Subunit Messenger RNA Research Articles

Ectopic expression of meiotic cohesin generates chromosome instability in cancer cell line

Many tumors express meiotic genes that could potentially drive somatic chromosome instability. While germline cohesin subunits SMC1B, STAG3, and REC8 are widely expressed in many cancers, messenger RNA and protein for RAD21L subunit are expressed at very low levels. To elucidate the potential of meiotic cohesins to contribute to genome instability, their expression was investigated in human cell lines, predominately in DLD-1. While the induction of the REC8 complex resulted in a mild mitotic phenotype, the expression of the RAD21L complex produced an arrested but viable cell pool, thus providing a source of DNA damage, mitotic chromosome missegregation, sporadic polyteny, and altered gene expression. We also found that genomic binding profiles of ectopically expressed meiotic cohesin complexes were reminiscent of their corresponding specific binding patterns in testis. Furthermore, meiotic cohesins were found to localize to the same sites as BORIS/CTCFL, rather than CTCF sites normally associated with the somatic cohesin complex. These findings highlight the existence of a germline epigenomic memory that is conserved in cells that normally do not express meiotic genes. Our results reveal a mechanism of action by unduly expressed meiotic cohesins that potentially links them to aneuploidy and chromosomal mutations in affected cells.

Human skeletal muscle acetylcholine receptor gene expression in elderly males performing heavy resistance exercise.

Muscle fiber denervation is a major contributor to the decline in muscle mass and function during aging. Heavy resistance exercise is an effective tool for increasing muscle mass and strength, but whether it can rescue denervated muscle fibers remains unclear. Therefore, the purpose of this study was to investigate the potential of heavy resistance exercise to modify indices of denervation in healthy elderly individuals. Thirty-eight healthy elderly men (72 ± 5 yr) underwent 16 wk of heavy resistance exercise, whereas 20 healthy elderly men (72 ± 6 yr) served as nonexercising sedentary controls. Muscle biopsies were obtained pre and post training, and midway at 8 wk. Biopsies were analyzed by immunofluorescence for the prevalence of myofibers expressing embryonic myosin [embryonic myosin heavy chain (MyHCe)], neonatal myosin [neonatal myosin heavy chain (MyHCn)], nestin, and neural cell adhesion molecule (NCAM), and by RT-qPCR for gene expression levels of acetylcholine receptor (AChR) subunits, MyHCn, MyHCe, p16, and Ki67. In addition to increases in strength and type II fiber hypertrophy, heavy resistance exercise training led to a decrease in AChR α1 and ε subunit messenger RNA (mRNA; at 8 wk). Changes in gene expression levels of the α1 and ε AChR subunits with 8 wk of heavy resistance exercise supports the role of this type of exercise in targeting stability of the neuromuscular junction. The number of fibers positive for NCAM, nestin, and MyHCn was not affected, suggesting that a longer timeframe is needed for adaptations to manifest at the protein level.

Salsolinol—a potential inhibitor of the gonadotropic axis in sheep during lactation

This study tested the hypothesis that salsolinol, a derivative of dopamine, affects GnRH and LH secretion in lactating sheep. In the in vivo experiment, the structural analogue of salsolinol, 1-methyl-3,4-dihydroisoquinoline (1-MeDIQ), was infused into the infundibular nucleus-median eminence of sheep at the fifth wk of lactation to antagonize salsolinol's action. Simultaneously, cerebrospinal fluid from the third brain ventricle, to determine GnRH concentration, and plasma samples, to measure LH concentration, were collected. In the in vitro experiment, the anterior pituitary (AP) explants from weaned sheep were incubated in culture medium containing 2 doses of salsolinol, 20 and 100 μg/mL (S20 and S100, respectively). The concentration of LH in the collected media and relative expression of LHβ subunit messenger RNA in the AP explants were determined. No significant difference was found in mean GnRH concentration in response to 1-MeDIQ infusion, but both mean plasma LH concentration and LH pulse frequency increased significantly (P < 0.001 and P < 0.05, respectively) compared with those in controls. Significantly higher LH concentrations occurred during the first (P < 0.001), second (P < 0.001), and fourth (P < 0.05) h of 1-MeDIQ infusion. In the in vitro study, both the S20 and S100 doses of salsolinol caused a significant decrease in the mean medium LH concentration compared with that in the control (P < 0.01 and P < 0.001, respectively). Salsolinol had no effect on the relative LHβ subunit messenger RNA expression in the incubated tissue. In conclusion, salsolinol is a potential inhibitor of the secretory activity of the gonadotropic axis in lactating sheep, at least at the AP level. Although no significant changes in GnRH release were directly confirmed, an increase in the frequency of LH pulses does not allow to exclude the central action of salsolinol.

Analgesic-antitumor peptide inhibits the migration and invasion of HepG2 cells by an upregulated VGSC β1 subunit.

Analgesic-antitumor peptide (AGAP), one of the scorpion toxin polypeptides, has been shown to have an antitumor activity. Recombinant AGAP (rAGAP) was shown to affect the migration and invasion of HepG2 cells via a voltage-gated sodium channel (VGSC) β1 subunit. The VGSC β1 subunit was validated as a cell adhesion molecule (CAM) in human hepatocellular carcinoma (HCC) cell lines. rAGAP suppresses the migration and invasion of HepG2 cells but has no significant effect of human liver HL7702 cells without β1 subunit expression. rAGAP inhibits the migration and invasion of the cells when the VGSC β1 subunit is overexpressed in HL7702 cells. To explain these findings, VGSC β1 subunit messenger RNA (mRNA) and protein levels were measured. The β1 subunit protein level was upregulated in a dose-dependent manner following treatment with rAGAP while there was no significant change in the mRNA level, so rAGAP might be an active component of the VGSC β1 subunit.

Up-regulation of integrin α6β4 expression by mitogens involved in dairy cow mammary development.

In dairy cows, the extracellular microenvironment varies significantly from the virgin state to lactation. The function of integrin α6β4 is dependent on cell type and extracellular microenvironment, and the precise expression profile of α6β4 and its effects on mammary development remain to be determined. In the present study, real-time PCR and immunohistochemistry were used to analyze the expression and localization of integrin α6β4 in Holstein dairy cow mammary glands. The effects of integrin α6β4 on the proliferation induced by mammogenic mitogens were identified by blocking integrin function in purified dairy cow mammary epithelial cells (DCMECs). The results showed that the localization of β4 subunit and its exclusive partner the α6 subunit were not consistent but were co-localized in basal luminal cells and myoepithelial cells, appearing to prefer the basal surface of the plasma membrane. Moreover, α6 and β4 subunit messenger RNA (mRNA) levels changed throughout the stages of dairy cow mammary development, reflected well by protein levels, and remained higher in the virgin and pregnancy states, with duct/alveolus morphogenesis and active cell proliferation, than during lactation, when growth arrest is essential for mammary epithelial cell differentiation. Finally, the upregulation of integrin expression by both mammogenic growth hormone and insulin-like growth factor-1 and the inhibited growth of DCMECs by function-blocking integrin antibodies confirmed that integrin α6β4 was indeed involved in dairy cow mammary development.

Ovariectomy increases L-type Ca(2+) channel activity in porcine coronary smooth muscle.

Sex hormone status has been demonstrated to play a role in the regulation of ion channel activity. We previously demonstrated increased L-type Ca channel current (ICa) in the coronary smooth muscle cells (SMCs) of male swine compared with female swine. In male swine, endogenous testosterone increases ICa in SMCs by enhanced expression of the pore-forming α1 subunit Cav1.2. Conversely, the role of sex hormones in female swine has not previously been investigated. Therefore, the purpose of the current study was to determine the effect of ovariectomy (OVX) on L-type Ca channel activity and expression in female Yucatan miniature swine. Sexually mature female swine were obtained from a breeder and either left intact (intact female [IF]; n = 5) or ovariectomized (n = 6). Sensitivity to depolarization-induced contractions was increased by OVX. Accordingly, mean (SEM) ICa was enhanced in the OVX group (-9.5 [0.6] pA/pF) compared with the IF group (-4.5 [0.3] pA/pF), although L-type Ca channel α1 subunit (Cav1.2; α1c) messenger RNA (mRNA) and protein expressions were unchanged.Among the L-type Ca channel β subunits, β1 (188 [31]) and β2a (561 [79]) had higher mRNA expression levels (target/18S) than β3 (9 [1]) and β4 (2 [0.1]). Although β2a, β3, and β4 mRNA and protein expressions were not different between groups, protein expression of the β1 subunit (Cavβ1) was decreased in the OVX group compared with the IF group. Endogenous female hormones inhibit L-type Ca channel activity in coronary SMCs potentially via the up-regulation of Cavβ1 subunit expression.

Vascular smooth muscle cells from small human omental arteries express P2X1 and P2X4 receptor subunits.

Stimulation of P2X receptors by ATP in vascular smooth muscle cells (VSMCs) is proposed to mediate vascular tone. However, understanding of P2X receptor-mediated actions in human blood vessels is limited, and therefore, the current work investigates the role of P2X receptors in freshly isolated small human gastro-omental arteries (HGOAs). Expression of P2X1 and P2X4 receptor subunit messenger RNA (mRNA) and protein was identified in individual HGOA VSMCs using RT-PCR and immunofluorescent analysis and using Western blot in multi-cellular preparations. ATP of 10μmol/l and αβ-meATP of 10μmol/l, a selective P2X receptor agonist, evoked robust increases in [Ca(2+)]i in fluo-3-loaded HGOA VSMCs. Pre-incubation with 1μmol/l NF279, a selective P2X receptor antagonist, reduced the amplitude of αβ-meATP-induced increase in [Ca(2+)]i by about 70%. ATP of 10μmol/l and αβ-meATP of 10μmol/l produced similar contractile responses in segments of HGOA, and these contractions were greatly reduced by 2μmol/l NF449, a selective P2X receptor inhibitor. These data suggest that VSMCs from HGOA express P2X1 and P2X4 receptor subunits with homomeric P2X1 receptors likely serving as the predominant target for extracellular ATP.

The effects of repeated zolpidem treatment on tolerance, withdrawal-like symptoms, and GABAA receptor mRNAs profile expression in mice: Comparison with diazepam

Zolpidem is a short-acting, non-benzodiazepine hypnotic that acts as a full agonist at α1-containing GABAA receptors. Overall, zolpidem purportedly has fewer instances of abuse and dependence than traditionally used benzodiazepines. However, several studies have shown that zolpidem may be more similar to benzodiazepines in terms of behavioral tolerance and withdrawal symptoms. In the current study, we examined whether subchronic zolpidem or diazepam administration produced deficits in zolpidem's locomotor-impairing effects, anxiety-like behaviors, and changes in GABAAR subunit messenger RNA (mRNA). Mice were given subchronic injections of either zolpidem (10mg/kg), diazepam (20mg/kg), or vehicle twice daily for 7days. On day 8, mice were given a challenge dose of zolpidem (2mg/kg) or vehicle before open field testing. Another set of mice underwent the same injection regimen but were sacrificed on day 8 for qRT-PCR analysis. We found that subchronic zolpidem and diazepam administration produced deficits in the acute locomotor-impairing effects of zolpidem and increased anxiety-like behaviors 1day after drug termination. In addition, we found that subchronic treatment of zolpidem and diazepam induced distinct but overlapping GABAAR subunit mRNA changes in the cortex but few changes in the hippocampus, amygdala, or prefrontal cortex. Levels of mRNA measured in separate mice after a single injection of either zolpidem or diazepam revealed no mRNA changes. In mice, subchronic treatment of zolpidem and diazepam can produce deficits in the locomotor-impairing effects of zolpidem, anxiety-like withdrawal symptoms, and subunit-specific mRNA changes.

Maturity-dependent fractionation of neutrophil progenitors: A new method to examine in vivo expression profiles of differentiation-regulating genes

To investigate differentiation-dependent gene expression during granulopoiesis, we established a new method to isolate six sequential differentiation stages of neutrophil progenitors from bone marrow. Neutrophil progenitors were divided into three populations by density centrifugation, followed by depletion of other lineages, and further separated by fluorescence-activated cell sorting based on the expressions of CD34, CD11b, and CD16: CD34(+) fraction from a low-density population (F1), CD11b(-)/CD16(-) (F2), CD11b(+)/CD16(-) (F3), and CD11b(+)/CD16(low) (F4) fractions with intermediate density, and CD11b(+)/CD16(int) (F5) and CD11b(+)/CD16(high) (F6) fractions from a high-density population. To examine whether this fractionation was applicable to the study of in vivo gene expression profiles during granulopoiesis, we analyzed messenger RNA levels of AML-1 and CCAAT/enhancer binding protein (EBP)-ε and two target genes of C/EBP-ε, granulocyte-macrophage colony-stimulating factor receptor common β subunit and lactoferrin, in the six fractions and peripheral blood-derived neutrophils (F7). Expression of AML-1 and C/EBP-ε peaked at F1 and F4, respectively, followed by a gradual decrease. Although granulocyte-macrophage colony-stimulating factor receptor common β subunit messenger RNA levels remained low from F1 through F6 and elevated at F7, lactoferrin messenger RNA showed a drastic increase at F3 and dropped at F5. The difference in the expression profiles of the two C/EBP-ε target genes suggests the involvement of regulators other than C/EBP-ε in the induction of the two genes. The new fractionation method is able to provide new information on maturation-dependent gene expression during granulopoiesis.

Selective Pyramidal Cell Reduction of GABAA Receptor α1 Subunit Messenger RNA Expression in Schizophrenia

Levels of messenger RNA (mRNA) for the α1 subunit of the GABA(A) receptor, which is present in 60% of cortical GABA(A) receptors, have been reported to be lower in layer 3 of the prefrontal cortex (PFC) in subjects with schizophrenia. This subunit is expressed in both pyramidal cells and interneurons, and thus lower α1 subunit levels in each cell population would have opposite effects on net cortical excitation. We used dual-label in situ hybridization to quantify GABA(A) α1 subunit mRNA expression in calcium/calmodulin-dependent kinase II α (CaMKIIα)-containing pyramidal cells and glutamic acid decarboxylase 65 kDa (GAD65)-containing interneurons in layer 3 of the PFC from matched schizophrenia and healthy comparison subjects. In subjects with schizophrenia, mean GABA(A) α1 subunit mRNA expression was significantly 40% lower in pyramidal cells, but was not altered in interneurons. Lower α1 subunit mRNA expression in pyramidal cells was not attributable to potential confounding factors, and thus appeared to reflect the disease process of schizophrenia. These results suggest that pyramidal cell inhibition is reduced in schizophrenia, whereas inhibition of GABA neurons is maintained. The cell type specificity of these findings may reflect a compensatory response to enhance layer 3 pyramidal cell activity in the face of the diminished excitatory drive associated with the lower dendritic spine density on these neurons.

The Effects of Aging and Genotype on NMDA Receptor Expression in Growth Hormone Receptor Knockout (GHRKO) Mice

Caloric restriction enhances N-methyl-D-aspartate (NMDA) receptor binding and upregulates messenger RNA expression of the GluN1 subunit during aging. Old growth hormone receptor knockout mice resemble old calorically restricted rodents in enhanced life span and brain function, as compared with aged controls. This study examined whether aged growth hormone receptor knockout mice also show enhanced expression of NMDA receptors. Six or 23- to 24-month-old male normal-sized control or dwarf growth hormone receptor knockout mice were assayed for NMDA-displaceable [(3)H]glutamate binding (autoradiography) and GluN1 subunit messenger RNA (in situ hybridization). There was slight sparing of NMDA receptor binding densities within aged medial prefrontal and motor cortices, similar to caloric restriction, but there were greater age-related declines in GluN1 messenger RNA in growth hormone receptor knockout versus control mice. These results suggest that some of the functional improvements in aged mice with altered growth hormone signaling may be due to enhancement of NMDA receptors, but not through the upregulation of messenger RNA for the GluN1 subunit.

Differential expression of glycine receptor subunit messenger RNA in the rat following spinal cord injury

Spinal cord injury (SCI) results in alterations in the regulation of many genes that may influence neuronal death and the subsequent loss of motor function and neuropathic pain. The subtype expression mRNA levels of glycine receptors (GlyRs) after SCI are unknown. Using real-time reverse transcription PCR, this study analyzed changes in the mRNA abundance of the four major GlyR subunits (α13 and β) at 6, 24 h and 3, 7 and 10 days after SCI in adult male rats. SCI was induced at the T9 level by transection. Our results show a complicated temporal and spatial pattern of alteration in GlyR mRNA expression levels after SCI. Temporal and spatial variations with different degrees and direction (up or downregulation) of expression alteration were observed. The greatest variation was seen in GlyRα1, whereas GlyRα2 was downregulated in all regions following SCI. This study shows that alteration in GlyR expression starts as early as 6 h after SCI. The most significant points of this research are temporal elevation of GlyRα1 and continuous reduction of GlyRα2. Alterations in GlyR expression within the spinal cord may have a key role in the development of pathological pain. Therefore, control of GlyR expression could represent a novel therapeutic avenue for the development of new painkiller agents in SCI.

Regulation of glutamate receptor subunit availability by microRNAs

The efficacy of synaptic transmission depends, to a large extent, on postsynaptic receptor abundance. The molecular mechanisms controlling receptor abundance are poorly understood. We tested whether abundance of postsynaptic glutamate receptors (GluRs) in Drosophila neuromuscular junctions is controlled by microRNAs, and provide evidence that it is. We show here that postsynaptic knockdown of dicer-1, the endoribonuclease necessary for microRNA synthesis, leads to large increases in postsynaptic GluR subunit messenger RNA and protein. Specifically, we measured increases in GluRIIA and GluRIIB but not GluRIIC. Further, knockout of MiR-284, a microRNA predicted to bind to GluRIIA and GluRIIB but not GluRIIC, increases expression of GluRIIA and GluRIIB but not GluRIIC proportional to the number of predicted binding sites in each transcript. Most of the de-repressed GluR protein, however, does not appear to be incorporated into functional receptors, and only minor changes in synaptic strength are observed, which suggests that microRNAs primarily regulate Drosophila receptor subunit composition rather than overall receptor abundance or synaptic strength.

Molecular Pathology of Genetic Epilepsies Associated with GABAA Receptor Subunit Mutations

Mutations in ligand-gated ion channel genes associated with idiopathic generalized epilepsies have been reported in excitatory acetylcholine receptor alpha4 and beta2 subunit genes linked to autosomal dominant nocturnal frontal lobe epilepsy and in inhibitory GABA(A) receptor alpha1, beta3, gamma2, and delta subunit genes associated with childhood absence epilepsy, juvenile myoclonic epilepsy, pure febrile seizures, generalized epilepsy with febrile seizures plus, and generalized epilepsy with tonic-clonic seizures. Recent studies suggest that these mutations alter receptor function or biogenesis, including impaired receptor subunit messenger RNA stability, receptor subunit protein folding and stability, receptor assembly, and receptor trafficking.

Expression of nicotinic acetylcholine receptors and subunit messenger RNAs in the enteric nervous system of the neonatal rat

In the enteric nervous system (ENS) excitatory nicotinic cholinergic transmission is mediated by neuronal nicotinic acetylcholine receptors (nAChR) and is critical for the regulation of gastric motility. nAChRs are ligand-gated pentameric ion channels found in the CNS and peripheral nervous system. The expression of heteromeric nAChR and receptor subunit mRNAs was investigated in the neonatal rat ENS using receptor autoradiography with the radiolabeled ligand 125I-epibatidine, and in situ hybridization with subtype specific probes for ligand binding alpha (α2, α3, α4, α5, α6) and structural beta (β2, β3, β4) subunits. The results showed strong nicotine sensitive binding of 125I-epibatidine around the stomach, and small and large intestines. The binding was partially displaced by A85380, a nicotinic ligand which differentiates between different heteromeric nAChR subtypes, suggesting a mixed receptor population. Radioactive in situ hybridization detected expression of α3, α5, α7, β2 and β4 mRNA in the myenteric plexus of the stomach, and small and large intestines. In the submucosal plexus of the small and large intestines expression of α3, α5 and β4 was found in some ganglia. There was no signal for α4, α6 and β3 in the ENS but positive hybridization signal for α2 transcripts was seen in some areas of the small intestines. However, the signal was not associated with any ganglion cells. The results confirm the presence of heteromeric nAChRs in the ENS similar to those found in the peripheral nervous system, with the majority being composed of α3(α5)β4, and a few α3β2 nAChRs. In addition, homomeric α7 nAChRs could be present.

Effects of Nano-Anatase on Ribulose-1, 5-Bisphosphate Carboxylase/Oxygenase mRNA Expression in Spinach

Being a proven photocatalyst, nano-anatase is capable of undergoing electron transfer reactions under light. In previous studies, we had proven that nano-anatase improved photosynthesis and greatly promoted spinach growth. However, the molecular mechanisms of carbon reaction promoted by nano-anatase are still not clearly understood. In the present paper, reverse transcription polymerase chain reaction and Northern blotting were carried out, and the results showed that the expression of ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco) small subunit (rbcS) and Rubisco large subunit (rbcL) messenger RNA (mRNA) in the nano-anatase-treated spinach were obviously promoted, respectively, while the effects of bulk TiO2 were not so significant. Accordingly, the protein expression of Rubisco from the nano-anatase-treated spinach was increased by 40% compared with control. Further analysis indicated that the activity of Rubisco in the nano-anatase-treated spinach was significantly higher than the control, by up to 2.33 times, and bulk TiO2 treatment had no such significant effects. Together, one of the molecular mechanisms of carbon reaction promoted by nano-anatase is that the nano-anatase treatment results in the enhancement of Rubisco mRNA amounts, the protein levels, and activity of Rubisco, thereby leading to the improvement of Rubisco carboxylation and the high rate of photosynthetic carbon reaction.

N-Methyl-D-Aspartate Receptor and Calbindin-Containing Neurons in the Anterior Cingulate Cortex in Schizophrenia and Bipolar Disorder

Glutamatergic modulation of gamma-aminobutyric acid (GABA) interneurons via the NR2A subunit of the N-methyl-D-aspartate (NMDA) receptor in the cerebral cortex contributes to the pathophysiology of schizophrenia and bipolar disorder. Previously, we found that, in the anterior cingulate cortex (ACCx), the number of GABA cells that expressed the messenger RNA (mRNA) for the NMDA NR2A subunit was significantly decreased in subjects with schizophrenia and bipolar disorder and that this decrease occurred most prominently in layer 2. In this study, we hypothesized that the subset of GABA interneurons that contained the calcium-binding protein calbindin (CB), by virtue of their preferential localization to layer 2, might be particularly affected. We simultaneously labeled the mRNA for the NMDA NR2A subunit with [(35)S] and the mRNA for CB with digoxigenin with an immunoperoxidase procedure. We found that, in the normal human ACCx, only approximately 10% of all CB-containing cells expressed NR2A mRNA. However, compared with the normal control subjects and subjects with bipolar disorder, the density of CB+/NR2A+ neurons in layer 2 was increased by 41% to 44 % in subjects with schizophrenia, whereas the amount of NR2A mRNA/CB+ neurons was unchanged. These observations suggest that, in schizophrenia, a number of CB-containing cells that normally do not express NR2A might become NR2A-expressing or, perhaps not mutually exclusively, the number of CB-expressing cells might be increased and these cells express NR2A. The findings of this study highlight the notion that glutamatergic innervation of subsets of GABA cells might be differentially altered in schizophrenia and bipolar disorder.

L-type calcium channel current up-regulation by chronic stress is associated with increased α1c subunit expression in rat ventricular myocytes

The L-type calcium channel plays a pivotal role in the regulation of a wide range of cellular processes, including membrane excitability, Ca(2+) homeostasis, protein phosphorylation, and gene regulation. Alterations in the density or function of the L-type calcium channel have been implicated in a variety of cardiovascular diseases. Our previous study found that acute restraint stress could cause an enhancement of the L-type calcium current (I (Ca-L))(,) which correlated with an up-regulation of activation characters of the calcium channel. In this study, we observed the change of I (Ca-L) in rat ventricular myocytes under chronic restraint stress using the whole-cell patch-clamp technique and further explored its modulation mechanisms. The results showed that chronic restraint stress could also enhance I (Ca-L), but increased I (Ca-L) was not accompanied by an alteration of the characteristics of activation and inactivation of the L-type calcium channel. Furthermore, results from reverse-transcription polymerase chain reaction and Northern blot showed that the abundance of alpha(1c) subunit messenger RNA of the L-type calcium channel in the ventricle was increased significantly after chronic stress, and Western blot analysis revealed the amount of alpha(1c) subunit protein also was elevated. These results suggest that the L-type calcium channel is involved in stress-induced cardiomyocyte injury, and the up-regulated expression of the L-type calcium channel alpha(1c) subunit might contribute to the I (Ca-L) change under chronic stress, which is different from the regulation mechanism of acute restraint stress that mostly relates to an alteration in protein kinase A-dependent channel activation. Thus, it would provide a new insight into the mechanism of cardiomyocyte injury induced by stress.

Intracellular calcium mobilization of the aganglionic intestine in the endothelin B receptor gene –deficient rat

Background and Aim Up to now, numerous reports have analyzed the pathogenesis of Hirschsprung's disease (HD) by means of physiologic, pathologic, or molecular biologic methods. However, very little is still known about the smooth muscle cell itself. The endothelin B receptor gene–deficient (EDNRB(−/−)) rat, which is suitable for research of HD, has an aganglionic segment of the total colon. Our purpose is to investigate the myogenic mechanisms using simultaneous measurements of the intracellular Ca 2+ concentration ([Ca 2+]i) and tension and reverse transcriptase polymerase chain reaction for L-type Ca 2+ channel (L-VOC) expression. Methods The muscle strips of the rat distal colon were loaded with a Ca 2+ indicator dye, fura-PE3/AM, for 3 to 4 hours. The changes in the fluorescence intensity of Ca 2+-fura-PE3 complex of the strips were monitored with a front surface fluorometer (CAM-230). The fluorescence intensities at 340- and 380-nm excitation and their ratio (F340/F380) were recorded as the level of [Ca 2+]i. The comparison of L-VOC α1c subunit messenger RNA (mRNA) expression in both wild and homozygous rat was performed by reverse transcriptase polymerase chain reaction. Results The peak levels of force development induced by carbachol were 139.1% ± 5.0% in EDNRB(−/−) rat, whereas the peak levels were 242.1% ± 27.7% in EDNRB(+/+) rat. The changes in the [Ca 2+]i elevation induced by carbachol were 101.7% ± 12.2% in the homozygous rat, whereas these were 143.8% ± 8.9% in the wild-type rat. Both results in the homozygous rat significantly decreased in comparison with those of the wild rat ( P < .05). The expression of the L-VOC channel mRNA also decreased in the homozygous rat. Conclusions This is the first report to show the [Ca 2+]i mobilization in the smooth muscles of the rat model of HD. The decrease in both [Ca 2+]i and force development was thus considered to be due to the decrease in the Ca 2+ channel expression.

Ketamine-Induced Neuronal Cell Death in the Perinatal Rhesus Monkey

Ketamine is widely used as a pediatric anesthetic. Studies in developing rodents have indicated that ketamine-induced anesthesia results in brain cell death. Additional studies are needed to determine if ketamine anesthesia results in brain cell death in the nonhuman primate and if so, to begin to define the stage of development and the duration of ketamine anesthesia necessary to produce brain cell death. Rhesus monkeys (N = 3 for each treatment and control group) at three stages of development (122 days of gestation and 5 and 35 postnatal days [PNDs]) were administered ketamine intravenously for 24 h to maintain a surgical anesthetic plane, followed by a 6-h withdrawal period. Similar studies were performed in PND 5 animals with 3 h of ketamine anesthesia. Animals were subsequently perfused and brain tissue processed for analyses. Ketamine (24-h infusion) produced a significant increase in the number of caspase 3-, Fluoro-Jade C- and silver stain-positive cells in the cortex of gestational and PND 5 animals but not in PND 35 animals. Electron microscopy indicated typical nuclear condensation and fragmentation in some neuronal cells, and cell body swelling was observed in others indicating that ketamine-induced neuronal cell death is most likely both apoptotic and necrotic in nature. Ketamine increased N-methyl-D-aspartate (NMDA) receptor NR1 subunit messenger RNA in the frontal cortex where enhanced cell death was apparent. Earlier developmental stages (122 days of gestation and 5 PNDs) appear more sensitive to ketamine-induced neuronal cell death than later in development (35 PNDs). However, a shorter duration of ketamine anesthesia (3 h) did not result in neuronal cell death in the 5-day-old monkey.