(1/253) The mode of action of contractile effects induced by external calcium and its related bivalent cations in the KCl-depolarized rat uterus.
The cumulative log dose-response curve (DRC) isometrically produced by CaCl2 on depolarized uteri of rats in Ca2+-free KCl Ringer's solution (pH 7.8) showed a plateau-shaped type, when responses were plotted as a percent of the maximal tension of 50 micrometer acetylcholine, whereas those produced by BaCl2 or SrCl2 were a simple sigmoid type with the maximal response near the height of the plateau induced by Ca2+. The plateau phase of Ca2+ was inhibited competitively by Mg2+ (0.5--50 mM) and non-competitively by Mn2+ (30 micrometer--1 mM), whereas the maximal contraction of Ca2+ was not inhibited by either ion. Dantrolene (0.1 mM) inhibited both the plateau and the maximal contraction of Ca 2+ and retained the plateau-shaped type. EGTA (2mM) potentiated the plateau to the maximal response level and changed the DRC induced by Ca2+ to the simple sigmoid type. From these results, the plateau of DRC induced by Ca2+ seems to be mainly due to actions of external Ca2+ on the calcium binding of internal surface of cell membrane, suggesting that the internal surface is the site of the bivalent cations tested. (+info)
(2/253) Seizure-induced cell death produced by repeated tetanic stimulation in vitro: possible role of endoplasmic reticulum calcium stores.
Seizures may cause brain damage due to mechanisms initiated by excessive excitatory synaptic transmission. One such mechanism is the activation of death-promoting intracellular cascades by the influx and the perturbed homeostasis of Ca2+. The neuroprotective effects of preventing the entry of Ca2+ from voltage-dependent Ca2+ channels, NMDA receptors, and non-NMDA receptors, is well known. Less clear is the contribution to excitotoxicity of Ca2+ released from endoplasmic reticulum (ER) stores. We produced epileptiform discharges in combined entorhinal cortex/hippocampus slices using repeated tetanic stimulation of the Schaffer collaterals and assessed cell death after 1, 3, or 12-14 h with gel electrophoresis of genomic DNA and immunohistologically using terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine 5'-triphosphate (dUTP) nick end labeling (TUNEL) staining. We manipulated ER Ca2+ stores using two conventional drugs, dantrolene, which blocks the Ca2+ release channel, and thapsigargin, which blocks sarco-endoplasmic reticulum Ca2+-ATPases resulting in depletion of ER Ca2+ stores. To monitor epileptogenesis, and to assess effects attributable to dantrolene and thapsigargin on normal synaptic transmission, extracellular potentials were recorded in stratum pyramidale of the CA1 region. Repeated tetanic stimulation reliably produced primary afterdischarge and spontaneous epileptiform discharges, which persisted for 14 h, the longest time recorded. We did not observe indications of cell death attributable to seizures with either method when assessed after 1 or 3 h; however, qualitatively more degraded DNA always was observed in tetanized slices from the 12- to 14-h group compared with time-matched controls. Consistent with these data was a significant, fourfold, increase in the percentage of TUNEL-positive cells in CA3, CA1, and entorhinal cortex in tetanized slices from the 12- to 14-h group (16. 5 +/- 4.4, 33.7 +/- 7.1, 11.6 +/- 2.1, respectively; means +/- SE; n = 7) compared with the appropriate time-matched control (4.1 +/- 2.2, 7.3 +/- 2.0, 2.8 +/- 0.9, respectively; n = 6). Dantrolene (30 microM; n = 5) and thapsigargin (1 microM; n = 4) did not affect significantly normal synaptic transmission, assessed by the amplitude of the population spike after 30 min of exposure. Dantrolene and thapsigargin also were without effect on the induction or the persistence of epileptiform discharges, but both drugs prevented seizure-induced cell death when assessed with gel electrophoresis. We suggest that Ca2+ entering a cell from the outside, in addition to the Ca2+ contributed from ryanodine-sensitive stores (i.e., Ca2+-induced Ca2+ release), may be necessary for seizure-induced cell death. (+info)
(3/253) Simultaneous evaluation of spatial working memory and motivation by the allocentric place discrimination task in the water maze in rats.
In order to evaluate learning and memory deficits separately from and simultaneously with motivational, motor and sensory impairments in identical animals, we developed the allocentric place discrimination task test using a water maze in rats. For this assessment task, two similar, visible platforms, one was fixed and the other was floating, were simultaneously present in a pool, and the working memory of the allocentric place discrimination task was evaluated. After training, the task accuracy was high about 85% correct and animals were used repeatedly. The accuracy decreased significantly when the pool was surrounded with a black curtain. Muscarinic receptor antagonist scopolamine 0.5 mg/kg selectively impaired the accuracy. Muscle relaxant dantrolene 10 mg/kg selectively decreased swimming speed. Under low motivational condition (warm water), still time increased and swimming speed decreased, but the accuracy was not affected. Similar to warm water, opioid receptor agonist morphine 15 mg/kg increased still time and decreased swimming speed. These results suggest that the allocentric place discrimination task is useful in evaluating spatial working memory ability independently of and concurrently with also visual, motor ability and motivation in identical animals. (+info)
(4/253) Mobilization of GLUT-4 from intracellular vesicles by insulin and K(+) depolarization in cultured H9c2 myotubes.
The insulin-responsive glucose transporter, GLUT-4, moves from an intracellular compartment to the cell surface in response to insulin and/or muscle contraction. Treatment of H9c2 myotubes with insulin significantly increased uptake of 2-deoxyglucose. Depolarization of the myotubes by increasing extracellular [K(+)], which mimics the initial phases of excitation-contraction coupling, also increased 2-deoxyglucose uptake. The K(+)- but not insulin-evoked increase was blocked by dantrolene, an inhibitor of Ca(2+) release from the sarcoplasmic reticulum. In contrast, wortmannin, an inhibitor of phosphatidylinositol 3-kinase, blocked insulin- but not K(+)-stimulated 2-deoxyglucose uptake. Increased glucose uptake in response to insulin or K(+) depolarization was associated with increased GLUT-4 in plasma membranes and depletion of a population of small intracellular GLUT-4-containing vesicles. Similarly, in H9c2 cells transfected with c-myc-tagged GLUT-4, translocation of c-myc GLUT-4 to the cell surface was increased after stimulation with insulin or K(+) depolarization. Taken together, these data demonstrate that insulin and K(+) depolarization increase glucose uptake by recruiting GLUT-4 from intracellular vesicles to the plasma membrane of H9c2 myotubes via distinct signaling mechanisms. (+info)
(5/253) Suspected recurrence of malignant hyperthermia after post-extubation shivering in the intensive care unit, 18 h after tonsillectomy.
A 25-yr-old man, subsequently shown to be malignant hyperthermia (MH) susceptible by in vitro contracture testing, developed MH during anaesthesia for tonsillectomy. Prompt treatment, including dantrolene, led to rapid resolution of the metabolic crisis. Eighteen hours later the patient's trachea was extubated in the ICU, when he had been stable and apyrexial overnight. Twenty minutes after extubation, an episode of shivering was followed by the onset of tachycardia, hypertension, tachypnoea and a rapid increase in temperature. Recurrence of MH was suspected and the patient was given another dose of dantrolene with good clinical effect. Shivering in this patient may have been an indicator or a causative factor of recurrence of MH. (+info)
(6/253) Calcium release from internal stores is required for the generation of spontaneous hyperpolarizations in dopaminergic neurons of neonatal rats.
We recently have demonstrated the existence of spontaneous hyperpolarizations in midbrain dopaminergic neurons of neonatal but not adult rats. These events are mediated by the opening of apamin-sensitive K(+) channels after a rise in the intracellular concentration of Ca(2+). They are resistant to tetrodotoxin in most cases and are probably endogenous (i.e., not synaptically activated). Here their mechanism was investigated. Cyclopiazonic acid (10 microM), a specific inhibitor of endoplasmic reticulum Ca(2+) ATPases, reversibly abolished the events. Caffeine, which promotes Ca(2+) release from intracellular stores, had concentration-dependent effects. At 1 mM, it markedly and steadily increased the frequency and the amplitude of the hyperpolarizations. At 10 mM, it induced a transient increase in their frequency followed by their cessation. All these effects were quickly reversible. Ryanodine (10 microM), which decreases the conductance of Ca(2+) release channels, irreversibly blocked the spontaneous hyperpolarizations. Dantrolene (100 microM), a blocker of Ca(2+) release from sarcoplasmic reticulum of striated muscle, did not affect the events. On the other hand, Cd(2+) (100-300 microM), a broad antagonist of membrane voltage-gated Ca(2+) channels, significantly reduced the amplitude and the frequency of the hyperpolarizations. However, when the frequency of the events was increased by 1 mM caffeine, Cd(2+) affected them to a smaller extent, whereas cyclopiazonic acid still abolished them. We conclude that internal stores are the major source of Ca(2+) ions that induce the K(+) channel openings underlying the spontaneous hyperpolarizations of these neurons. (+info)
(7/253) Presenilin-1 mutation increases neuronal vulnerability to focal ischemia in vivo and to hypoxia and glucose deprivation in cell culture: involvement of perturbed calcium homeostasis.
Many cases of early-onset inherited Alzheimer's disease (AD) are caused by mutations in the presenilin-1 (PS1) gene. Studies of cultured neural cells suggest that PS1 mutations result in perturbed cellular calcium homeostasis and may thereby render neurons vulnerable to apoptosis. In light of evidence that metabolic impairment plays a role in AD, that cerebral ischemia may be a risk factor for AD, and that individuals with AD have increased morbidity and mortality after stroke, we examined the impact of a PS1 mutation on neuronal vulnerability to ischemic injury. We report that the extent of brain injury after focal cerebral ischemia reperfusion is increased, and behavioral outcome is worsened, in PS1 mutant knock-in mice compared to wild-type mice. Cultured cortical neurons from PS1 mutant mice exhibit increased vulnerability to glucose deprivation and chemical hypoxia compared to their wild-type counterparts. Calcium imaging studies demonstrated enhanced elevation of intracellular calcium levels after glucose deprivation and chemical hypoxia in neurons from PS1 mutant mice. Agents that block calcium release from IP(3)- and ryanodine-sensitive stores (xestospongin and dantrolene, respectively) protected against the endangering action of the PS1 mutation. Our data suggest that presenilin mutations may promote neuronal degeneration in AD by increasing the sensitivity of neurons to age-related ischemia-like conditions. The data further suggest that drugs that stabilize endoplasmic reticulum calcium homeostasis may prove effective in suppressing the neurodegenerative process in AD patients. (+info)
(8/253) Investigation of the role of intracellular Ca(2+) stores in generation of the muscarinic agonist-induced slow afterdepolarization (sADP) in guinea-pig olfactory cortical neurones in vitro.
1. Intracellular recordings were made from guinea-pig olfactory cortical brain slice neurones to assess the possible role of intracellular Ca(2+) stores in the generation of the slow post-stimulus afterdepolarization (sADP) and its underlying tail current (I(ADP)), induced by muscarinic receptor activation. 2. Caffeine or theophylline (0.5 - 3 mM) reduced the amplitude of the I(ADP) (measured under 'hybrid' voltage clamp) induced in the presence of the muscarinic agonist oxotremorine-M (OXO-M, 10 microM) by up to 96%, without affecting membrane properties or muscarinic depolarization of these neurones. 3. The L-type Ca(2+) channel blocker nifedipine (1, 10 microM) also inhibited I(ADP) (by up to 46%), while ryanodine (10 microM) (a blocker of Ca(2+) release from internal stores) produced a small ( approximately 10%) reduction in I(ADP) amplitude; however, neither 10 microM dantrolene (another internal Ca(2+) release blocker) nor the intracellular Ca(2+) store re-uptake inhibitors thapsigargin (3 microM) or cyclopiazonic acid (CPA, 15 microM) affected I(ADP) amplitude. 4. IBMX (100 microM), a phosphodiesterase inhibitor, also had no effect on I(ADP). Furthermore, inhibition of I(ADP) by caffeine was not reversed by co-application of 100 microM adenosine. 5. Caffeine (3 mM) or nifedipine (10 microM) reduced the duration of presumed Ca(2+) spikes revealed by intracellular Cs(+) loading. When applied in combination, nifedipine and caffeine effects were occlusive, rather than additive, suggesting a common site of action on L-type calcium channels. 6. We conclude that Ca(2+)-induced Ca(2+) release (CICR) from internal stores does not contribute significantly to muscarinic I(ADP) generation in olfactory cortical neurones. However caffeine and theophylline, which enhance CICR in other systems, blocked I(ADP) induction. We suggest that this action might involve a combination of L-type voltage-gated Ca(2+) channel blockade, and a direct inhibitory action on the putative I(ADP) K(+) conductance. (+info)