Involvement of Na+-Ca2+ exchanger on metabotropic glutamate receptor 1-mediated [Ca2+]i transients in rat cerebellar Purkinje neurons.

Cerebellar Purkinje neurons have intracellular regulatory systems including Ca2+-binding proteins, intracellular Ca2+ stores, Ca2+-ATPase and Na+-Ca2+ exchanger (NCX) that keep intracellular Ca2+ concentration ([Ca2+]i) in physiological range. Among these, NCX interacts with AMPA receptors, activation of which induces cerebellar synaptic plasticity. And the activation of metabotropic glutamate receptor 1 (mGluR1) is also involved in the induction of cerebellar long-term depression. The interaction of NCX with mGluR1 is not known yet. Thus, in this study, the functional relationship between NCX and mGluR1 in modulating the [Ca2+]i in rat Purkinje neurons was investigated. The interaction between NCX and mGluR1 in Purkinje neurons was studied by measuring intracellular Ca2+ transients induced by an agonist of group I mGluRs, 3,5-dihydroxyphenylglycine (DHPG). The DHPG-induced Ca2+ transient was significantly reduced by treatments of NCX inhibitors, bepridil and KB-R7943. When cells were pretreated with antisense oligodeoxynucleotides of NCX, the DHPG-induced Ca2+ transient was also inhibited. These results suggest that NCX modulates the activity of mGluR1 in cerebellar Purkinje neurons. Therefore, NCX appears to play an important role in the physiological function of cerebellar Purkinje neurons such as synaptic plasticity.

Development of the visual pathway is disrupted in mice with a targeted disruption of the calcium channel beta(3)-subunit gene.

Refinement of the retinal pathways to the superior colliculus (SC) and dorsal lateral geniculate nucleus (dLGN) is mediated by nitric oxide (NO). Long-term depression (LTD) can also be induced in SC and LGN during the time at which these pathways are refined, and this LTD is partially dependent on NO and L-type Ca(2+) channel function. In an effort to determine whether NO-mediated pathway refinement is also mediated by Ca(2+) channel function, we have examined the refinement of the retinocollicular and retinogeniculate pathways in mice which lack the gene for the Ca(2+) channel beta(3) subunit (CCKO) and which have significantly reduced L-type Ca(2+) currents. Injections of the anterograde tracer cholera toxin subunit B/HRP were made into one eye of these knockout animals and in wild-type mice ages postnatal day (P) 13, P19, and P26. After 48 hours, mice were perfused and sections processed by using tetramethylbenzidine histochemistry. Labeling distribution in some animals was analyzed quantitatively. Obvious differences in the distribution of the ipsilateral retinocollicular pathway were observed at P15, with the pathway being more exuberant in CCKO mice. This difference was statistically significant. More subtle differences were seen at P21 and P28. Obvious differences were also seen in the contralateral retinogeniculate pathway which in CCKO mice filled most of the domain normally occupied by ipsilateral eye fibers. This difference was also statistically significant. We conclude that reduction in L-type Ca(2+) currents has an effect on axonal refinement similar to that which occurs in NO knockout mice, which supports the possibility that L-type Ca(2+) channel-dependent LTD mediates NO-dependent axonal refinement.

Requirement for the L-type Ca(2+) channel alpha(1D) subunit in postnatal pancreatic beta cell generation.

Pancreatic beta cells are the source of insulin, which directly lowers blood glucose levels in the body. Our analyses of alpha(1D) gene-knockout (alpha(1D)(-/-)) mice show that the L-type calcium channel, alpha(1D), is required for proper beta cell generation in the postnatal pancreas. Knockout mice were characteristically slightly smaller than their littermates and exhibited hypoinsulinemia and glucose intolerance. However, isolated alpha(1D)(-/-) islets persisted in glucose sensing and insulin secretion, with compensatory overexpression of another L-type channel gene, alpha(1C). Histologically, newborn alpha(1D)(-/-) mice had an equivalent number of islets to wild-type mice. In contrast, adult alpha(1D)(-/-) mice showed a decrease in the number and size of islets, compared with littermate wild-type mice due to a decrease in beta cell generation. TUNEL staining showed that there was no increase in cell death in alpha(1D)(-/-) islets, and a 5-bromo-2' deoxyuridine-labeling (BrdU-labeling) assay illustrated significant reduction in the proliferation rate of beta cells in alpha(1D)(-/-) islets.

Identity of mouse IA-2 and PTP35 genes of the tyrosine phosphatase family, and their expression in neuroendocrine tissues.

Recently, IA-2, one of the major diabetic autoantigens, and PTP35 cDNA were independently isolated by subtraction cloning using insulinoma cells and a polymerase chain reaction (PCR)-based search for conserved sequences using NIH3T3 fibroblast cell line, respectively. By Southern blot analysis and nucleotide sequence determination of reverse transcription PCR products, we showed that IA-2 and PTP35 are identical and exist as a single gene in a mouse genome. The expression of IA-2/PTP35 messages was detected by northern blot analysis in MIN6N8 cells, an insulinoma cell line derived from non-obese diabetic mice, but its expression level was not affected by the ambient glucose level, phorbol-12-myristate 13-acetate or tumour necrosis factor-alpha. We also generated polyclonal antibodies to murine IA-2/PTP35 by immunization with recombinant proteins. Subsequent immunohistochemical analysis using these polyclonal antibodies disclosed that IA-2/PTP35 is strongly expressed in mouse neuroendocrine tissues such as pancreatic islets and the hypothalamus-pituitary gland. These results suggest that IA-2/PTP35 functions primarily in neuroendocrine tissues.

Neuronal voltage-activated calcium channels: on the roles of the alpha 1E and beta 3 subunits.

Many neurons of the central and peripheral nervous systems display multiple high voltage-activated (HVA) Ca2+ currents, often classified as L-, N-, P-, Q, and R-type. The heterogeneous properties of these channels have been attributed to diversity in their pore-forming alpha 1, subunits, in association with various beta subunits. However, there are large gaps in understanding how individual subunits contribute to Ca2+ channel diversity. Here we describe experiments to investigate the roles of alpha 1E and beta 3 subunits in mammalian neurons. The alpha 1E subunit is the leading candidate to account for the R-type channel, the least understood of the various types of high voltage-activated Ca2+ channels. Incubation with alpha 1E antisense oligonucleotide caused a 53% decrease in the peak R-type current density, while no significant changes in the current expression were seen in sense oligonucleotide-treated cells. The specificity of the alpha 1E antisense oligonucleotides was supported by the lack of change in the amplitude of P/Q current. These results upheld the hypothesis that members of the E class of alpha 1 subunits support the high voltage-activated R-type current in cerebellar granule cells. We studied the role of the Ca2+ channel beta 3 subunit using a gene targeting strategy. In sympathetic beta 3-/- neurons, the L-type current was significantly reduced relative to wild type (wt). In addition, N-type Ca2+ channels made up a smaller proportion of the total Ca2+ current than in wt due to a lower N-type current density in a group of neurons with small total currents. Voltage-dependent activation of P/Q-type Ca2+ channels was described by two Boltzmann components with different voltage dependence. The absence of the beta 3 subunit was associated with a shift in the more depolarized component of the activation along the voltage axis toward more negative potentials. The overall conclusion is that deletion of the beta 3 subunit affects at least three distinct types of HVA Ca2+ channel, but no single type of channel is solely dependent on beta 3.

Targeted disruption of the Ca2+ channel beta3 subunit reduces N- and L-type Ca2+ channel activity and alters the voltage-dependent activation of P/Q-type Ca2+ channels in neurons.

In comparison to the well characterized role of the principal subunit of voltage-gated Ca2+ channels, the pore-forming, antagonist-binding alpha1 subunit, considerably less is understood about how beta subunits contribute to neuronal Ca2+ channel function. We studied the role of the Ca2+ channel beta3 subunit, the major Ca2+ channel beta subunit in neurons, by using a gene-targeting strategy. The beta3 deficient (beta3-/-) animals were indistinguishable from the wild type (wt) with no gross morphological or histological differences. However, in sympathetic beta3-/- neurons, the L- and N-type current was significantly reduced relative to wt. Voltage-dependent activation of P/Q-type Ca2+ channels was described by two Boltzmann components with different voltage dependence, analogous to the "reluctant" and "willing" states reported for N-type channels. The absence of the beta3 subunit was associated with a hyperpolarizing shift of the "reluctant" component of activation. Norepinephrine inhibited wt and beta3-/- neurons similarly but the voltage sensitive component was greater for N-type than P/Q-type Ca2+ channels. The reduction in the expression of N-type Ca2+ channels in the beta3-/- mice may be expected to impair Ca2+ entry and therefore synaptic transmission in these animals. This effect may be reversed, at least in part, by the increase in the proportion of P/Q channels activated at less depolarized voltage levels.