Toomas Neuman

Nerve growth factor increases the cyclic GMP level and activates the cyclic GMP phosphodiesterase in PC12 cells

Nerve growth factor (NGF) rapidly increases the cyclic GMP (cGMP) level about 2–3‐fold and enhances the cGMP phosphodiesterase (PDE) activity about 2‐fold in rat pheochromocytoma PC12 cells. No changes in the level of cyclic AMP (cAMP) and in the activity of cAMP PDE were found. GTP and a nonhydrolysable analog of GTP, GMP‐PCP, at 100 μM, were able to mimic the effect of NGF on the cGMP PDE activity. These results suggest that the cGMP system may be one of the second messengers of NGF action in PC12 cells.

Nerve growth factor induces rapid redistribution of F-actin in PC12 cells

Nerve growth factor (NGF) induces the redistribution of F‐actin in rat pheochromocytoma PC12 cells within 2–10 min, whereas epidermal growth factor (EGF) has no effect on microfilament organization. This redistribution of F‐actin in PC12 cells is not protein synthesis dependent, but can be blocked by methyltransferase inhibitors.

Nerve growth factor-induced rapid reorganization of microfilaments in PC12 cells: Possible roles of different second messenger systems

Nerve growth factor (NGF) induces in 2 to 10 min the redistribution of F-actin in rat pheochromocytoma PC12 cells. The NGF specificity of this phenomenon was shown by blocking it with anti-NGF antibodies. We used the rapid F-actin redistribution as an assay to study NGF second messenger systems and their inhibition or activation by specific agents. The results show that the NGF-induced effect on the microfilament system of PC12 cells can be specifically inhibited by lithium chloride and neomycin, inhibitors of the phosphoinositol system, but cannot be mimicked by TPA and acetylcholine, the activators of the phosphoinositol system. An increase in the intracellular concentration of cyclic AMP by addition of dBcAMP (but not dBcGMP) caused rapid F-actin redistribution that nonetheless differed from the NGF-induced effect. Changes in the intracellular calcium level did not have any influence on the microfilament system of PC12 cells. The specificity of the inhibition of NGF-induced effects by methylase inhibitors was questionable, since MTA- or SAH-treated PC12 cells acquired an altered morphology even in the absence of NGF or dBcAMP. Using the microfilament- and microtubule-disrupting drugs cytochalasin B and colchicine, we showed that the microtubule system in PC12 cells is required for the initiation of neurite outgrowth and that microfilament-associated filopodial activity does not appear to be necessary.

The muscarinic receptor-mediated action of acetylcholine in the gastrulating chick embryo.

Some of the muscarinic receptor-mediated effects of acetylcholine in early chick embryo cells at stages three-four by Hamburger and Hamilton were studied. Acetylcholine increased the intracellular level of cGMP about two-fold. Acetylcholine raised the intracellular level of free calcium from the basal level of 120 nM to 140 nM. Atropine, a muscarinic antagonist, blocked both the above-mentioned responses.

Changes in the acetylcholinesterase and choline acetyltransferase activities in the early development of the chick embryo

SummaryAcetylcholinesterase (AChE, EC 3.1.1.7) and choline acetyltransferase (CAT, EC 2.3.1.6) activities where studied in the early development of the chick embryo. A sharp increase in AChE activity occurred in the gastrulating embryo. The highest AChE activity was associated with hypoblast cells. By sucrose density gradient centrifugation three molecular forms of AChE with sedimentation coefficients 4.7 S, 6.8 S and 10.9 S were determined. During the gastrulation there was no remarkable change in the activity of CAT. A two-fold decrease in the CAT activity occurred at the end of gastrulation.

Isolation and characterization of nerve growth factor from Vipera berus (common viper) venom

Abstract 1. 1. Nerve growth factor from Vipera berus berus venom was purified by gel filtration on Sephadex G-100 (superfine), ion-exchange-chromatography on DEAE-Sephadex A-50 and chromatofocusing on PBE 118. 2. 2. The Vipera berus berus venom NGF consists of multiple molecular forms with pIs in the interval 9.1–9.7. All isoforms have identical mol. wts ≈35,000 ± 3000 (in gel filtration) and 17,000 ± 2000 , 15,000 ± 2000 (by SDS electrophoresis with β-mercaptoethanol). 3. 3. V. berus berus venom NGF reacted with monoclonal antibodies against Vipera lebetina NGF and caused differentiation of pheochromocytoma PC12 cells.

Monoclonal antibody immunoaffinity chromatography of the nerve growth factor from snake venoms

1. Pure monoclonal antibodies to Vipera lebetina venom nerve growth factor have been isolated by affinity chromatography using CNBr-agarose bound antigen. 2. Nerve growth factors from ten snake venoms (Vipera lebetina, Vipera russellii, Vipera berus berus, Vipera ursini, Echis carinatus, Agkistrodon halys, Bungarus caeruleus, Naja naja oxiana, Naja naja, Naja naja atra) were purified using monoclonal antibodies against NGF linked to BrCN-activated agarose.

Monoclonal antibodies against Vipera lebetina venom nerve growth factor cross-react with other snake venom nerve growth factors

Nerve growth factor (NGF) was isolated from the venom of Vipera lebetina and was purified to homogeneity as judged by SDS gel electrophoresis. The biologically active NGF was used to immunize BALB/c mouse, and the spleen cells from immunized mouse were fused with mouse PAI myeloma cells. Forty-seven hybrid cell lines, secreting monoclonal antibodies to V. lebetina NGF, were isolated and nine of them purified from ascitic fluids. The isolated antibodies define two partially overlapping epitopes of the V. lebetina NGF which are not involved in the biological activity of the molecule. Both epitopes are also present on the beta-NGF from the mouse salivary gland and on the NGFs from the following snake venoms: V. lebetina, V, ursini, V, berus berus, Echis carinatus, Bungarus caeruleus, Agkistrodon halys, Naja naja oxiana, Naja naja atra and Naja naja, but not on the bovine seminal plasma NGF. The mol. wts of the NGFs in these snake venoms were determined by Western immunoblot with monoclonal antibodies. The mol. wts of the NGFs from V. ursini (37,000), E. carinatus (36,000, 44,000) and A. halys (29,000) were determined for the first time.

Sensitive time-resolved fluoroimmunoassay of nerve growth factor and the disappearance of nerve growth factor from rat pheochromocytoma PC12 cell culture medium

A sensitive time-resolved fluoroimmunoassay of nerve growth factor (NGF) has been developed. The method is based on the unique property of the lanthanides for delayed fluorescence, which reduces substantially the endogenous fluorescence of biological substances, because the excitation of the sample and detection of the fluorescence signal are separated in time and in wavelength. Using the europium-conjugated antibodies to the NGF from Vipera lebetina (snake) venom and to the beta NGF from mouse submandibular gland in a solid-phase quantitative two-site fluoroimmunoassay, we obtained a maximal sensitivity of 10 pg/ml (0.38 pM)for mouse NGF and 40 pg/ml (1.2 pM) for snake NGF. Using this method, we investigated the disappearance of NGF from rat pheochromocytoma PC12 cell culture medium. Mouse beta NGF (5-10 ng/ml) disappeared completely after 12 h of incubation, whereas snake NGF was not substantially internalized even after 48 h.

Ultrastructural localization of adenylate cyclase and cAMP phosphodiesterase in the gastrulating chick embryo

SummaryThe ultrastructural localization of adenylate cyclase (E.C. 4.6.1.1.) and cAMP phosphodiesterase (PDE) (E.C. 3.1.4.17.) in the ectoderm of the developmental stage 4 chick embryo was studied. Adenylate cyclase was localized in the lateral surfaces of the ectodermal cells. In the primitive streak cells the enzymatic activity was observed on all the lateral surfaces, whereas in the periphery of the blastoderm the reaction product was localized in the apical parts of the lateral plasma membranes only.cAMP PDE localized in the apical cytoplasm of the ectodermal cells, with highest activity in the globular projections.