herbimycin and staurosporine-aglycone

Intrahippocampal administration of kainic acid (KA) induces synaptic release of neurotrophins, mainly brain-derived neurotrophic factor, which contributes to the acute neuronal excitation produced by the toxin. Two protein tyrosine kinase inhibitors, herbimycin A and K252a, were administered intracerebroventricularly, in a single dose, to attenuate neurotrophin signaling during the acute effects of KA, and their role in epileptogenesis was evaluated in adult, male Wistar rats weighing 250-300 g. The latency for the first Racine stage V seizure was 90 +/- 8 min in saline controls (N = 4) which increased to 369 +/- 71 and 322 +/- 63 min in animals receiving herbimycin A (1.74 nmol, N = 4) and K252a (10 pmol, N = 4), respectively. Behavioral alterations were accompanied by diminished duration of EEG paroxysms in herbimycin A- and K252a-treated animals. Notwithstanding the reduction in seizure severity, cell death (60-90% of cell loss in KA-treated animals) in limbic regions was unchanged by herbimycin A and K252a. However, aberrant mossy fiber sprouting was significantly reduced in the ipsilateral dorsal hippocampus of K252a-treated animals. In this model of temporal lobe epilepsy, both protein kinase inhibitors diminished the acute epileptic activity triggered by KA and the ensuing morphological alterations in the dentate gyrus without diminishing cell loss. Our current data indicating that K252a, but not herbimycin, has an influence over KA-induced mossy fiber sprouting further suggest that protein tyrosine kinase receptors are not the only factors which control this plasticity. Further experiments are necessary to elucidate the exact signaling systems associated with this K252a effect.

Topics: Analysis of Variance; Animals; Benzoquinones; Carbazoles; Cell Death; Electroencephalography; Enzyme Inhibitors; Epilepsy, Temporal Lobe; Excitatory Amino Acid Agonists; Indole Alkaloids; Kainic Acid; Lactams, Macrocyclic; Limbic System; Male; Mossy Fibers, Hippocampal; Nerve Growth Factors; Protein-Tyrosine Kinases; Rats; Rats, Wistar; Rifabutin; Seizures; Statistics, Nonparametric

Damnacanthal is an anthraquinone compound isolated from the root of Morinda citrifolia and was reported to have a potent inhibitory activity towards tyrosine kinases such as Lck, Src, Lyn and EGF receptor. In the present study, we have examined the effects of damnacanthal on ultraviolet ray-induced apoptosis in ultraviolet-resistant human UVr-1 cells. When the cells were treated with damnacanthal prior to ultraviolet irradiation, DNA fragmentation was more pronounced as compared to the case of ultraviolet irradiation alone. The other tyrosine kinase inhibitors, herbimycin A and genistein, also caused similar effects on ultraviolet-induced apoptosis but to a lesser extent. Serine/threonine kinase inhibitors, K252a, staurosporine and GF109203X, rather suppressed the ultraviolet-induced DNA cleavage. Immunoblot analysis showed that pretreatment with damnacanthal followed by ultraviolet irradiation increased the levels of phosphorylated extracellular signal-regulated kinases and stress-activated protein kinases. However, the other tyrosine kinase inhibitors did not increase the phosphorylation of extracellular signal-regulated kinases but stimulated phosphorylation of stress-activated protein kinases. Consequently, the ultraviolet-induced concurrent increase in both phosphorylated extracellular signal-regulated kinases and stress-activated protein kinases after pretreatment with damnacanthal might be characteristically related to the stimulatory effect of damnacanthal on ultraviolet-induced apoptosis.

Topics: Anthraquinones; Apoptosis; Benzoquinones; Calcium-Calmodulin-Dependent Protein Kinases; Carbazoles; Cell Line; DNA Fragmentation; Enzyme Inhibitors; Genistein; Humans; Indole Alkaloids; Indoles; Lactams, Macrocyclic; Maleimides; Microscopy, Phase-Contrast; Nuclear Proteins; Phosphorylation; Protein-Tyrosine Kinases; Quinones; Rifabutin; Staurosporine; Ultraviolet Rays

In vitro phosphorylation reactions using extracts of Streptomyces griseus cells and gamma-[32P]ATP revealed the presence of multiple phosphorylated proteins. Most of the phosphorylations were distinctly inhibited by staurosporine and K-252a which are known to be eukaryotic protein kinase inhibitors. The in vitro experiments also showed that phosphorylation was greatly enhanced by manganese and inhibition of phosphorylation by staurosporine and K-252a was partially circumvented by 10 mM manganese. A calcium-activated protein kinase(s) was little affected by these inhibitors. Herbimycin and radicicol, known to be tyrosine kinase inhibitors, completely inhibited the phosphorylation of one protein. Consistent with their in vitro effects the protein kinase inhibitors inhibited aerial mycelium formation and pigment production by S. griseus. All these data suggest that S. griseus possesses several protein kinases of eukaryotic type which are essential for morphogenesis and secondary metabolism. In vitro phosphorylation of some proteins in a staurosporine-producing Streptomyces sp. was also inhibited by staurosporine, K-252a and herbimycin, which suggests the presence of a mechanism for self-protection in this microorganism.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Alkaloids; Benzoquinones; Carbazoles; Cell Differentiation; Fungal Proteins; Indole Alkaloids; Isoquinolines; Lactams, Macrocyclic; Manganese; Peptide Fragments; Phosphorylation; Piperazines; Protein Kinase C; Protein Kinase Inhibitors; Protein-Tyrosine Kinases; Quinones; Rifabutin; Staurosporine; Streptomyces griseus

An involvement of protein tyrosine kinase in the transduction of the signals initiated by nerve growth factor (NGF) was investigated. A tyrosine kinase inhibitor, herbimycin, inhibited neurite outgrowth of rat pheochromocytoma PC12 cells induced by NGF but not that by dibutyryl-cAMP. Herbimycin and genistein blocked NGF-dependent activation of ras p21 whose essential function in neuronal differentiation has been reported. These observations suggested that tyrosine kinase activity is involved in the signaling pathways. K-252a, by contrast, inhibited NGF-induced but not EGF-dependent activation of ras p21. Tyrosine kinase activity of gp140trk, a constituent of NGF receptor, is activated by NGF for much a longer period compared to the activation of EGF receptor autokinase activity by EGF. We further demonstrated that autophosphorylation of gp140trk is selectively inhibited by K-252a.

Topics: Adrenal Gland Neoplasms; Amino Acid Sequence; Animals; Benzoquinones; Bucladesine; Carbazoles; Glycoproteins; Indole Alkaloids; Lactams, Macrocyclic; Molecular Sequence Data; Nerve Growth Factors; Oncogene Protein p21(ras); Pheochromocytoma; Phosphorylation; Protein-Tyrosine Kinases; Quinones; Rats; Receptors, Cell Surface; Receptors, Nerve Growth Factor; Rifabutin; Signal Transduction; Tumor Cells, Cultured