilatin/atrial natriuretic peptide (rCDD/ANP 99-126). hURO, rURO, and rCDD/ANP produced a concentration-dependent increase in sodium excretion. However, differences in natriuretic efficiency are observed. rCDD/ANP causes a decrease in fractional sodium reabsorption from 93.6 (SD 2.6, n = 5) to 86.8% (SD 2.4, n = 5), similar to hURO. rURO shows a significantly higher natriuretic effect decreasing fractional sodium reabsorption from 92.0 (SD 0.7, n = 5) to 79.1% (SD 1.2, n = 5). From these results we conclude that the response of the isolated kidney to URO is critically dependent on the perfusion pressure and that URO exhibits tubular action on renal sodium excretion. The observed differences among the tested peptides on renal function may be due to species differences in the peptide sequence.

Urodilatin: a new peptide with beneficial effects in the postoperative therapy of cardiac transplant recipients.

Renal failure after heart transplantation (HTx) still remains a serious problem, especially when cyclosporin A is used for immunosuppression in the early postoperative therapy. To preserve good renal function without reducing immunosuppressive cyclosporin A treatment, we administered urodilatin (CDD/ANP-95-126) in a long-term, low-dose infusion in addition to the usual medication after heart transplantation. From November 1990 to June 1991, 51 patients (46 male and 5 female; mean age 48 years) were treated with a 6-20 ng/kg bw.min infusion for 96 h after HTx. The renal function and hemodynamic parameters of these urodilatin-treated patients were compared in this sequential study with 40 patients (33 male and 7 female; mean age 49 years) who had undergone HTx previously from May to November, 1990, as controls. In this phase IIa study, both groups did not differ significantly with respect to age, sex, indication for HTx, and preoperative renal function. In comparison with controls patients treated with urodilatin had a significantly better renal function: a reduction in the peak plasma creatinine (PC values day 4: 1.5 +/- 0.11 vs. 2.19 +/- 0.19 mg/dl; P = 0.002), a lower peak serum urea (SU values day 4: 109 +/- 8 vs. 154.7 +/- 8.94 mg/dl; P = 0.0036), and a lower incidence of hemodialysis (6% vs. 10%) were observed. Adequate diuresis was maintained in spite of the reduction of furosemide by more than 60% (P = 0.005) on each day of urodilatin infusion in comparison with controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Uptake of antitetanus F(ab')2 fragments into eukaryotic cells.

1. In order to introduce antitetanus immunoglobulin fragments into eukaryotic cells, either antitetanus F(ab')2 or Fab' fragments have been linked to carrier molecules. Aciclovir, horseradish peroxidase, wheat germ agglutinin, and transferrin were tried as carriers. 2. F(ab')2-aciclovir and Fab'-horseradish peroxidase were not internalized by NG108-15 neurohybridoma cells. 3. [Fab']2-wheat germ agglutinin and F(ab')2-transferrin conjugates were internalized into various cells. 4. F(ab')2-transferrin conjugates were made with three different linkers: N-succinimidyl 3-(2-pyridyldithio) propionate, bis-maleimido hexane, and bis-maleimidoethoxy propane. All three conjugates were internalized but had a different fate inside the cells.