guanosine-diphosphate and staurosporine-aglycone

The effects of 5-hydroxytryptamine (5-HT) on an inward current activated by extracellular ATP were investigated in rat pheochromocytoma PC12 cells. Under whole-cell voltage-clamp conditions 5-HT (10 microM) reversibly enhanced the amplitude of the current activated by 30 microM ATP. The enhancement may not be due to an increase in the number of functional channels because the current activated by 300 microM ATP was not remarkably augmented compared with the current activated by 30 microM ATP. The current enhancement by 100 microM 5-HT was less obvious than that by 10 microM 5-HT. When the current kinetics were compared, activation of the ATP-evoked current was accelerated to the same extent by either 10 or 100 microM 5-HT, whereas deactivation was largely more accelerated by 100 microM 5-HT. Propranolol (10 microM), a 5-HT1 receptor antagonist, or LY53857 (10 microM), a 5-HT2 receptor antagonist, exerted an agonistic effect: the ATP-activated current was facilitated by these compounds. Metoclopramide (10 microM), a 5-HT3 receptor antagonist, neither facilitated the ATP-activated current, nor blocked the current facilitation by 5-HT. Guanosine 5'-O-(2-thiodiphosphate) (GDP[beta S]) (2 mM), the non-hydrolysable analog of guanosine 5'-triphosphate (GTP), or K-252a (2 microM), a protein kinase inhibitor, did not affect the facilitation by 5-HT of the ATP-activated current when they were included in the intracellular solution. The ATP-activated current pre-facilitated by 10 microM dopamine was not enhanced by 10 microM 5-HT. Similarly, the pre-facilitation by 5-HT attenuated the current enhancement by dopamine.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenosine Triphosphate; Animals; Carbazoles; Dopamine; Guanosine Diphosphate; Indole Alkaloids; Ion Channels; Kinetics; Patch-Clamp Techniques; PC12 Cells; Protein Kinase C; Rats; Receptors, Serotonin; Serotonin; Serotonin Antagonists

Movements of membrane-bounded organelles through cytoplasm frequently occur along microtubules, as in the neuron-specific case of fast axonal transport. To shed light on how microtubule-based organelle motility is regulated, pharmacological probes for GTP-binding proteins, or protein kinases or phosphatases were perfused into axoplasm extruded from squid (Loligo pealei) giant axons, and effects on fast axonal transport were monitored by quantitative video-enhanced light microscopy. GTP gamma S caused concentration-dependent and time-dependent declines in organelle transport velocities. GDP beta S was a less potent inhibitor. Excess GTP, but not GDP, masked the effects of coperfused GTP gamma S. The effects of GTP gamma S on transport were not mimicked by broad spectrum inhibitors of protein kinases (K-252a) or phosphatases (microcystin LR and okadaic acid), or as shown earlier, by ATP gamma S. Therefore, suppression of organelle motility by GTP gamma S was guanine nucleotide-specific and evidently did not involve irreversible transfer of thiophosphate groups to protein. Instead, the data imply that organelle transport in the axon is modulated by cycles of GTP hydrolysis and nucleotide exchange by one or more GTP-binding proteins. Fast axonal transport was not perturbed by AlF4-, indicating that the GTP gamma S-sensitive factors do not include heterotrimeric G-proteins. Potential axoplasmic targets of GTP gamma S include dynamin and multiple small GTP-binding proteins, which were shown to be present in squid axoplasm. These collective findings suggest a novel strategy for regulating microtubule-based organelle transport and a new role for GTP-binding proteins.

Topics: Animals; Axonal Transport; Axons; Carbazoles; Decapodiformes; Ethers, Cyclic; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Indole Alkaloids; Kinetics; Microcystins; Microtubules; Okadaic Acid; Peptides, Cyclic; Phosphoprotein Phosphatases; Protein Kinase Inhibitors; Thionucleotides

NGF and BDNF elevate cAMP and IP3 levels in membranes of PC12 cells within a subsecond time period. The cAMP formation induced by NGF and BDNF pretreatment for 2 s was reduced by GDP-beta-S and PTX, but not the trkNGFR inhibitor K 252a. NGF, but not BDNF, induced IP3 formation. IP3 formation was reduced by K 252a, but not by GDP-beta-S and PTX. Using p75NGFR expressing, but trkNGFR-deficient PCNA cell membranes, NGF and BDNF induced cAMP formation, but not IP3 formation. We suggest that NGF and BDNF induced cAMP formation is mediated via a p75NGFR/G-protein mediated mechanism, and IP3 formation via a K 252a sensitive pathway.

Topics: Animals; Carbazoles; Cell Membrane; Cyclic AMP; Guanosine Diphosphate; Indole Alkaloids; Inositol 1,4,5-Trisphosphate; Naphthalenes; Nerve Growth Factors; Nerve Tissue Proteins; PC12 Cells; Polycyclic Compounds; Receptors, Nerve Growth Factor; Thionucleotides

L-Glutamate inhibits the K+ conductance that dominates the electrical behavior of a Müller glial cell. The effect of glutamate is enhanced by simultaneous exposure to dopamine. L-Glutamate acts at a metabotropic receptor that controls the K+ conductance through two pathways. A rapid pathway produces a partial inhibition in less than 2 s. Thereafter, a slow pathway progressively inhibits the conductance with a half-time of minutes. Pathways initiated by L-glutamate and dopamine appear to converge on and stimulate adenylyl cyclase. A subsequent step is the activation of a cAMP-dependent protein kinase, PKA. The local overflow of L-glutamate from active synapses may functionally remove K+ channels from nearby glial membranes. A uniform rise in extracellular L-glutamate concentration, as might occur during pathological conditions, should suppress a glial cell's K+ conductance and allow other voltage-dependent processes to be influenced by depolarization.

Topics: Adenylate Cyclase Toxin; Ambystoma; Animals; Carbazoles; Electric Conductivity; Electric Stimulation; Glutamates; Glutamic Acid; Guanosine Diphosphate; In Vitro Techniques; Indole Alkaloids; Kinetics; Membrane Potentials; Models, Neurological; Neuroglia; Potassium Channels; Protein Kinase C; Retina; Thionucleotides; Time Factors; Virulence Factors, Bordetella

Activation of p21ras, demonstrated directly as an increase in p21ras-associated GTP, was induced rapidly but transiently by both nerve growth factor (NGF) and epidermal growth factor (EGF) in PC12 cells. The factors activate p21ras to equal extents and with virtually identical time courses. Growth factor-induced p21ras activation and tyrosine phosphorylation have similar time courses and sensitivities to genistein inhibition, indicating that p21ras activation is a result of tyrosine kinase activity. Furthermore, PC12 mutants lacking the Trk NGF receptor tyrosine kinase also lack NGF-inducible p21ras activation. The protein kinase inhibitor K252a and the methyltransferase inhibitor MTA abolish NGF-induced, but not EGF-induced, p21ras activation--effects correlated with inhibition only of NGF-induced tyrosine phosphorylation. In spite of differences in sensitivity to genistein, MTA, and K252a, EGF- and NGF-stimulated p21ras activation are not additive, implying that they do share at least one step in common.

Topics: Animals; Carbazoles; Epidermal Growth Factor; Genistein; Guanosine Diphosphate; Guanosine Triphosphate; Indole Alkaloids; Isoflavones; Kinetics; Nerve Growth Factors; PC12 Cells; Phosphorylation; Protein-Tyrosine Kinases; Proto-Oncogene Proteins p21(ras); Tyrosine