cytochalasin-d and rhod-2

Increased levels of mitochondrial-free calcium have been associated with several cell-death paradigms, such as excitotoxicity and ceramide-mediated neuronal death. In the latter, calcium is transferred from the endoplasmic reticulum to mitochondria by a mechanism that is only partly understood. We show here that CDK5 (cyclin-dependent kinase 5) plays a role. Free calcium levels in the endoplasmic reticulum and mitochondria were measured with fluorescent markers in C2-ceramide-treated primary cultures of mesencephalic neurons and differentiated pheochromocytoma PC12 cells. Calcium levels decreased in the endoplasmic reticulum as they increased in mitochondria. Both changes were blocked by the pharmacological and molecular CDK5 inhibitors roscovitine and a dominant-negative form of CDK5. Although the kinase did not mediate the transfer of calcium per se, which required the proapoptotic Bcl-2 family protein t-Bid (the truncated form of Bid), it facilitated the transfer by inducing the clustering of endoplasmic reticulum and mitochondria around the centrosome where they formed close contacts, as shown by immunocytochemistry and electron microscopy. Organelle clustering resulted from CDK5-dependent phosphorylation of the microtubule-associated protein tau on threonine 231. This caused its release from microtubules into the soluble fraction of cellular proteins, which appears to favor retrograde transport of the organelles. Mutation of threonine 231 to alanine, so that tau could not be phosphorylated at this site, prevented the ceramide-induced release of tau from microtubules, organelle clustering, the increase in mitochondrial-free calcium levels, and neuronal death, demonstrating the importance of the CDK5-dependent signaling cascade in this calcium-dependent cell-death mechanism.

Topics: Analysis of Variance; Animals; Blotting, Western; Calcium; Cell Count; Cell Death; Cells, Cultured; Ceramides; Cloning, Molecular; Cyclin-Dependent Kinase 5; Cytochalasin D; Drug Interactions; Embryo, Mammalian; Endoplasmic Reticulum; Enzyme Activation; Enzyme Inhibitors; Female; Gene Expression Regulation; Heterocyclic Compounds, 3-Ring; Immunohistochemistry; Male; Mesencephalon; Microscopy, Electron, Transmission; Mitochondria; Neurons; Nocodazole; Phosphorylation; Pregnancy; Purines; Rats; Rats, Wistar; Roscovitine; tau Proteins; Time Factors; Transfection; Tubulin

Chemical uncouplers diacetyl monoxime (DAM) and cytochalasin D (cyto-D) are used to abolish cardiac contractions in optical studies, yet alter intracellular Ca(2+) concentration ([Ca(2+)](i)) handling and vulnerability to arrhythmias in a species-dependent manner. The effects of uncouplers were investigated in perfused mouse hearts labeled with rhod-2/AM or 4-[beta-[2-(di-n-butylamino)-6-naphthyl]vinyl]pyridinium (di-4-ANEPPS) to map [Ca(2+)](i) transients (emission wavelength = 585 +/- 20 nm) and action potentials (APs) (emission wavelength > 610 nm; excitation wavelength = 530 +/- 20 nm). Confocal images showed that rhod-2 is primarily in the cytosol. DAM (15 mM) and cyto-D (5 microM) increased AP durations (APD(75) = 20.0 +/- 3 to 46.6 +/- 5 ms and 39.9 +/- 8 ms, respectively, n = 4) and refractory periods (45.14 +/- 12.1 to 82.5 +/- 3.5 ms and 78 +/- 4.24 ms, respectively). Cyto-D reduced conduction velocity by 20% within 5 min and DAM by 10% gradually in 1 h (n = 5 each). Uncouplers did not alter the direction and gradient of repolarization, which progressed from apex to base in 15 +/- 3 ms. Peak systolic [Ca(2+)](i) increased with cyto-D from 743 +/- 47 (n = 8) to 944 +/- 17 nM (n = 3, P = 0.01) but decreased with DAM to 398 +/- 44 nM (n = 3, P < 0.01). Diastolic [Ca(2+)](i) was higher with cyto-D (544 +/- 80 nM, n = 3) and lower with DAM (224 +/- 31, n = 3) compared with controls (257 +/- 30 nM, n = 3). DAM prolonged [Ca(2+)](i) transients at 75% recovery (54.3 +/- 5 to 83.6 +/- 1.9 ms), whereas cyto-D had no effect (58.6 +/- 1.2 ms; n = 3). Burst pacing routinely elicited long-lasting ventricular tachycardia but not fibrillation. Uncouplers flattened the slope of AP restitution kinetic curves and blocked ventricular tachycardia induced by burst pacing.

Topics: Action Potentials; Animals; Arrhythmias, Cardiac; Calcium; Cytochalasin D; Diacetyl; Electrophysiology; Fluorescent Dyes; Heart; Heterocyclic Compounds, 3-Ring; In Vitro Techniques; Kinetics; Mice; Mice, Inbred Strains; Myocardial Contraction; Nucleic Acid Synthesis Inhibitors; Organ Preservation Solutions; Perfusion

The ability to image calcium signals at subcellular levels within the intact depolarizing heart could provide valuable information toward a more integrated understanding of cardiac function. Accordingly, a system combining two-photon excitation with laser-scanning microscopy was developed to monitor electrically evoked [Ca(2+)](i) transients in individual cardiomyocytes within noncontracting Langendorff-perfused mouse hearts. [Ca(2+)](i) transients were recorded at depths

Topics: Animals; Calcium; Calcium Signaling; Chelating Agents; Cytochalasin D; Diacetyl; Diagnostic Imaging; Egtazic Acid; Enzyme Inhibitors; Fluorescent Dyes; Fura-2; Heart; Heterocyclic Compounds, 3-Ring; In Vitro Techniques; Mice; Mice, Inbred Strains; Mice, Transgenic; Muscle Cells; Muscle Contraction; Nucleic Acid Synthesis Inhibitors; Perfusion; Photons; Transforming Growth Factor beta; Transforming Growth Factor beta1