Up-regulation of α1D Ca channel subunit mRNA expression in the hippocampus of aged F344 rats

There is growing evidence that alterations in calcium (Ca²⁺) homeostasis may play a role in processes of brain aging and neurodegeneration. There also is evidence that some of the altered Ca²⁺ homeostasis in hippocampal neurons may arise from an increased density of L-type voltage sensitive Ca²⁺ channels (L-VSCC). In the present studies, we tested the possibility that previously observed increases in functional L-VSCC with aging might be related to up-regulated gene/mRNA expression for Ca²⁺ channel subunits. A significant aging-related increase in mRNA content for the α_1D subunit of the L-type VSCC was observed in hippocampus of aged F344 rats (25 months old) relative to young (4 months old) and middle-aged animals (13 months old), as assessed by both in situ hybridization analyses (densitometry and grain density) and ribonuclease protection assay (RPA). In RPA analyses, the α_1C subunit mRNA also showed a significant increase in 25-month-old rats. No age changes were seen in mRNA for the β_1b subunit of VSCC or for GAPDH, a standard control. The clearest increases in α_1D mRNA expression were observed in subfield CA1, with little or no change seen in dentate gyrus. Although these results alone do not demonstrate that mRNA/gene expression changes contribute directly to changes in functional Ca²⁺ channels, they clearly fulfill an important prediction of that hypothesis. Therefore, these studies may have important implications for the role of gene expression in aging-dependent alterations in brain Ca²⁺ homeostasis.     

Deficits in the expression of the NR2B subunit in the hippocampus of aged Fisher 344 rats

The NMDA receptor (NMDAR) has been implicated in the induction of LTP at hippocampal synapses, and has been proposed to play a significant role in the involvement of the hippocampus with learning and memory. Aged rats are known to have deficits in LTP, learning and memory. We tested the hypothesis that aged rats might have deficits in expression of NMDAR subunits. Aged rats have significantly lower levels of NR2B mRNA and protein compared to young animals. This complements a recent report which showed improved learning and memory in mice which overexpress NR2B. No changes were seen in either the mRNA or the protein levels of the NMDAR subunit NR2A, nor in the alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionate receptor (AMPAR) subunit GluR2. Our data support the hypothesis that age related alterations in the expression of the NMDAR NR2B subunit might underlie deficits in LTP and learning and memory in aged animals.     

Postnatal expression of alpha2 nicotinic acetylcholine receptor subunit mRNA in developing cortex and hippocampus

Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated cation channels composed of alpha and beta subunits. nAChR subunit expression is highly regulated during development. Previous studies have revealed increased expression of alpha3, alpha5, alpha7, and beta4 subunit mRNAs and alpha7 binding sites during hippocampal and cortical development. Here, we examined the expression of alpha2 subunit mRNA in rat cortex and hippocampus using highly sensitive radioactive in situ hybridization. alpha2 Subunit mRNA expression was first detected at P3 in cortex and hippocampus. During postnatal development the distribution of alpha2 subunit mRNA expression was spatially similar to the one found in adult, exhibiting highly restricted expression in scattered cells mostly in cortical layer V and retrosplenial cortex, and in scattered cells in CA1/CA3 stratum oriens and CA3 stratum radiatum. However, the expression intensity and number of alpha2 positive cells strongly increased to reach peak levels in both cortex and hippocampus at P7 and decreased thereafter to moderate to low to levels. Double in situ hybridization revealed that most, but not all, alpha2 mRNA expression was located in non-pyramidal GAD-positive cortical and hippocampal interneurons. Thus, similar to other nAChR subunits, alpha2 mRNA expression is transiently upregulated during postnatal development and nAChRs containing alpha2 subunits could regulate GABAergic activity during a critical period of network formation.     

钙拮抗剂对高血压大鼠心房肌L-型钙通道和钠-钙离子交换器mRNA的影响

目的:观察钙拮抗剂地尔硫卓对Goldblatt高血压大鼠模型心房肌L-型钙通道α_1C亚单位(CaL-α_1C)和Na^＋-Ca²⁺交换器(NCX)mRNA的变化影响,在基因水平探讨其潜在的意义。方法:Sprague-Dawley大鼠,高血压组用U型银夹夹住左肾动脉,右肾保留;假手术组(S组)只分离左肾动脉,不夹银夹,套尾法监测尾动脉血压。高血压组术后1周开始治疗,分为高剂量地尔硫艹卓组(HD组)、低剂量地尔硫艹卓组(LD组)和对照组(C组),分别于治疗后4周与S组同时处死动物(各6只),RT-PCR半定量分析CaL-α_1C和NCXmRNA的表达量(以GAPDH为内参照)。结果:C组心房肌CaL-α_1C的mRNA水平分别是S组、HD组、LD组的2.5倍、2.4倍、2.1倍(P＜0.01),S组、HD组、LD组之间无显著差别。C组心房肌NCX的mRNA水平分别是S组、HD组、LD组的1.9倍、1.6倍、2.1倍(P＜0.01),S组、HD组、LD组之间无显著差别。结论:肾性高血压大鼠心房肌CaL-α_1C、NCX的mRNA水平均表现为上调,应用钙拮抗剂能够抑制其上调,抑制作用不依赖于血压水平的降低。     

Up-regulation of P/Q-type voltage-gated Ca2+ channel immunoreactivity within parvalbumin positive neurons in the rat hippocampus following status epilepticus

To identify the roles of VGCC subtypes in damages/impairs of inhibitory transmission during epileptogenesis, we investigated temporal- and spatial-specific alterations in voltage-gated Ca(2+) channel (VGCC) immunoreactivities within parvalbumin (PV, a Ca(2+) binding protein) positive neurons in the rat hippocampus following status epilepticus (SE). Compared to controls, only P/Q-type (alpha1A) VGCC immunoreactivity was enhanced in PV positive neurons at the early point following SE. The alteration in P/Q-type (alpha1A) VGCC immunoreactivity showed an inverse proportionality to that in PV immunoreactivity in the dentate gyrus and the CA1 region. These findings suggest that SE may induce prolonged up-regulation in P/Q-type VGCC expression within PV positive neurons.     

Differential expression of alpha 1 and beta subunits of voltage dependent Ca2+ channel at the neuromuscular junction of normal and P/Q Ca2+ channel knockout mouse

Voltage-dependent calcium channels (VDCC) have a key role in neuronal function transforming the voltage signals into intracellular calcium signals. They are composed of the pore-forming alpha(1) and the regulatory alpha(2)delta, gamma and beta subunits. Molecular and functional studies have revealed which alpha(1) subunit gene product is the molecular constituent of each class of native calcium channel (L, N, P/Q, R and T type). Electrophysiological and immunocytochemical studies have suggested that at adult mouse motor nerve terminal (MNT) only P/Q type channels, formed by alpha(1A) subunit, mediate evoked transmitter release. The generation of alpha(1A)-null mutant mice offers an opportunity to study the expression and localization of calcium channels at a synapse with complete loss of P/Q calcium channel. We have investigated the expression and localization of VDCCs alpha(1) and beta subunits at the wild type (WT) and knockout (KO) mouse neuromuscular junction (NMJ) using fluorescence immunocytochemistry. The alpha(1A) subunit was observed only at WT NMJ and was absent at denervated muscles and at KO NMJ. The subunits alpha(1B), alpha(1D) and alpha(1E) were also present at WT NMJ and they were over- expressed at KO NMJ suggesting a compensatory expression due to the lack of the alpha(1A). On the other hand, the beta(1b), beta(2a) and beta(4) were present at the same levels in both genotypes. The presence of other types of VDCC at WT NMJ indicate that they may play other roles in the signaling process which have not been elucidated and also shows that other types of VDCC are able to substitute the alpha(1A) subunit, P/Q channel under certain pathological conditions.     

Serum from aged F344 rats conditions the activation of young macrophages

There is considerable controversy about the molecular mechanisms responsible for the variations in innate immunity associated with age. While in vivo, aged animals and humans react to an inflammatory signal with an excessive production of pro-inflammatory cytokines, studies in vitro generally show that this response is attenuated in macrophages from old individuals. In an effort to examine possible extrinsic factors that might affect the response of macrophages to lipopolysaccharide (LPS), we have challenged peritoneal macrophages obtained from young rats with sera obtained from rats of different ages. Our results indicate that the serum from aged rats significantly impairs the capacity of young macrophages to induce tumor necrosis factor-alpha (TNF-alpha) production, while at the same time it increases the basal levels of interleukin-6 (IL-6). The effect of serum from aged donors on TNF-alpha secretion requires pre-incubation and is sensitive to heat inactivation. In contrast, the stimulating effect on IL-6 is resistant to heat, and thus should not be due to a protein factor. Therefore, our results indicate that the age-related changes in macrophage activity are not only the consequence of intrinsic changes, but there also appears to be a modulatory effect imparted by the external milieu.     

Pain sensitivity in mice lacking the Ca(v)2.1alpha1 subunit of P/Q-type Ca2+ channels

The role of voltage-gated Ca(2+) (Ca(V)) channels in pain mechanisms has been the object of intense investigation using pharmacological approaches and, more recently, using mutant mouse models lacking the Ca(V)alpha(l) pore-forming subunit of N-, R- and T-type channels. The role of P/Q-type channels in nociception and pain transmission has been investigated by pharmacological approaches but remains to be fully elucidated. To address this issue, we have analyzed pain-related behavioral responses of null mutant mice for the Ca(V)2.1alpha(1) subunit of P/Q-type channels. Homozygous null mutant Ca(V)2.1alpha(1)-/- mice developed dystonia at 10-12 days after birth and did not survive past weaning. Tested at ages where motor deficit was either absent or very mild, Ca(V)2.1alpha(1)-/- mice showed reduced tail withdrawal latencies in the tail-flick test and reduced abdominal writhes in the acetic acid writhing test. Adult heterozygous Ca(V)2.1alpha(1)+/- mice did not show motor deficits in the rotarod and activity cage tests and did not show alterations in pain responses in the tail-flick test and the acetic acid writhing test. Strikingly, they showed a reduced licking response during the second phase of formalin-induced inflammatory pain and a reduced mechanical allodynia in the chronic constriction injury model of neuropathic pain. Our findings show that P/Q-type channels play an antinociceptive role in sensitivity to non-injurious noxious thermal stimuli and a pronociceptive role in inflammatory and neuropathic pain states, pointing to an important role of Ca(V)2.1 channels in central sensitization.     

A tctex1-Ca2+ channel complex for selective surface expression of Ca2+ channels in neurons

Voltage-gated Ca(2+) channels (VGCCs) are important in regulating a variety of cellular functions in neurons. It remains poorly understood how VGCCs with different functions are sorted within neurons. Here we show that the t-complex testis-expressed 1 (tctex1) protein, a light-chain subunit of the dynein motor complex, interacts directly and selectively with N- and P/Q-type Ca(2+) channels, but not L-type Ca(2+) channels. The interaction is insensitive to Ca(2+). Overexpression in hippocampal neurons of a channel fragment containing the binding domain for tctex1 significantly decreases the surface expression of endogenous N- and P/Q-type Ca(2+) channels but not L-type Ca(2+) channels, as determined by immunostaining. Furthermore, disruption of the tctex1-Ca(2+) channel interaction significantly reduces the Ca(2+) current density in hippocampal neurons. These results underscore the importance of the specific tctex1-channel interaction in determining sorting and trafficking of neuronal Ca(2+) channels with different functionalities.     

Sorcin modulates cardiac L-type Ca2+ current by functional interaction with the alpha1C subunit in rabbits

We examined the modulation of the cardiac L-type Ca(2+) channel (LTCC) by the regulatory protein sorcin and tested the hypothesis that modulation occurred by direct interaction. Whole-cell patch-clamp recordings were made on native rabbit ventricular myocytes and HEK 293 cells expressing cardiac alpha(1C) subunits. In ventricular cells, sorcin increased peak current when using either Ca(2+) or Ba(2+) as charge carriers. In HEK 293 cells, sorcin increased peak current density when using Ba(2+) as a charge carrier but not when using Ca(2+). In ventricular myocytes, current inactivation (tau(fast), in ms) was slowed by sorcin with Ca(2+) as the charge carrier, whilst in the presence of Ba(2+) it was enhanced. In HEK 293 cells, sorcin significantly enhanced tau(fast), but no significant change was observed with Ba(2+). This trend was mimicked by the truncated peptide, sorcin Ca(2+)-binding domain, which lacks the N-terminal domain. These data suggest that sorcin interacts with LTCC via its C-terminal domain, which alters current magnitude and tau(fast). These effects appear to be influenced by the prevailing experimental conditions.     

Beta1-subunits increase surface expression of a large-conductance Ca2+-activated K+ channel isoform

Auxiliary (beta) subunits of large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels regulate the gating properties of the functional channel complex. Here we show that an avian beta1-subunit also stimulates the trafficking of BK(Ca) channels to the plasma membrane in HEK293T cells and in a native population of developing vertebrate neurons. One C-terminal variant of BK(Ca) alpha-subunits, called the VEDEC isoform after its five last residues, is largely retained in intracellular compartments when it is heterologously expressed in HEK293T cells. A closely related splice variant, called QEERL, shows high levels of constitutive trafficking to the plasma membrane. Co-expression of beta1-subunits with the VEDEC isoform resulted in a large increase in surface BK(Ca) channels as assessed by cell-surface biotinylation assays, whole cell recordings of membrane current, and confocal microscopy in HEK293T cells. Co-expression of beta1-subunits slowed the gating kinetics of BK(Ca) channels, as reported previously. Consistent with this, overexpression of beta1-subunits in a native cell type that expresses intracellular VEDEC channels, embryonic day 9 chick ciliary ganglion neurons, resulted in a significant increase in macroscopic Ca(2+)-activated K(+) current. Both the cytoplasmic N- and C-terminal domains of avian beta1 are able to bind directly to VEDEC and QEERL channels. However, overexpression of the N-terminal domain by itself is sufficient to stimulate trafficking of VEDEC channels to the plasma membrane, whereas overexpression of either the cytoplasmic C-terminal domain or the extracellular loop domain did not affect surface expression of VEDEC.     

Expression of the alpha1D subunit of the L-type voltage gated calcium channel in human liver

Calcium channel blocker is useful for a variety of purposes and is effective for preventing hepatitis elicited by different inducers, suggesting its possible clinical application for treating hepatitis. The alpha1-subunit of the dihydropyridine-sensitive L-type calcium channel is a target of calcium channel blocker. For clinical application of calcium channel blocker, it is important to analyze the expression of the L-type calcium channel in the liver. However, the subtype of the L-type calcium channel alpha1-subunit expressed in the liver was not known. In the present study, the alpha1-subunit of the calcium channel expressed in human liver was systematically analyzed. The alpha1D subunit of the dihydropyridine-sensitive L-type voltage gated calcium channel is expressed relatively strongly in the liver and may play an important role in the liver.     

Increased glucose tolerance in N-type Ca2+ channel alpha(1B)-subunit gene-deficient mice

The voltage-dependent N-type Ca2+ channel is localized in the plasma membrane of insulin-releasing beta-cells and glucagon-releasing alpha-cells in the islets of Langerhans in the pancreas. To examine the contribution of N-type Ca2+ channel to glucose homeostasis, we performed glucose tolerance and insulin tolerance tests with N-type Ca2+ channel alpha(1B)-subunit-deficient mice on a normal or high-fat diet. The fasting glucose level in homozygous mice on the normal diet was significantly lower than those in wild and heterozygous mice. In glucose tolerance tests, the homozygous mice showed a higher glucose clearance rate and a similar pattern of insulin levels to those of wild and heterozygous mice. In insulin tolerance tests, glucose clearance rates showed no significant difference among wild, heterozygous and homozygous mice. In animals on the high-fat diet, food consumption was the same among wild, heterozygous and homozygous mice, but body weight gain was reduced in homozygous mice. After 8 weeks of the high-fat diet, homozygous mice showed lower fasting glucose levels and exhibited higher glucose clearance and lower insulin levels than wild or heterozygous mice in glucose tolerance tests. Glucose clearance rates showed no significant difference among wild, heterozygous and homozygous mice in insulin tolerance tests. After 10 weeks of the high-fat diet, the alpha(1B)-deficient homozygous mice showed lower lipid deposition in liver and lower plasma glucagon, leptin and triglyceride levels than wild or heterozygous mice. These results suggest that N-type Ca2+ channels play a role in insulin and glucagon release, and that N-type Ca2+ channel alpha(1B)-subunit deficient mice show improved glucose tolerance without any change in insulin sensitivity. Thus, N-type Ca2+ channel blockers might be candidate anti-diabetic/anti-obesity agents.     

Reduction in levels of amphiphysin 1 mRNA in the hippocampus of aged rats subjected to repeated variable stress

Various neurobiological studies of aging indicate that elevated levels of circulating glucocorticoids lead to hippocampal vulnerability to stress, though little is known about the molecular mechanism underlying stress vulnerability in the elderly. We have compared the gene expression profiles in the hippocampus of aged (20 months) and adult (3 months) rats in response to repeated variable stress (RVS) for 4 days, using a cDNA array technique and real-time quantitative PCR, to identify putative genes involved in the mechanism of stress vulnerability in the elderly. We found a significant decrease in the levels of amphiphysin 1 mRNA in aged rats subjected to RVS compared with treated and untreated adult rats or to untreated aged rats. Similarly, we found a significant decrease in hippocampal levels of amphiphysin 1 mRNA in aged rats subjected to RVS for 8 days, but not in those subjected to a single VS. These findings suggest that the decrease in the hippocampal levels of amphiphysin 1 mRNA in response to repeated stress may be involved in the stress vulnerability in the elderly, and may lead to the disturbance of learning and memory under stressful conditions in the elderly.     

Human skeletal muscle calcium channel alpha1S is expressed in the basal ganglia: distinctive expression pattern among L-type Ca2+ channels

Voltage-gated calcium channels (VGCCs) are essential molecules for neuronal function. VGCCs consist of five subunits, alpha1, alpha2, beta, gamma, and delta. Among the ten subtypes of the alpha1 subunit (alpha1A-I and S), expression of alpha1S was previously believed to be restricted to the skeletal muscle. We report here, however, that alpha1S is also expressed in human and rat central nervous system. First, we performed PCR screening for VGCC alpha1 subunits in human nervous system using degenerate primers, and identified alpha1S as well as all the eight alpha1 subunits with previously described expression. Intriguingly, alpha1S was selectively localized to the basal ganglia, particularly the caudate nucleus. In situ hybridization showed that alpha1S was expressed in medium-sized caudate neurons. Quantitative analysis using real time RT-PCR revealed a distinct pattern of alpha1S expression among L-type calcium channels. Furthermore, RT-PCR using laser-mediated manipulation of single cells suggested that human alpha1S was coexpressed with ryanodine receptors (RYRs) in GABAergic neurons. Our results suggest the potential relevance of alpha1S to dopaminergic signal transduction and calcium-induced calcium release in caudate neurons.     

Hypothalamo-pituitary-adrenocortical dysregulation in aging F344/Brown-Norway F1 hybrid rats

Hypothalamo-pituitary-adrenocortical (HPA) axis aging was studied in young (3 mo), middle aged (15 mo) and aged (30 mo) F344/Brown Norway hybrid rats. This strain was selected to obviate HPA-relevant pathologies found in other aging models. Aged, unstressed rats showed enhanced central HPA drive, marked by elevated ACTH release and decreased pituitary proopiomelanocortin and corticotropin-releasing factor receptor 1 (CRH-R1) mRNAs. Acute corticosterone responses to spatial novelty were exacerbated in aged rats; however, responses to restraint or hypoxia were not affected. Chronic stress exposure also differentially increased HPA drive in aged animals, marked by elevated paraventricular nucleus CRH peptide levels and pituitary proopiomelanocortin mRNA. Plasma ACTH and pituitary POMC and CRH-R1 mRNA expression in middle-aged rats were intermediate those of young and aged animals. Middle-aged animals responded to chronic stress with disproportionate increases in CRH mRNA levels, and increased corticosterone secretion following hypoxia but not novelty. The results suggest a gradual increase in HPA tone across the aging process, culminating in marked hyperresponsivity to both acute and chronic stress in senescence.     

Direct activation of the ion channel TRPA1 by Ca2+

TRPA1 is an ion channel expressed by nociceptors and activated by irritant compounds such as mustard oil. The endogenous function of TRPA1 has remained unclear, a fact highlighted by ongoing debate over its potential role as a sensor of noxious cold. Here we show that intracellular Ca(2+) activates human TRPA1 via an EF-hand domain and that cold sensitivity occurs indirectly (and nonphysiologically) through increased [Ca(2+)](i) during cooling in heterologous systems.     

Impaired acquisition of novel locomotor tasks in aged and norepinephrine-depleted F344 rats.

Performance of rats on a motor learning paradigm that has been demonstrated to be dependent upon cerebellar norepinephrine (NE) was studied in 20-month-old F344 rats. The behavioral task is identical to that described by Watson and McElligott: Rats are trained on a runway consisting of aluminum pegs arranged in a regular pattern. Rats receive a water reward at either end of the runway. Subsequent to training, rats are tested for running times on a runway with irregularly spaced rods. The ability of rats to improve their performance (decrease their running times) on this novel motor task is diminished in young rats that have received 6-hydroxydopamine lesions. Rats at 20 months of age are known to have deficits in cerebellar noradrenergic transmission; thus, the hypothesis to be tested was to determine if aged rats demonstrated performance deficits similar to young rats depleted of central stores of NE. The rate of acquisition of the task was determined by the decrease in running times with successive days of training. The ability of 20-month-old F344 rats to acquire proficiency on the novel motor task was impaired and the rate of acquisition of the novel motor task was not different from the young 6-hydroxydopamine-lesioned rats. In an attempt to distinguish between alterations in motor coordination and motor learning, additional tests of psychomotor performance were assessed for all groups of rats. These tests included a walking on 2.5- and 5-cm rods, speed of running on the motor task, and number and types of mistakes made on the motor learning task.(ABSTRACT TRUNCATED AT 250 WORDS)