Murine RARbeta4 displays reduced transactivation activity, lower affinity for retinoic acid, and no anti-AP1 activity

The biological actions of retinoic acid (RA) are mediated by retinoic acid receptors (RARalpha, beta, and gamma) and retinoid X receptors (RXR alpha, beta, and gamma). Each of the RARs is expressed as four to seven different isoforms. Four isoforms of RAR beta (beta1, beta2, beta3, and beta4), which differ only in their N-terminal sequence (A domain) have been described. These RARbeta isoforms display a specific pattern of expression in developing and adult animals and are highly evolutionarily conserved suggesting that they mediate distinct cellular effects of vitamin A. Experiments were performed to examine directly the RA-binding activity, transactivation activity, and anti-AP1 activity of each of these four RARbeta isoforms. The results demonstrate that RARbeta1, beta2, and beta3 bind RA with a similar K(d) value, have a similar EC(50) value in RA-dependent transactivation assays and inhibit AP1 activity to a similar level. By contrast, RARbeta4 has an elevated K(d) for RA, an increased EC(50) value in RA-dependent transactivation assays and does not display the ability to inhibit AP1 activity. This provides additional evidence that at least one RAR isoform, RARbeta4, may mediate distinct activities within a cell. Furthermore, these data suggest that the presence of an A domain in RARbeta is important for modulating these activities of RARs.     

Kainic acid-induced status epilepticus alters GABA receptor subunit mRNA and protein expression in the developing rat hippocampus

Kainic acid-induced status epilepticus leads to structural and functional changes in inhibitory GABAA receptors in the adult rat hippocampus, but whether similar changes occur in the developing rat is not known. We have used in situ hybridization to study status epilepticus-induced changes in the GABAAalpha1-alpha5, beta1-beta3, gamma1 and gamma2 subunit mRNA expression in the hippocampus of 9-day-old rats during 1 week after the treatment. Immunocytochemistry was applied to detect the alpha1, alpha2 and beta3 subunit proteins in the control and treated rats. In the saline-injected control rats, the alpha1 and alpha4 subunit mRNA expression significantly increased between the postnatal days 9-16, whereas those of alpha2, beta3 and gamma2 subunits decreased. The normal developmental changes in the expression of alpha1, alpha2, beta3 and gamma2 subunit mRNAs were altered after the treatment. The immunostainings with antibodies to alpha1, alpha2 and beta3 subunits confirmed the in situ hybridization findings. No neuronal death was detected in any hippocampal subregion in the treated rats. Our results show that status epilepticus disturbs the normal developmental expression pattern of GABAA receptor subunit in the rat hippocampus during the sensitive postnatal period of brain development. These perturbations could result in altered functional and pharmacological properties of GABAA receptors.     

Altered expression of retinoic acid (RA) receptor mRNAs in the fetal mouse secondary palate by all-trans and 13-cis RAs: implications for RA-induced teratogenesis

Retinoic acid (RA) is mandatory for various biological processes and normal embryonic development but is teratogenic at high concentrations. In rodents, one of the major malformations induced by RA is cleft palate (CP). RA mediates its effects by RA receptors (RARs), but the expression patterns of RARs in the developing palate are still unclear. We investigated the normal expression of RAR alpha, beta, and gamma messenger RNAs (mRNAs) in the fetal mouse secondary palate and the effects of all-trans and 13-cis RAs on the expression of RAR mRNAs by Northern blot analysis. RAR alpha (2.8, 3.8 kb), RAR beta (3.3 kb), and RAR gamma (3.7 kb) mRNAs were detected in the fetal palate on gestational days (GD) 12.5-14.5. The expression of RAR alpha and gamma mRNAs did not show apparent sequential changes, but that of RAR beta mRNA increased at GD 13.5. Treatment of pregnant mice with 100 mg/kg all-trans RA induced CP in 94% of the fetuses and elevated the levels of RAR beta and gamma mRNAs in the fetal palate. The up-regulation of RAR beta mRNA by all-trans RA was more marked than that of RAR gamma mRNA. Treatment with 100 mg/kg 13-cis RA induced CP in only 19% of the fetuses. Although 13-cis RA elevated the RAR beta and gamma mRNA levels in fetal palates, its up-regulation was slower and less marked than that induced by all-trans RA. These findings indicate that the induction of RAR beta mRNA in the fetal palate correlates well with the tissue concentration of all-trans RA after RA treatment, and RAR beta may be one of the most influential candidate molecules for RA-induced teratogenesis.     

Distribution and Regulation of ENaC Subunit and CFTR mRNA Expression in Murine Female Reproductive Tract

The present study investigated the regional distribution and cyclic changes in the mRNA expression of epithelial Na+ channel (ENaC) subunit and cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated Cl- channel, in adult female mouse reproductive tract. In situ hybridization revealed that in contrast to the abundant expression of CFTR, ENaC (alpha, beta, gamma) mRNA signal was not detected throughout the estrus cycle in the ovary and oviduct. Messenger RNA for all ENaC subunits was abundantly detected in the cervical and vaginal epithelia throughout the estrus cycle but for CFTR, mRNA was found only at proestrus. In the uterine epithelium, alphaENaC mRNA was detected at diestrus but not found at any other stage, while CFTR mRNA was only detected at early estrus but not other stages. Semi-quantitative RT-PCR detected mRNA for all ENaC subunits in the uterus throughout the cycle with maximal expression at diestrus and CFTR mRNA was only found in the early stages of the cycle. The involvement of ENaC and CFTR in Na+ absorption and Cl- secretion was demonstrated in cultured endometrial epithelia using the short-circuit current technique and found to be influenced by ovarian hormones. Taken together, these data indicate a main secretory role of the ovary and oviduct and a predominantly absorptive role of the cervix and vagina. The present results also suggest an ability of the uterus to secrete and absorb at different stages of the estrus cycle. Variations in the fluid profiles may be dictated by the regional and cyclic variations in expression of ENaC and CFTR and are likely to contribute to various reproductive events in different regions of the female reproductive tract.     

Expression of mRNA of beta 1- and beta 2-adrenergic receptors in Xenopus oocytes results from structurally distinct receptor mRNAs

Poly(A)+-selected RNA prepared from cells or tissues that express a homogeneous population of either beta 1- or beta 2-adrenergic receptors was isolated and then microinjected into Xenopus laevis oocytes. Following microinjection, the expression of beta-adrenergic receptors was assessed by equilibrium radioligand binding analysis using the antagonist ligand [3H]dihydroalprenolol. The pharmacology of the newly- expressed beta-adrenergic receptors in oocyte membranes was the same as that of the original tissue used as a source of RNA. Hybridization of nick-translated cDNA of hamster beta 2-adrenergic receptor to poly(A)+-selected RNA from tissues containing beta 2-adrenergic receptors was to a mRNA species of 2.2 kilobases. In contrast, hybridization of the cDNA probe to poly(A)+-selected RNA from tissues containing beta 1-adrenergic receptors was to a mRNA species of 2.0 kilobases. A single-stranded fragment of hamster beta 2-adrenergic receptor cDNA corresponding to nucleotides 730-886 was isolated and uniformly radiolabeled. This region of the gene is predicted to encode for the entire second exofacial loop (L4-5), the entire fifth transmembrane-spanning region, and the first 5 amino acid residues of the third cytoplasmic loop (L5-6) of the beta 2-adrenergic receptor. Hybridization at 48 and 56 degrees C of poly(A)+-selected RNA prepared from sources that express either beta 1 or beta 2-adrenergic receptors to the antisense orientation strand of this region of the beta 2-adrenergic receptor cDNA was followed by S1 endonuclease digestion of nonhybridized sequences. At 48 degrees C, S1-resistant hybrids from both sources of RNA protected the probe from S1 endonuclease digestion. At 56 degrees C, however, only the RNA prepared from the source of beta 2-adrenergic receptors protected the probe from S1 endonuclease digestion. These results demonstrate that the mRNAs encoding for the structurally homologous beta 1- and beta 2-adrenergic receptors are distinct in the pharmacological specificity of their translation products and in their size and structure.     

Differential expression of prostaglandin receptor mRNAs during adipose cell differentiation.

To clarify the molecular basis for the prostaglandin (PG) mediated effects in adipose cells at various stages of their development, expression of mRNAs encoding receptors specific for prostaglandin E2, F2alpha and I2 (i.e. EP, FP, and IP receptors) was investigated in differentiating clonal Ob1771 pre-adipocytes, as well as in mouse primary adipose precursor cells and mature adipocytes. We have further characterized the differential expression of mRNAs encoding three subtypes of the EP receptor, i.e. EP1, EP3, and EP4, and examined the expression of mRNAs encoding the three isoforms (alpha, beta, and gamma) of the EP3 receptor. Altogether the results show that the expression of IP, FP, EP1, and EP4 receptor mRNAs was considerably more pronounced in pre-adipose cells than in adipose cells, mRNAs encoding the alpha, beta, and gamma isoforms of the EP3 receptor were all exclusively expressed in freshly isolated mature adipocytes. These data may indicate that PGI2, PGF2alpha, and PGE2 may interact directly with specific receptors in pre-adipose cells, whose transduction mechanisms are known to affect maturation related changes. In mature adipocytes, however, the equipment of mRNAs encoding the EP3 receptor isoforms is in agreement with the well known effect of PGE2 on adenylate cyclase and lipolysis in mature adipocytes.     

Distribution of mRNA and binding sites of adrenoceptors and muscarinic receptors in the rat heart

Since there exist some obscurities in the expression of mRNAs and their receptors in the heart, we have investigated the gene expression (mRNA levels) of adrenoceptors (alpha1A-, alpha1B-, beta1-, beta2-, beta3-) and muscarinic receptors (M2) and the density of receptor binding sites (alpha1A-, alpha1B-, beta1-, beta2-adrenoceptors, muscarinic receptors). Moreover, the heart regions consist of tissue rich in ganglion cells (that are of importance in heart neural circuits) and those virtually free of them (myocytes). Therefore, we have examined the differences in the distribution of mRNAs/receptor binding sites in the atrial samples of the heart rich in ganglion cells vs. those are virtually free of them. Binding sites and mRNAs of muscarinic receptors and alpha1B-adrenoceptors differ in their distribution in different heart regions. The mRNAs for beta1- and beta2-adrenoceptors were almost equally distributed herein, while the amount of beta-adrenoceptors significantly differs in the heart regions. The alpha1A- and beta3-adrenoceptors mRNAs were also found in all investigated heart regions, but at significantly lower level and have not shown region differences. This is a new finding, especially to beta3-adrenoceptors, as they were not regularly found in each heart regions. alpha1B-adrenoceptors have similar distribution of their mRNAs and binding sites in some heart parts. Thus, we can conclude that there are noticeable differences in the presence of receptors in heart regions that contain ganglion cells in comparison to those are virtually free of them.