Cytoplasmic location of α1A voltage-gated calcium channel C-terminal fragment (Cav2.1-CTF) aggregate is sufficient to cause cell death.

The human α1A voltage-dependent calcium channel (Cav2.1) is a pore-forming essential subunit embedded in the plasma membrane. Its cytoplasmic carboxyl(C)-tail contains a small poly-glutamine (Q) tract, whose length is normally 4∼19 Q, but when expanded up to 20∼33Q, the tract causes an autosomal-dominant neurodegenerative disorder, spinocerebellar ataxia type 6 (SCA6). A recent study has shown that a 75-kDa C-terminal fragment (CTF) containing the polyQ tract remains soluble in normal brains, but becomes insoluble mainly in the cytoplasm with additional localization to the nuclei of human SCA6 Purkinje cells. However, the mechanism by which the CTF aggregation leads to neurodegeneration is completely elusive, particularly whether the CTF exerts more toxicity in the nucleus or in the cytoplasm. We tagged recombinant (r)CTF with either nuclear-localization or nuclear-export signal, created doxycyclin-inducible rat pheochromocytoma (PC12) cell lines, and found that the CTF is more toxic in the cytoplasm than in the nucleus, the observations being more obvious with Q28 (disease range) than with Q13 (normal-length). Surprisingly, the CTF aggregates co-localized both with cAMP response element-binding protein (CREB) and phosphorylated-CREB (p-CREB) in the cytoplasm, and Western blot analysis showed that the quantity of CREB and p-CREB were both decreased in the nucleus when the rCTF formed aggregates in the cytoplasm. In human brains, polyQ aggregates also co-localized with CREB in the cytoplasm of SCA6 Purkinje cells, but not in other conditions. Collectively, the cytoplasmic Cav2.1-CTF aggregates are sufficient to cause cell death, and one of the pathogenic mechanisms may be abnormal CREB trafficking in the cytoplasm and reduced CREB and p-CREB levels in the nuclei.

Cellular mechanisms of lead neurotoxicity.

Lead (Pb2+), a heavy metal, has been used by humans for many technological purposes, which is the main reason for its present widespread distribution. Although various actions have been taken to decrease the use and distribution of lead in the environment, it remains a significant health hazard. The toxic mechanism of lead is caused by its ability to substitute for other polyvalent cations (particularly divalent cations, such as calcium [Ca2+] and zinc [Zn2+]) in the molecular machinery of living organisms. These interactions allow lead to affect different biologically significant processes, including metal transport, energy metabolism, apoptosis, ionic conduction, cell adhesion, inter- and intracellular signaling, diverse enzymatic processes, protein maturation, and genetic regulation. Membrane ionic channels and signaling molecules seem to be one of the most relevant molecular targets contributing to lead's neurotoxicity; the developing central nervous system is particularly susceptible. At critical times in development, lead may have a disorganizing influence with long-lasting effects that may continue into teenage years and beyond. Pediatric lead poisoning is more common than adult lead poisoning, and its effects may occur at reduced blood levels with subclinical symptoms, thus a high index of suspicion is necessary for physicians when dealing with pediatric patients. Long-term effects of lead poisoning may produce cognitive and motor impairment, with behavioral alterations. This review is centered on the description of the molecular mechanisms of lead toxicity and its repercussions on cellular functions.

Activación de la calcio calmodulina quinasa II, CaMKII, por el uridin nucleósido difosfato, UDP, en neuronas granulares de cerebelo / Calcium Calmoduline Kinase II, CaMKII, activation by the uridine nucleoside diphosphate, UPD, in cerebellar granule neurons

Las neuronas granulares de cerebelo en cultivo presentan receptores metabotrópicos de nucleótidos de tipo P2Y6, cuyo agonista fisiológico específico es el nucleótido, uridina difosfato, UDP. Estudios de PCR muestran la presencia de este receptor y el incremento de la expresión con el tiempo. Los estudios de respuesta en célula individual mediante microfluorimetría muestran un incremento del calcio citosólico al estimular con UDP, siendo los incrementos mas significativos en el soma. El incremento del calcio citosólico produce la activación de diversos enzimas dependientes de calcio y concretamente de la calcio-calmodulina quinasa II, CAMKII, enzima que se encuentra ampliamente distribuida en toda la topografía de la neurona granular. Este enzima al activarse se auto-fosforila y mediante anticuerpos contra la forma fosforilada se pueden detectar las zonas mas activas en la célula. Cuando se estimula con UDP, la CaMKII fosforilada aparece fundamentalmente asociada al soma neural, con mucha menor actividad en las prolongaciones axodendríticas, lo que se corresponde con la distribución de los receptores P2Y6 funcionales

Cultured granule cells from cerebellum exhibit nucleotide metabotropic receptors such as the P2Y6 subtype, which physiological agonist is the uridine diphosphate, UDP. The PCR analysis show the presence of P2Y6 messenger RNA, increasing with the days in culture. Single cell microfluorimetric studies show citosolic calcium increase in response to UDP, this being more significant at the soma level. The cytosolic calcium increase triggers cellular responses mediated by calcium dependent enzymes. This is the case for calcium-calmodulin kinase II, CaMKII, which is extensively distributed through the granule neuron, according the immunocytochemical studies. This enzyme once activated is able to autophosphorylate and by using antibodies against the phosphorylated form the active zones can be detected. After UDP stimulation, the location of the phosphorylated form of CaMKII appears to be mainly at the neural soma, with lower presence at the axodendritic prolongations, which correlates with the functional P2Y6 subtype receptor distribution

Effect of contrast media on vascular smooth muscle cells.

PURPOSE: In order to alleviate the adverse effects of contrast media (CMs) on the vascular system, the role of Ca2+ in the viability of vascular smooth muscle cells (VSMCs) was investigated. MATERIAL AND METHODS: VSMCs were obtained from swine thoracic aorta. The number of VSMCs was counted under a microscope using the trypan blue dye-exclusion method 24 h after culture in RPMI containing physiological saline (SAL) as control, iothalamate (IOT), or iohexol (IOH) at 10% by volume with CaCl2 added at 0 to 2.0 mmol/l. Free Ca2+ in the above media was measured using an ion-selective electrode. RESULTS: Free Ca2+ was 0.4 to 1.5 mmol/l with ionic IOT and 0.4 to 1.8 mmol/l with non-ionic IOH as well as with control. The ratio of viable cells grown in the presence of CMs to those grown in the control was optimal at approximately 0.60 near 1 mmol/l Ca2+ and decreased markedly to 0.00 at 1.5 mmol/l Ca2+ in the presence of IOT and to 0.39 at 1.8 mmol/l Ca2+ in the presence of IOH, while the ratios decreased gradually to 0.28 in the presence of IOT and 0.53 in the presence of IOH at 0.4 mmol/l Ca2+. CONCLUSION: Ionic IOT is more cytotoxic to VSMCs than non-ionic IOH. However, the cytotoxicity was minimal and similar between both CMs at 1 mmol/l Ca2+ in accordance with the sodium-calcium balance.

Proteolytic cleavage and cellular toxicity of the human alpha1A calcium channel in spinocerebellar ataxia type 6.

Spinocerebellar ataxia type 6 (SCA6) is a neurodegenerative disease caused by small CAG repeat expansion in the alpha1A calcium channel gene. We found that the human alpha1A calcium channel protein expressed in human embryonic kidney 293T cells produces a 75 kDa C-terminal fragment. This fragment is more toxic to cells than the full-length alpha1A calcium channel, regardless of polyglutamine tract length. In cells stably transfected with plasmids of full-length alpha1A calcium channel cDNAs, the C-terminal fragment protein is present in the mutant transformant but not in the wild-type one, indicative that this C-terminal fragment with the expanded polyglutamine tract is more resistant to proteolysis than that with the normal sized polyglutamine tract. We speculate that the toxic C-terminal fragment, in which resistance to proteolysis is rendered by the expanded polyglutamine, has a key role in the pathological mechanism of SCA6.