Heme oxygenase-2 acts to prevent neuronal death in brain cultures and following transient cerebral ischemia.

Heme oxygenase (HO) cleaves the heme ring to form biliverdin, which is rapidly reduced to bilirubin, carbon monoxide, and iron. HO1, the first form of the enzyme discovered, is an inducible protein, concentrated in tissues that are exposed to degrading red blood cells and stimulated by hemolysis and numerous other toxic perturbations to eliminate potentially toxic heme. By contrast, HO2 is constitutive and most highly concentrated in neural tissues. Carbon monoxide, formed from HO2, is a putative neurotransmitter in the brain and peripheral autonomic nervous system. HO1 regulates the efflux of potentially toxic iron from cells, as iron efflux is deficient in mice with targeted deletion of HO1 (HO1(-/-)), and transfection of HO1 facilitates iron efflux. Bilirubin appears to be a physiologic neuroprotectant. Activation of HO2 by phorbol esters, that stimulate protein kinase C to phosphorylate HO2, augments production of bilirubin which protects brain cultures from oxidative stress. Bilirubin itself in nanomolar concentrations is neuroprotective, while HO2 deletion (HO2(-/-)) leads to increased neurotoxicity in brain cultures and increased neural damage following transient cerebral ischemia in intact mice. Mechanisms whereby HO2 provides neuroprotection have not been clarified including whether protection is primarily associated with apoptotic or necrotic cell death. Moreover, the generality of neurotoxic stimuli influenced by HO2 has been unclear. We now demonstrate increased neuronal death in cerebellar granule cultures of HO2(-/-) mice with a selective augmentation of apoptotic death. We also demonstrate that HO2 transfection rescues apoptotic death. In intact mice, we show an increased incidence of apoptotic morphology in the penumbra area surrounding the infarct core in HO2(-/-) mice undergoing transient focal ischemia.

Bilirubin, formed by activation of heme oxygenase-2, protects neurons against oxidative stress injury.

Heme oxygenase (HO) catalyzes the conversion of heme to carbon monoxide, iron, and biliverdin, which is immediately reduced to bilirubin (BR). Two HO active isozymes exist: HO1, an inducible heat shock protein, and HO2, which is constitutive and highly concentrated in neurons. We demonstrate a neuroprotective role for BR formed from HO2. Neurotoxicity elicited by hydrogen peroxide in hippocampal and cortical neuronal cultures is prevented by the phorbol ester, phorbol 12-myristate 13-acetate (PMA) via stimulation of protein kinase C. We observe phosphorylation of HO2 through the protein kinase C pathway with enhancement of HO2 catalytic activity and accumulation of BR in neuronal cultures. The neuroprotective effects of PMA are prevented by the HO inhibitor tin protoporphyrin IX and in cultures from mice with deletion of HO2 gene. Moreover, BR, an antioxidant, is neuroprotective at nanomolar concentrations.

Glyceraldehyde-3-phosphate dehydrogenase: nuclear translocation participates in neuronal and nonneuronal cell death.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein levels increase in particulate fractions in association with cell death in HEK293 cells, S49 cells, primary thymocytes, PC12 cells, and primary cerebral cortical neuronal cultures. Subcellular fractionation and immunocytochemistry reveal that this increase primarily reflects nuclear translocation. Nuclear GAPDH is tightly bound, resisting extraction by DNase or salt treatment. Treating primary thymocytes, PC12 cells, and primary cortical neurons with antisense but not sense oligonucleotides to GAPDH prevents cell death. Because cell-death-associated nuclear translocation of GAPDH and antisense protection occur in multiple neuronal and nonneuronal systems, we propose that GAPDH is a general mediator of cell death and uses nuclear translocation as a signaling mechanism.

Cloned and expressed rat Ca2+-sensing receptor.

We have stably expressed cDNA for the rat brain Ca2+ sensing receptor in Chinese hamster ovary cells. Stimulation of phosphatidylinositol hydrolysis and arachidonic acid (AA) release displayed markedly cooperative responses to Ca2+ with Hill coefficients of 4-5. Both phosphatidylinositol and AA responses were not detected below a threshold of 1.5 mM Ca2+. Mg2+ behaved as a partial agonist with only half the maximal inositol phosphate and AA responses displayed by Ca2+ and with a more shallow concentration-response slope. The potency of Mg2+ in augmenting inositol phosphate and AA responses, in the presence of 1.5 mM Ca2+, implies that serum Mg2+ concentrations attained in clinical conditions will influence the Ca2+-sensing receptor.

Human cerebral cortical cell lines from patients with unilateral megalencephaly and Rasmussen's encephalitis.

Continuous cerebral cortical cell lines have been developed from two patients, an 11-month-old with unilateral megalencephaly and a seven-year-old with Rasmussen's encephalitis, designated HCN-1 and HCN-2, respectively. The two cell lines stain for neuronal markers such as neurofilament and neuron-specific enolase but not for non-neuronal markers such as glial fibrillary acidic protein and S-100 protein. In the presence of appropriate growth factors, the cells extend long, branched processes resembling neurons. Differentiation of HCN-1 cells can be induced with nerve growth factor, dibutyryl cyclic AMP and isobutylmethylxanthine, while for HCN-2 cells nerve growth factor, isobutylmethylxanthine and the phorbol ester 12-O-tetradecaoylphorbol-13-acetate are most effective. Immunohistochemical staining of both differentiated cell lines reveals intense staining for GABA, glutamate, somatostatin, cholecystokinin-8 and methionine enkephalin. Two human cortical neuronal cell lines have been developed which represent neuronal precursors. These cell lines propagate in culture and are capable of differentiating upon the addition of a variety of growth factors and chemical agents. These cell lines should prove to be useful models for the study of in vitro neuronal processes.

Odorants differentially enhance phosphoinositide turnover and adenylyl cyclase in olfactory receptor neuronal cultures.

Both the cAMP and the phosphoinositide (PI) second messenger systems have been implicated in olfactory signal transduction. We have developed a primary culture system of mammalian olfactory receptor neurons (ORNs; Ronnett et al., 1991a) to permit analysis of odorant-induced second messenger system activation in the intact ORN. The ability of a series of odorants to stimulate PI turnover and adenylyl cyclase was examined. All odorants stimulated both second messenger systems, although with differential potencies. Stimulation of PI turnover desensitized upon reexposure of cultures to odorant. The enhancement by single odorants of both adenylyl cyclase and PI turnover, but to varying degrees, affords a mechanism for increased specificity in olfactory signal transduction.

Primary culture of neonatal rat olfactory neurons.

We have prepared primary cultures of purified neonatal rat olfactory neurons. Dissociated olfactory epithelial cells are maintained in modified Eagle's medium with D-valine, cytosine arabinoside, and NGF. NGF is required for neuronal survival. Immunohistochemical staining is positive for the neuronal markers vimentin, olfactory marker protein, and neuron-specific enolase, but negative for the glial markers, glial fibrillary acidic protein, and S-100 protein. Physiologic concentrations of odorants stimulate cAMP accumulation in the cells. Because of their morphology, biochemical composition, and responsiveness to odorants, these cells should enhance olfactory investigations.

Odorant-sensitive adenylate cyclase: rapid, potent activation and desensitization in primary olfactory neuronal cultures.

Using primary olfactory neuronal cultures, we have demonstrated rapid, potent increases in cAMP levels and adenylate cyclase [AC; ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] activity in response to odorants. Isobutyl-methoxypyrazine is active at 1 nM. Odorant enhancement is dependent on Ca2+ concentration with maximal effects at 10-100 microM. Biphasic temporal and concentration-related effects occur with all odorants. All odorants examined elicit desensitization with AC responses abolished when odorants are reapplied immediately after removal. When reapplied 1 min after removal, odorants elicit an AC response greater than on first exposure, implying a cellular "memory" for odorants.

Human cortical neuronal cell line: establishment from a patient with unilateral megalencephaly.

A cell line has been established in continuous culture of human cerebral cortical neurons obtained from a patient with unilateral megalencephaly, a disorder associated with continued proliferation of immature neuronal cells. When differentiated in the presence of nerve growth factor, 1-isobutyl-3-methylxanthine, and dibutyryl adenosine 3',5'-monophosphate (cAMP), the cells display mature neuronal morphology with numerous long, extensively branched processes with spines and varicosities. The cells stain positively for neurofilament protein and neuron-specific enolase (selective neuronal markers) but are negative for glial markers, such as glial fibrillary acidic protein, S-100, and myelin basic protein. The cells also stain positively for the neurotransmitters gamma-aminobutyric acid (GABA), glutamate, somatostatin, cholecystokinin-8, and vasoactive intestinal polypeptide. These cells may facilitate characterization of neurons in the human central nervous system.

Phenylalkylamine-sensitive calcium channels in osteoblast-like osteosarcoma cells. Characterization by ligand binding and single channel recordings.

(-)-[3H]Desmethoxyverapamil ((-)-DMV) binds saturably to homogenates of the osteoblast-like cell lines UMR 106 and ROS 17/2.8 with KD values of 45 and 61 nM and Bmax values of 6.0 and 5 pmol/mg protein, respectively. Binding is stereoselective with (-)-DMV 8-10 times more potent than (+)-DMV. None of the dihydropyridine or benzothiazepine Ca2+ antagonists examined affect (-)-[3H]DMV binding. Monovalent cations such as Li+, Na+, and K+ inhibit (-)[3H]DMV binding in the 100-400 mM range. Divalent cations such as Ba2+, Sr2+, Ca2+, and Mg2+ are effective binding inhibitors in the 2-5 mM range. ROS 17/2.8 cells express a channel on the apical plasma membrane which conducts Ba2+ and Ca2+. With 110 mM BaCl2 or CaCl2 as charge carriers the single channel conductance is 3-5 picosiemens. In cell-excised patches the channel selects for Ba2+ over Na+ 3.3:1. In the absence of divalent ions the channel conducts Na+ ions with a single channel conductance of 13 picosiemens. This Na+ conductance decreases with physiological levels of Ca2+. The channel appears related to the (-)-[3H]DMV binding site, since its conductance is blocked by verapamil in a dose-dependent manner. Moreover, DMV blocks the channel stereoselectively with relative potencies of the isomers corresponding to their affinities for the binding site. The dihydropyridine drugs BAY K 8644 or (+)-202-791 do not affect channel opening. These binding and biophysical data indicate that osteoblast cells have a phenylalkylamine receptor associated with a Ca2+ channel.

Monoclonal antibody production by receptor-mediated electrically induced cell fusion.

Fusion of myeloma cells and B lymphocytes to form hybridomas which produce monoclonal antibodies has been a major advance, but the poor efficiency and randomness of viral or polyethylene glycol fusion techniques generally gives poor yields of specific, high affinity antibodies. High voltage electrical fields with dielectrophoresis to ensure cell alignment can fuse a limited number of cells under direct microscopic examination, but it is not possible to identify B-cells destined to secrete relevant antibodies. However, B-cells express, on their surface, antigen receptor immunoglobulins of the same antigenic specificity as the secreted antibodies. Binding of antigen to surface immunoglobulins stimulates proliferation and differentiation of B-cells into plasma cells. Here we report the use of the selective, high affinity interaction of antigen with surface immunoglobulins on B-cells to facilitate a close adherence to myeloma cells. The antigen, covalently conjugated to avidin, binds to the surface immunoglobulins on B-cells. This B-cell-antigen-avidin complex binds to biotin covalently attached to the surface of myeloma cells. An intense electric field across a bulk cell suspension then produces selective fusion of cells in contact, that is, of myeloma cells with B-cells which make the appropriate antibody. We have used this technique with several antigens, and all resultant hybridomas secrete appropriate antibodies with very high affinity.

Diffuse enkephalin innervation from caudate to globus pallidus.

The source of enkephalin in neuronal terminals of the rat globus pallidus was evaluated with electrolytic and knife cut lesions. Straight knife cuts medial to the globus pallidus or a curved knife cut just below the corpus callosum do not reduce immunofluorescence staining in the globus pallidus, indicating that cerebral cortex is not a major source of pallidal enkephalin. Electrolytic lesions of dorsal and ventral portions of the globus pallidus, and even lesions which sever medial from lateral globus, fail to reduce pallidal staining, ruling out long enkephalin neurons traversing the globus. Large electrolytic lesions of the head of the caudate do decrease dorsal pallidal fluorescence, though multiple small caudate lesions are without observable effect. Large pallidal lesions elicit a build-up of enkephalin fluorescence in the caudate. These results suggest that the caudate is the sole source of pallidal enkephalin and that the innervation diffusely converges on the smaller globus pallidus.

Temporal acquistion of enhanced fibrinolytic activity by syrian hamster embryo cells following treatment with benzo(a)pyrene.

Following treatment of Syrian hamster embryo cells with benzo(a)pyrene, the time required for the expression of enhanced fibrinolytic activity was examined. For this study, the fibrin-agarose overlay method was developed to distinguish the activity of normal and transformed colonies of hamster cells. Colonies possessing enhanced fibrinolytic activity were not observed one passage (2 weeks after treatment). Morphologically transformed colonies, which exhibited no enhanced fibrinolytic activity, were observed 8 days following treatment. In contrast to these two early changes, cells capable of growth in soft agar were observed much later (6 to 8 weeks after treatment). Untreated Syrian hamster embryo cells generally senesced and did not exhibit enhanced fibrinolytic activity. Approximately 1 of 10 untreated cultures escaped senescence and evolved as a continuous cell line; such cultures frequently exhibited enhanced fibrinolytic activity. These results suggest that the acquisition of enhanced fibrinolytic activity, while perhaps not a cause of neoplastic transformation, may reflect a loss of control of the normal function of the cellular genetic apparatus during the process of transformation.