Α-subunit mRNA Research Articles

Effects of treatment with GABA A receptor subunit antisense oligodeoxynucleotides on GABA-stimulated 36Cl − influx in the rat cerebral cortex

GABA A receptor function was studied in cerebral cortical vesicles prepared from rats after intracerebroventricular microinjections of antisense oligodeoxynucleotides (aODNs) for α1, γ2, β1, β2 subunits. GABA A receptor α1 subunit aODNs decreased α1 subunit mRNA by 59±10%. Specific [ 3H]GABA binding was decreased by α1 or β2 subunit aODNs (to 63±3% and 64±9%, respectively) but not changed by γ2 subunit aODNs (94±5%). Specific [ 3H]flunitrazepam binding was increased by α1 or β2 subunit aODNs (122±8% and 126±11%, respectively) and decreased by γ2 subunit aODNs (50±13%). The “knockdown” of specific subunits of the GABA A receptor significantly influenced GABA-stimulated 36Cl − influx. Injection of α1 subunit aODNs decreased basal 36Cl − influx and the GABA E max; enhanced GABA modulation by diazepam; and decreased antagonism of GABA activity by bicuculline. Injection of γ2 subunit aODNs increased the GABA E max; reversed the modulatory efficacy of diazepam from enhancement to inhibition of GABA-stimulation; and reduced the antagonist effect of bicuculline. Injection of β2 subunit aODNs reduced the effect of diazepam whereas treatment with β1 subunit aODNs had no effect on the drugs studied. Conclusions from our studies are: (1) α1 subunits promote, β2 subunits maintain, and γ2 subunits suppress GABA stimulation of 36Cl − influx; (2) α1 subunits suppress, whereas β2, and γ2 subunits promote allosteric modulation by benzodiazepines; (3) diazepam can act as an agonist or inverse agonist depending on the relative composition of the receptor subunits; and (4) the mixed competitive/non-competitive effects of bicuculline result from activity at α1 and γ2 subunits and the lack of activity at β1 and β2 subunits.

Gonadotropin Regulation of Inhibin α-Subunit mRNA and Immunoreactive Protein in Cultured Chicken Granulosa Cells

Gonadotropin regulation of the inhibin α-subunit was investigated in chicken granulosa cell cultures. Granulosa layers were isolated from the F1 and F3 + F4 follicles from three to four hens, pooled according to size, dispersed, and cultured (n=3 replications for each experiment). In Experiments 1 and 2 either ovine LH or FSH was added to the cultures at doses of 0, 5, and 25 ng/ml. The cultures were terminated at 4, 24, and 48 h after plating. For both follicle sizes the expression of mRNA for the inhibin α-subunit was less (P<0.05) at 24 and 48 h in untreated cells than in those treated with both doses of LH. Expression of the mRNA for the inhibin α-subunit was significantly increased only by the 25 ng/ml dose of FSH and only in the F1 follicle at 24 and 48 h compared to the untreated cells. After 48 h of culture, immunoreactive α-subunit protein accumulation was greater for both follicle types in the media of cells treated with the highest dose of LH and FSH than in the media from untreated cells. In Experiment 3, doses of 0, 5, 25, or 50 ng/ml of either LH or FSH were added to F1 and F3 + F4 granulosa cells. All cultures were terminated at 48 h. LH and FSH increased the expression of the mRNA and immunoreactive protein for the inhibin α-subunit equally in a time-dependent manner. These experiments indicate that gonadotropins enhance the expression of both the mRNA and the protein for the inhibin α-subunit in chicken granulosa cells.

Physiological modulation of GABA A receptor plasticity by progesterone metabolites

The possible functional relation between changes in brain and plasma concentrations of neurosteroids and the plasticity of γ-aminobutyric acid type A (GABA A) receptors in the brain during pregnancy and after delivery was investigated in rats. The concentrations in the cerebral cortex and plasma of pregnenolone as well as of progesterone and its neuroactive derivatives allopregnanolone (3α-hydroxy-5α-pregnan-20-one) and allotetrahydrodeoxycorticosterone (5α-hydroxy-3α,21-diol-20-one) increased during pregnancy, peaking around day 19, before returning to control (estrus) values immediately before delivery (day 21). In the postpartum period, steroid concentrations in plasma and brain did not differ from control values. The densities of [ 3 H ]GABA, [ 3 H ]flunitrazepam, and t-[ 35 S ]butylbiciclophosphorotionate (TBPS) binding sites in the cerebral cortex also increased during pregnancy, again peaking on day 19 and returning to control values on day 21; receptor density was decreased further 2 days after delivery and again returned to control values within 7 days. These changes were accompanied by a decrease in the apparent affinity of the binding sites for the corresponding ligand on day 19 of pregnancy. The amount of the γ2L subunit mRNA decreased progressively during pregnancy, in the cerebral cortex and hippocampus, returned to control value around the time of delivery and did not change in the postpartum period. On the contrary, the amount of α4 subunit mRNA was not modified during pregnancy both in the cerebral cortex and hippocampus whereas significantly increased 7 days after delivery only in the hippocampus. No significant changes were apparent for α1, α2, α3, β1, β2, β3 and γ2S subunit mRNAs. Administration of finasteride, a specific 5α-reductase inhibitor, to pregnant rats from days 12 to 18 markedly reduced the increases in the plasma and brain concentrations of allopregnanolone and allotetrahydrodeoxycorticosterone as well as prevented both the increase in the densities of [ 3 H ]flunitrazepam and [ 35 S ]TBPS binding sites and the decrease of γ2L mRNA normally observed during pregnancy. The results demonstrate that the changes in the plasticity of GABA A receptors that occur in rat brain during pregnancy and after delivery are related to the physiological changes in plasma and brain concentrations of neurosteroids.

Differential Expression of α1, α2, α3, and α5GABAAReceptor Subunits in Seizure-Prone and Seizure-Resistant Rat Models of Temporal Lobe Epilepsy

Temporal lobe epilepsy remains one of the most widespread seizure disorders in man, the etiology of which is controversial. Using new rat models of temporal lobe epilepsy that are either prone or resistant to develop complex partial seizures, we provide evidence that this seizure susceptibility may arise from arrested development of the GABAA receptor system. In seizure-prone (Fast kindling) and seizure-resistant (Slow kindling) rat models, both the mRNA and protein levels of the major alpha subunit expressed in adult brain (alpha1), as well as those highly expressed during development (alpha2, alpha3, and alpha5), were differentially expressed in both models compared with normal controls. We found that alpha1 subunit mRNA expression in the Fast kindling strain was approximately half the abundance of control rats, whereas in the Slow kindling strain, it was approximately 70% greater than that of controls. However, Fast rats overexpressed the alpha2, alpha3, and alpha5 ("embryonic") subunits, having a density 50-70% greater than controls depending on brain area, whereas the converse was true of Slow rats. Using subunit-specific antibodies to alpha1 and alpha5 subunits, quantitative immunoblots and immunocytochemistry revealed a concordance with the mRNA levels. alpha1 protein expression was approximately 50% less than controls in the Fast strain, whereas it was 200% greater in the Slow strain. In contrast, alpha5 subunit protein expression was greater in the Fast strain than either the control or Slow strain. These data suggest that a major predispositional factor in the development of temporal lobe epilepsy could be a failure to complete the normal switch from the GABAA receptor alpha subunits highly expressed during development (alpha2, alpha3, and alpha5) to those highly expressed in adulthood (alpha1).

KGF prevents hyperoxia-induced reduction of active ion transport in alveolar epithelial cells.

We evaluated the effects of acute hyperoxic exposure on alveolar epithelial cell (AEC) active ion transport and on expression of Na+ pump (Na+-K+-ATPase) and rat epithelial Na+ channel subunits. Rat AEC were cultivated in minimal defined serum-free medium (MDSF) on polycarbonate filters. Beginning on day 5, confluent monolayers were exposed to either 95% air-5% CO2 (normoxia) or 95% O2-5% CO2 (hyperoxia) for 48 h. Transepithelial resistance (Rt) and short-circuit current (Isc) were determined before and after exposure. Na+ channel alpha-, beta-, and gamma-subunit and Na+-K+-ATPase alpha1- and beta1-subunit mRNA levels were quantified by Northern analysis. Na+ pump alpha1- and beta1-subunit protein abundance was quantified by Western blotting. After hyperoxic exposure, Isc across AEC monolayers decreased by approximately 60% at 48 h relative to monolayers maintained under normoxic conditions. Na+ channel beta-subunit mRNA expression was reduced by hyperoxia, whereas alpha- and gamma-subunit mRNA expression was unchanged. Na+ pump alpha1-subunit mRNA was unchanged, whereas beta1-subunit mRNA was decreased approximately 80% by hyperoxia in parallel with a reduction in beta1-subunit protein. Because keratinocyte growth factor (KGF) has recently been shown to upregulate AEC active ion transport and expression of Na+-K+-ATPase under normoxic conditions, we assessed the ability of KGF to prevent hyperoxia-induced changes in active ion transport by supplementing medium with KGF (10 ng/ml) from day 2. The presence of KGF prevented the effects of hyperoxia on ion transport (as measured by Isc) relative to normoxic controls. Levels of beta1 mRNA and protein were relatively preserved in monolayers maintained in MDSF and KGF compared with those cultivated in MDSF alone. These results indicate that AEC net active ion transport is decreased after 48 h of hyperoxia, likely as a result of a decrease in the number of functional Na+ pumps per cell. KGF largely prevents this decrease in active ion transport, at least in part, by preserving Na+ pump expression.

Nitric oxide inhibits transcription of the Na+-K+-ATPase alpha1-subunit gene in an MTAL cell line.

Nitric oxide (NO) has been implicated as an autocrine modulator of active sodium transport. To determine whether tonic exposure to NO influences active sodium transport in epithelial cells, we established transfected medullary thick ascending limb of Henle (MTAL) cell lines that overexpressed NO synthase-2 (NOS2) and analyzed the effects of deficient or continuous NO production [with or without NG-nitro-L-arginine methyl ester (L-NAME) in the culture medium, respectively] on Na+-K+-ATPase function and expression. The NOS2-transfected cells exhibited high-level NOS2 expression and NO generation, which did not affect cell viability or cloning efficiency. NOS2-transfected cells were grown in the presence of vehicle, NG-nitro-D-arginine methyl ester (D-NAME), or L-NAME for 16 h, after which 86Rb+ uptake assays, Northern analysis, or nuclear run-on transcription assays were performed. The NOS2-transfected cells allowed to produce NO continuously (vehicle or D-NAME) exhibited lower rates of ouabain-sensitive 86Rb+ uptake ( approximately 65%), lower levels of Na+-K+-ATPase alpha1-subunit mRNA ( approximately 60%), and reduced rates of de novo Na+-K+-ATPase alpha1-subunit transcription compared with L-NAME-treated cells. These results have uncovered a novel effect of NO to inhibit transcription of the Na+-K+-ATPase alpha1-subunit gene.

Hormonal modulation of branchial Na +-K +-ATPase subunit mRNA in a marine teleost Sparus sarba

The effect of hormone treatment on the abundance of Na +-K +-ATPase α- and β-subunit mRNA in Sparus sarba branchial tissue was investigated. Groups of seawater (33‰) and hypo-osmotic (6‰) acclimated fish were injected daily, with either saline, cortisol, recombinant bream growth hormone (rbGH) or ovine prolactin (oPRL). Total RNA from branchial tissue was analyzed by Northern blotting using PCR amplified Na +-K +-ATPase α- and β-subunit cDNA clones. Na +-K +-ATPase α- and β- subunit transcripts of 3.3kb and 2.4kb respectively, were detected and their abundance, after hormone treatment was assessed using RNA dot blots. The abundance of subunit mRNAs increased 1.4–1.9 fold, relative to controls, after cortisol treatment. The α:β mRNA ratio also increased in cortisol treated seawater acclimated fish. Growth hormone treatment did not cause any significant changes in Na +-K +-ATPase subunit mRNA, whereas prolactin significantly reduced α-subunit mRNA levels by approximately 0.5 fold in both seawater and hypo-osmotic conditions. The data from this study add further support to the generally accepted roles that cortisol and prolactin have in the modulation of Na +-K +-ATPase activity. It can be concluded from this study that S. sarba branchial Na +-K +-ATPase subunit expression is multihormonally regulated.

Aldosterone directly induces Na, K-ATPase alpha 1-subunit mRNA in the renal cortex of rat.

The change of blood pressure and the induction of Na, K-ATPase alpha 1-subunit mRNA have been investigated in the renal cortex of aldosterone-treated hypertensive rat. The increase of blood pressure by aldosterone-treatment for 25 days was decreased by the treatment of amiloride or spironolactone. The level of Na, K-ATPase alpha 1-subunit mRNA of the renal cortex in aldosterone-treated rat was increased than that in the control, and its increase was repressed by treatment of spironolactone, but not altered by the treatment of amiloride. This result suggests that the increase of Na, K-ATPase alpha 1-subunit mRNA in the renal cortex of aldosterone-treated hypertensive rat may be related with the direct induction of Na, K-ATPase mRNA without the increase of Na-traffic through Na-channel.

Laminin 2 (merosin), but not laminin 1 (EHS-laminin), may be a major laminin isoform present in normal and fibrotic rat liver

Recently several laminin isoforms were newly identified and found to distribute in a tissue and cell type-specific manner. We have studied the expression of several laminin isoforms, including α1(A), α2(M), β1(B1), β2(S), and γ1(B2) subunits, in normal and CCl 4-induced fibrotic rat liver by RT-PCR and performed immunohistochemical study with newly developed monoclonal antibody to merosin (laminin-2). RT-PCR study for laminin subunits revealed that α2(M) subunit mRNA transcript was dominant chain present in normal rat liver tissue, but α1(A) subunit mRNA was not. Laminin β1(B1) and γ1(B2) subunit mRNA were detected strongly and β2(S) subunit mRNA was detected weakly. During the CCl 4-induced rat hepatic fibrosis, α2, β1, β2, and γ1 subunit mRNA showed increased expression, but α1 transcript could not be detected during fibrotic process even in the condition where α1 subunit mRNA was detectable in rat placental tissue. Immunohistochemical studies with a novel monoclonal antibody to merosin also revealed that deposition of merosin increased during CCl 4-induced rat fibrotic liver. These data strongly suggest that laminin isoform containing α2 subunit (merosin) may be a major laminin present in normal and fibrotic rat liver.

Increased abundance of GABA A receptor subunit mRNAs in the brain of Long–Evans Cinnamon rats, an animal model of Wilson's disease

The abundance of mRNAs encoding various subunits of the γ-aminobutyric acid type A (GABA A) receptor was examined in different regions of the brain of Long–Evans Cinnamon (LEC) rats, an animal model of Wilson's disease (WD). The measurements were performed at two different stages of disease: at 9 weeks of age, when no symptoms are evident, and at 15 weeks of age, when 90% of the animals develop jaundice. The amounts of the γ2L and γ2S subunit mRNAs in the striatum, cerebellum, and cerebral cortex of LEC rats at 9 weeks of age were increased (+25 to +35%) compared with those in LE rats of the same age; these differences were no longer apparent in 15-week old animals. The amount of α1 subunit mRNA was also significantly increased (+30%) in the cerebellum of LEC rats at 9 weeks of age; although a smaller increase (+20%) was still evident at 15 weeks of age, this was not statistically significant. The amount of β2 subunit mRNA was increased in the cerebellum (+32%) and hippocampus (+21%) of LEC rats at 9 weeks of age, but no differences with LE rats were apparent at 15 weeks. The onset of isoniazid-induced seizures in LEC rats at 9 weeks of age was significantly delayed compared with that in LE rats. These results demonstrate abnormal expression of GABA A receptor subunit genes in the brain of LEC rats, and they suggest that this altered expression is associated with an increase in GABAergic tone.

Single-Cell RT-PCR and Functional Characterization of Ca2+Channels in Motoneurons of the Rat Facial Nucleus

Voltage-dependent Ca2+ channels are a major pathway for Ca2+ entry in neurons. We have studied the electrophysiological, pharmacological, and molecular properties of voltage-gated Ca2+ channels in motoneurons of the rat facial nucleus in slices of the brainstem. Most facial motoneurons express both low voltage-activated (LVA) and high voltage-activated (HVA) Ca2+ channel currents. The HVA current is composed of a number of pharmacologically separable components, including 30% of N-type and approximately 5% of L-type. Despite the dominating role of P-type Ca2+ channels in transmitter release at facial motoneuron terminals described in previous studies, these channels were not present in the cell body. Remarkably, most of the HVA current was carried through a new type of Ca2+ channel that is resistant to toxin and dihydropyridine block but distinct from the R-type currents described in other neurons. Using reverse transcription followed by PCR amplification (RT-PCR) with a powerful set of primers designed to amplify all HVA subtypes of the alpha1-subunit, we identified a highly heterogeneous expression pattern of Ca2+ channel alpha1-subunit mRNA in individual neurons consistent with the Ca2+ current components found in the cell bodies and axon terminals. We detected mRNA for alpha1A in 86% of neurons, alpha1B in 59%, alpha1C in 18%, alpha1D in 18%, and alpha1E in 59%. Either alpha1A or alpha1B mRNAs (or both) were present in all neurons, together with various other alpha1-subunit mRNAs. The most frequently occurring combination was alpha1A with alpha1B and alpha1E. Taken together, these results demonstrate that the Ca2+ channel pattern found in facial motoneurons is highly distinct from that found in other brainstem motoneurons.

Escherichia coliLipopolysaccharide Downregulates Soluble Guanylate Cyclase in Pulmonary Artery Smooth Muscle

The soluble isoform of guanylate cyclase (sGC) is activated by nitric oxide (NO) to form guanosine 3′:5′-cyclic monophosphate (cGMP). Cyclic GMP levels cause smooth muscle relaxation and regulate vascular tone to various vascular beds, including the lung. Under conditions of cytokine excess the inducible synthesis of NO may result in cGMP overproduction, generalized vasodilation, and septic shock. In the pulmonary bed the opposite response may occur, pulmonary hypertension. We hypothesized that sGC activity becomes downregulated in the face ofEscherichia colilipopolysaccharide (LPS). We tested the effects of LPS on α1-subunit sGC mRNA abundance, Western analysis, and enzyme activity in cultured rat pulmonary artery smooth muscle cells. LPS increased extracellular cGMP production by pulmonary artery smooth muscle cells, with increased levels being first detectable at 3–6 h (10 μg/ml LPS) and exceeding 140 pmol/ml by 24 h (P< 0.05). The response was inhibited by 0.05 mMl-NG-monomethyl-l-arginine (l-NMA) and, in turn, restored by 1 mMl-arginine, indicating a NO synthase-dependent response. Pretreating cells with LPS for ≥ 3 h inhibited subsequent cGMP synthesis in response to 10−4M SNAP for 60 min. Coincubating cells with 0.05 mMl-NMA also reversed this effect. Soluble GC enzyme activity in cells exposed to basal medium alone measured 0.74 pmol cGMP/ml per minute; activity in cells exposed to 10 μg/ml LPS for 24 h decreased to 0.04 pmol cGMP/ml per minute (P< 0.05). LPS pretreatment decreased sGC mRNA abundance and protein mass, but did not totally eliminate them. It is concluded that LPS affects cGMP synthesis at the level of enzyme activity, enzyme mass, and mRNA abundance. Over the short term (<24 h) LPS causes the synthesis of large amounts of cGMP. As the duration of exposure progresses (≥3 h), mechanisms come into play that decrease cGMP production significantly and include decreases in mRNA abundance, enzyme mass, and enzyme activity.

Alterations in GABAA receptor alpha 1 and alpha 4 subunit mRNA levels in thalamic relay nuclei following absence-like seizures in rats.

Modification of GABAA receptor mRNA levels by seizure activity can regulate general neuronal excitability. The possibility of absence seizure-induced alteration in GABAA receptor alpha 1, alpha 4, beta 2, and gamma 2 subunit gene expression in thalamic relay nuclei was studied in a rat model of absence seizures induced by gamma-hydroxybutyric acid (GHB). We observed a marked increase in alpha 1 mRNA and a corresponding decrease in alpha 4 mRNA in thalamic relay nuclei 2-4 h after the onset of GHB-induced absence seizures (when the seizures were terminating). These changes were selective to these alpha isoforms as neither beta 2 nor gamma 2 mRNA changed following seizures and occurred only in thalamic relay nuclei but not in hippocampus, a structure from which absence seizures do not evolve. The alterations in alpha 1 and alpha 4 mRNA persisted until about 12 h, and by 24 h after the seizure-onset the mRNA levels normalized. Blocking GHB-seizures produced no change in the levels of alpha 1 and alpha 4 mRNA in thalamic relay nuclei, suggesting that seizures themselves were responsible for mRNA alterations. In order to determine if absence seizure-induced changes in alpha 1 and alpha 4 mRNA had any physiological significance, GHB was readministered in rats 6 and 24 h after the onset of seizures. The total duration of GHB-seizures was found to be significantly decreased when GHB was readministered at 6 h but not 24 h after the seizure-onset. These results suggest that absence seizures regulate GABAA receptor alpha 1 and alpha 4 gene expression in thalamic relay nuclei as a compensatory mechanism by which absence seizures are terminated.

Expression of the nicotinic acetylcholine receptor α4 subunit mRNA in the rat cerebellar cortex

In order to compare the previous immunohistochemical and immunocytochemical data on the distribution of nicotinic acetylcholine receptor α4 subunit-like immunoreactivity with the expression of α4 mRNA in the rat cerebellar cortex, the present study determined the cellular distribution of α4 mRNA in the rat cerebellar cortex. Northern blot analysis revealed two α4 mRNA bands in the rat cerebellum and three in the cerebral cortex, hippocampus, hypothalamus and striatum. The total level of these transcripts was lower in the cerebellum than in the other four regions. The expression of α4 mRNA was high in Purkinje cells and granular cells, whereas low expression was detected in the molecular layer. These results suggest that the expression of α4 mRNA is closely related to the α4-like immunoreactivity in the molecular and Purkinje cell layers. In the granular layer, α4 mRNA was very highly and broadly expressed in comparison with the α4-like immunoreactivity.

Role of the target organ in determining susceptibility to experimental autoimmune myasthenia gravis

Injection of anti-AChR antibodies in passive transfer experimental autoimmune myasthenia gravis (EAMG) results in increased degradation of acetylcholine receptor (AChR) and increased synthesis of AChR α-subunit mRNA. Passive transfer of anti-Main Immunogenic Region (MIR) mAb 35 in aged rats does not induce clinical signs of disease nor AChR loss. The expression of the AChR subunit genes was analyzed in susceptible and resistant rats. In aged EAMG resistant rats, no increase in the amount of AChR α-subunit mRNA was measured. In vivo AChR degradation experiments did not show any increase in AChR degradation rates in aged resistant rats, in contrast to young susceptible rats. Taken together, these data demonstrate that resistance of the AChR protein to antibody-mediated degradation is the primary mechanism that accounts for the resistance to passive transfer EAMG in aged rats.

Distribution of mRNA for the α4 subunit of the nicotinic acetylcholine receptor in the human fetal brain

Neuronal nicotinic acetylcholine receptors (nAChRs) present in the central nervous system (CNS), are multimeric proteins constituted of two different subunits, α and β, with different subtype arrangements and different pharmacological and functional properties. By in situ hybridization, we studied the distribution of the mRNA for the α4 subunit of nAChRs in brains of human 25-week old normal and fragile X fetuses. A strong hybridization signal was detected throughout the thalamus, cortex, pyramidal layer of the Ammon's horn, and the granular layer of the dentate gyrus. Several other areas including the claustrum, caudate nucleus, putamen, globus pallidus, subthalamic nucleus, subiculum, entorhinal cortex, and Purkinje cell layer displayed a low to moderate radiosignal. With few exceptions, our data in the human brain agree those previously reported in the rat. Also, our data indicate that the α4 subunit mRNA is produced early in the development, in the more differentiated cells, and in a site-specific manner. Additionally, the α4 mRNA is produced in the brain of fragile X fetuses with the same pattern and same intensity than in the normal fetal brain suggesting that α4 subunit mRNA production is not altered in the fragile X syndrome. High levels of α4 subunit mRNA in human fetal brain support the hypothesis of a morphogenic role of nAChRs during the early CNS development.

Increased expression of dendritic mRNA following the induction of long-term potentiation

A small number of mRNAs, including Ca 2+/calmodulin-dependent protein kinase II α-subunit (CamKII α) mRNA and microtubule-associated protein 2 (MAP2) mRNA, are present in the dendrites of neurones as well as in the cell bodies. We show here that the induction of long-term potentiation (LTP) in the hippocampal perforant path/granule cell synapses in anaesthetised rats is associated with increased levels of CamKII α mRNA and MAP2 mRNA in the granule cell dendrites after 2 h. Similarly, induction of LTP in the Schaffer collateral/CA1 pyramidal cell synapses in hippocampal slices maintained in vitro also results in elevated dendritic levels of CamKII α mRNA and MAP2 mRNA 2 h later. In both models, the levels of various other mRNA species restricted to the cell body region were unaffected by the induction of LTP. Increased expression of dendritic CamKII α mRNA and MAP2 mRNA appears to be a general feature of hippocampal plasticity, since it occurs following LTP induction in both the dentate gyrus and the CA1 region. The elevation of mRNA levels in a restricted region close to the afferent synapses would allow a highly-localised enhancement of the synthesis of the corresponding proteins, providing an elegant mechanism for protein-synthesis-dependent synaptic plasticity to maintain a high degree of anatomical specificity.

5' heterogeneity in epithelial sodium channel alpha-subunit mRNA leads to distinct NH2-terminal variant proteins.

The amiloride-sensitive epithelial sodium channel (ENaC) is composed of three subunits: alpha, beta, and gamma. The human alpha-ENaC subunit is expressed as at least two transcripts (N. Voilley, E. Lingueglia, G. Champigny, M. G. Mattei, R. Waldmann, M. Lazdunski, and P. Barbry. Proc. Natl. Acad. Sci. USA 91: 247-251, 1994). To determine the origin of these transcripts, we characterized the 5' end of the alpha-ENaC gene. Four transcripts that differ at their first exon were identified. Exon 1A splices to exon 2 to form the 5' end of alpha-ENaC1, whereas exon 1B arises separately and continues into exon 2 to form alpha-ENaC2. Other variant mRNAs, alpha-ENaC3 and alpha-ENaC4, are formed by activating 5' splice sites within exon 1B. Although alpha-ENaC3 and -4 did not change the open reading frame for alpha-ENaC, alpha-ENaC2 contains upstream ATGs that add 59 amino acids to the previous (alpha-ENaC1) protein. To address the significance of these isoforms, both proteins were expressed in Xenopus oocytes. The cRNA for each alpha-ENaC transcript when combined with beta- and gamma-ENaC cRNA reconstituted a low-conductance ion channel with amiloride-sensitive currents of similar characteristics. We have thus identified variant alpha-ENaC mRNAs that lead to functional ENaC peptides.

Sustained Hyperoxia Inhibits the Postnatal Increase in Na,K-ATPase Activity in Distal Lung Epithelial Cells From Chronically Ventilated Preterm Baboons• 1649

Lung epithelial cell Na,K-ATPase activity increases at birth in association with clearance of fluid from the potential airspaces. We hypothesized that the pulmonary edema that occurs in preterm baboons after sustained hyperoxia and mechanical ventilation might be associated with inhibition of the perinatal increase in Na,K-ATPase activity. To test this hypothesis, we measured Na,K-ATPase activity in distal lung epithelial cells, and we assessed Na,K-ATPase immunoreactivity and α1 and β1subunit mRNA content in whole lung tissue harvested from neonatal baboons that were delivered prematurely (140d gestation, term=180d) and mechanically ventilated for 6-10d with either 100% O2 (hyperoxia) or sufficient O2 to maintain normal PaO2 (normoxia). Results were compared with those from gestation-matched control fetuses. Na,K-ATPase activity, determined by measurement of ouabain-sensitive 86Rb uptake (nmol/106 cells/h), averaged 14 ± 3 in cells from 8 control fetuses, 24 ± 3(significantly different from control, p<0.05) in cells from 10 newborns with normoxia, and 14 ± 2 in lung epithelial cells from 14 newborns with hyperoxia. These results demonstrate that ventilation with 100% O2 is associated with inhibition of the normal postnatal increase in lung epithelial cell Na,K-ATPase activity that occurs after premature birth without subsequent hyperoxia. Immunohistochemistry, performed with antisera to Na,K-ATPase holoenzyme, showed localization to the epithelial membrane in all groups, but there was decreased signal intensity in lung sections from the hyperoxia group compared to the normoxia group and to fetal controls. mRNA content for the α1 and β1 Na,K-ATPase subunits was determined in whole lung tissue by northern blot analysis and quantified by phosphorimagery. A 6-fold increase (p<0.05, n=3) in α1 subunit mRNA content was observed in the hyperoxic group relative to normoxic newborns and control fetuses. No significant difference was observed between the groups in mRNA content for the β1subunit. Thus, sustained postnatal hyperoxia enhanced α1 subunit mRNA abundance, but inhibited Na,K-ATPase activity in the lungs of preterm baboons. We speculate that the failure of epithelial cell Na,K-ATPase activity to increase postnatally in non-human primates that are mechanically ventilated with 100% O2 probably contributes to the postnatal lung edema and associated respiratory distress that occurs in these animals.

Age-related changes in nicotinic acetylcholine receptor subunits α4 and β2 messenger RNA expression in postmortem human frontal cortex and hippocampus

Age-related changes in nicotinic acetylcholine receptor (nAChR) subunit α4 and β2 messenger RNA (mRNA) expression in the postmortem human frontal cortex and hippocampus was investigated using the reverse transcription-polymerase chain reaction (RT-PCR). In the frontal cortex, both α4 and β2 subunit mRNA expression decreased with age. In the hippocampus, α4 subunit mRNA expression was unaltered, while β2 subunit mRNA expression significantly decreased with age. These findings suggest that nAChR transcription decreases during aging with differing vulnerability between subunits and brain regions, which could in part contribute to the reduction in cognitive functions seen in the elderly.