thapsigargin and 3-4-dihydroxyphenylglycol

Disruption of neuronal Ca(2+) homeostasis plays a well-established role in cell death in a number of neurodegenerative disorders. Recent evidence suggests that proteolysis of the type 1 inositol 1,4,5-trisphosphate receptor (InsP(3)R1), a Ca(2+) release channel on the endoplasmic reticulum, generates a dysregulated channel, which may contribute to aberrant Ca(2+) signaling and neurodegeneration in disease states. However, the specific effects of InsP(3)R1 proteolysis on neuronal Ca(2+) homeostasis are unknown, as are the functional contributions of this pathway to neuronal death. This study evaluates the consequences of calpain-mediated InsP(3)R1 proteolysis on neuronal Ca(2+) signaling and survival using adeno-associated viruses to express a recombinant cleaved form of the channel (capn-InsP(3)R1) in rat primary cortical neurons. Here, we demonstrate that expression of capn-InsP(3)R1 in cortical cultures reduced cellular viability. This effect was associated with increased resting cytoplasmic Ca(2+) concentration ([Ca(2+)](i)), increased [Ca(2+)](i) response to glutamate, and enhanced sensitivity to excitotoxic stimuli. Together, our results demonstrate that InsP(3)R1 proteolysis disrupts neuronal Ca(2+) homeostasis, and potentially acts as a feed-forward pathway to initiate or execute neuronal death.

Topics: Analysis of Variance; Animals; Calcium; Calcium Signaling; Cell Line, Transformed; Cell Survival; Cells, Cultured; Cerebral Cortex; Dose-Response Relationship, Drug; Embryo, Mammalian; Enzyme Inhibitors; Female; Gene Expression Regulation; Glutamic Acid; Humans; Inositol 1,4,5-Trisphosphate Receptors; Methoxyhydroxyphenylglycol; Microtubule-Associated Proteins; Mutagenesis, Site-Directed; Nerve Degeneration; Neurons; Point Mutation; Pregnancy; Rats; Thapsigargin; Transfection

Morphological and electrophysiological evidence has been accumulated in recent years to indicate that a functional gradient exists along the septo-temporal axis of the hippocampus such that spatial memory is associated primarily with the dorsal (septal) sector while the functions of the ventral sector are not yet clearly defined. Also, the ventral hippocampus (VH) is reported to express a much smaller long term potentiation of responses to afferent stimulation than the dorsal sector. In the present study, we first confirmed that CA1 region of VH slices expresses significantly smaller LTP than the dorsal hippocampus. Strikingly, much larger LTP was obtained in VH slices following low frequency priming stimulation applied prior to the tetanic stimulation. DHPG ((S)-3,5-Dihydroxyphenylglycine hydrate) a metabotropic glutamate receptor agonist, produced a similar potentiating effect on LTP as that produced by the priming stimulation. In both cases, the spectral analysis of spontaneous electrical activity recorded from the same location in the slice revealed an increase in peak amplitude around 30 Hz. MCPG, a metabotropic glutamate receptor antagonist, and both thapsigargin and cyclopiazonic acid, inhibitors of Ca(2+) release from stores, blocked the potentiating action of both DHPG and the priming stimulation. These results indicate that the ventral hippocampus possesses different network properties compared to the dorsal hippocampus and that its ability to undergo plastic changes is controlled by a metabotropic glutamate receptor.

Topics: Animals; Brain Mapping; Dose-Response Relationship, Radiation; Drug Interactions; Electric Stimulation; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Glycine; Hippocampus; Indoles; Long-Term Potentiation; Male; Methoxyhydroxyphenylglycol; Neural Pathways; Rats; Rats, Wistar; Thapsigargin; Time Factors

The response of the semicircular canal (SCC) to the group I mGluR-selective agonist dihydroxyphenylglycine (DHPG; 300 microM) - facilitation of afferent discharge rate - was dose-dependently reduced by the phospholipase C inhibitor U-73122 (1-100 microM; IC(50): 22 microM), the smooth endoplasmic reticulum Ca(++) ATPase inhibitor thapsigargin (100 nM-3 microM; IC(50): 500 nM), and xestospongin C (100 pM-1 microM; IC(50): 11 nM), an inositol trisphosphate receptor (IP(3)R) antagonist. Ryanodine, a modulator of Ca(++)-induced Ca(++) release, biphasically facilitated, then suppressed this response (1 nM-1 mM; approximate IC(50): 50 microM). 5 mM caffeine increased the amplitude (34.6+/-13.4%) and duration (453+/-169.8%; n=4) of the response of the SCC to DHPG, while 50 mM caffeine eliminated this response (n=2). The protein kinase C inhibitor bisindolylmaleimide I-HCl (10-100 microM; n=3) and the cyclic-ADP ribose antagonist 8-Br-cyclic-ADP ribose (1-10 microM; n=3) had no effect on the response of the SCC to DHPG. These data suggest that the increase in transmitter release following activation of group I mGluRs on vestibular hair cells is associated with intracellular Ca(++) release from both IP(3)-sensitive and ryanodine/caffeine-sensitive intracellular Ca(++) stores. Such positive feedback on transmitter release may serve to enhance the contrast between the spontaneous and stimulus-evoked modes of hair cell transmitter release, thereby optimizing signal discrimination at the synapse between hair cells and vestibular afferent fibers.

Topics: Animals; Auditory Pathways; Calcium Signaling; Electrophysiology; Hair Cells, Auditory; In Vitro Techniques; Macrocyclic Compounds; Methoxyhydroxyphenylglycol; Neurotransmitter Agents; Oxazoles; Rana pipiens; Receptors, Metabotropic Glutamate; Ryanodine; Semicircular Canals; Thapsigargin

Brain macroglia are known to express a diverse array of neurotransmitter receptors whose signal transduction pathways may be subject to heteroreceptor 'cross-talk'. In the current study we have examined group 1 mGlu receptor-evoked [Ca2+]i signalling, and possible heteroreceptor cross-talk, in cultured type 2 astrocytes. The selective group 1 metabotropic glutamate (mGlu) receptor agonist (S)-3,5-dihydroxyphenylglycine (DHPG) elevated [Ca2+]i (EC50 = 1.7 +/- 0.6 microM); an effect reversed by the selective mGlu receptor antagonist (S)-alpha-methyl-4-carboxyphenylglycine (IC50 = 52.7 +/- 8.7 microM). DHPG-evoked [Ca2+]i responses were abolished by (1) thapsigargin (100 nM), implicating the involvement of internal Ca2+ stores in group 1 mGlu [Ca2+]i responses and (2) the removal of extracellular Ca2+. When applied alone, the selective adenosine A1 receptor agonist, N6-cyclopentyladenosine (CPA, 100 nM) failed to influence [Ca2+]i. However, in the presence of 1 microM DHPG, CPA potently (EC50 = 12.3 +/- 1.9 nM) increased [Ca2+]i responses. In the presence of 100 nM CPA, the efficacy of DHPG was doubled without any significant change in the DHPG EC50 value. This effect was reversed by either the selective adenosine A1 receptor antagonist, 8-cyclopentyltheophylline (IC50 = 50.3 +/- 19.9 nM) or overnight incubation with Pertussis toxin (100 ng/ml). We conclude that (1) type 2 astrocytes contain group 1 mGlu receptors coupled to [Ca2+]i signalling and (2) co-activation of adenosine A1 receptors enhances group 1 mGlu-evoked [Ca2+]i responses in these cells via a Gi/o G protein-mediated mechanism.

Topics: Adenosine; Animals; Animals, Newborn; Astrocytes; Calcium; Cells, Cultured; Cerebral Cortex; Excitatory Amino Acid Antagonists; Methoxyhydroxyphenylglycol; Purinergic P1 Receptor Agonists; Rats; Rats, Wistar; Receptor Cross-Talk; Receptors, Metabotropic Glutamate; Receptors, Purinergic P1; Thapsigargin