Distribution of somatostatin receptor messenger RNAs in the rat gastrointestinal tract

BACKGROUND & AIMS: The gastrointestinal (GI) tract is a major source and target of somatostatin (SRIF). Recently, five pharmacologically different SRIF receptors (sst1-5) were cloned. The cellular and tissue distribution of the sst1-5 messenger RNAs (mRNAs) were studied in the rat GI tract using in situ hybridization histochemistry (ISHH). METHODS: Two sets of (35)S-uridine triphosphate (UTP)-labeled antisense and sense riboprobes were prepared for each sst. ISHH was conducted on frozen tissue samples from rat stomach, duodenum, jejunum, ileum, colon, and pancreas. RESULTS: mRNAs of all five sst-s are widely expressed in the rat GI tract. The distribution pattern for each sst mRNA was identical with both antisense probes. No specific signal was found with any of the sense probes. Each layer of the different parts of the gut expressed mRNAs of multiple sst subtypes. All organs expressed sst3 mRNA very intensely. The lowest levels of mRNA expression for all five subtypes within the GI tract were found in the pancreas. CONCLUSIONS: The widespread expression of sst mRNAs suggests a significant role for SRIF in the regulation of GI function.     

Status of somatostatin receptor messenger RNAs and binding sites in rat brain during kindling epileptogenesis

In situ hybridization histochemistry with somatostatin sst1-sst5 receptor messenger RNA-selective oligoprobes and quantitative receptor autoradiographic binding studies using [125I]Tyr3-octreotide, [Leu2,D-Trp22,125I-Tyr25]somatostatin-28 and [125I]CGP 23996 ([125I]c[Asn-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Tyr-Thr-Ser]) were performed to determine the level of expression of somatostatin receptor messenger RNA and receptor binding sites in the hippocampal formation, limbic system and cerebral cortex of adult rats electrically kindled in the dorsal hippocampus. In control rats (implanted with electrodes but not electrically stimulated), the somatostatin-1 receptor-selective [125I]Tyr3-octreotide and the non-subtype-selective [Leu3,D-Trp22,125I-Tyr25]somatostatin-28 preferentially labelled the strata oriens and radiatum of the CA1 subfield of the hippocampus, the molecular layer of the dentate gyrus, the subiculum and presubiculum of the hippocampal formation, the inner layer of the frontal cortex, and the lateral and basolateral nuclei of the amygdala. The non-subtype-selective radioligand [125I]CGP 23996 (in 5 mM Mg2+ buffer) preferentially labelled the strata oriens and radiatum of the CA1 subfield of the hippocampus, the subiculum and the basolateral nucleus of the amygdala. Under conditions where primarily somatostatin-2 receptors were labelled, [125I]CGP 23996 (in 120 mM Na+ buffer) showed strong binding in the strata oriens and radiatum of the CA1 subfield of the hippocampus and the frontal cortex, whereas the dentate gyrus, subiculum and amygdala showed only weak signals. During and after kindling, no significant differences were observed between the ipsi- and contralateral sides of the hippocampus. A significant decrease (about 40%) of somatostatin receptor binding sites was observed in the molecular layer of the dentate gyrus with all radioligands (except [125I]CGP 23996 in Na+ buffer, which did not label this area) at stage 2 (pre-convulsive stage) and one week, but not one month, after stage 5 (generalized motor seizures). In contrast to somatostatin receptor binding, no alterations of the messenger RNA levels for sst1-sst5 receptors were found either at stage 2 or at stage 5. Similarly, no changes in receptor binding or messenger RNA levels were observed in the brain of rats which experienced a single afterdischarge. The present study shows a significant and selective decrease of somatostatin-1 receptor binding sites in the dentate gyrus of kindled rats. This is part of the plastic changes induced by kindling and may contribute to the increased sensitivity for the induction of generalized seizures during kindling.     

Distribution of pre-pro-glucagon and glucagon-like peptide-1 receptor messenger RNAs in the rat central nervous system

Glucagon-like peptide-1 (GLP-1) is derived from the peptide precursor pre-pro-glucagon (PPG) by enzymatic cleavage and acts via its receptor, glucagon-like peptide-1 receptor (GLP-1R). By using riboprobes complementary to PPG and GLP-1R, we described the distribution of PPG and GLP-1R messenger RNAs (mRNAs) in the central nervous system of the rat. PPG mRNA-expressing perikarya were restricted to the nucleus of the solitary tact or to the dorsal and ventral medulla and olfactory bulb. GLP-1R mRNA was detected in numerous brain regions, including the mitral cell layer of the olfactory bulb; temporal cortex; caudal hippocampus; lateral septum; amygdala; nucleus accumbens; ventral pallium; nucleus basalis Meynert; bed nucleus of the stria terminalis; preoptic area; paraventricular, supraoptic, arcuate, and dorsomedial nuclei of the hypothalamus; lateral habenula; zona incerta; substantia innominata; posterior thalamic nuclei; ventral tegmental area; dorsal tegmental, posterodorsal tegmental, and interpeduncular nuclei; substantia nigra, central gray; raphe nuclei; parabrachial nuclei; locus ceruleus, nucleus of the solitary tract; area postrema; dorsal nucleus of the vagus; lateral reticular nucleus; and spinal cord. These studies, in addition to describing the sites of GLP-1 and GLP-1R synthesis, suggest that the efferent connections from the nucleus of the solitary tract are more widespread than previously reported. Although the current role of GLP-1 in regulating neuronal physiology is not known, these studies provide detailed information about the sites of GLP-1 synthesis and potential sites of action, an important first step in evaluating the function of GLP-1 in the brain. The widespread distribution of GLP-1R mRNA-containing cells strongly suggests that GLP-1 not only functions as a satiety factor but also acts as a neurotransmitter or neuromodulator in anatomically and functionally distinct areas of the central nervous system.     

Specific reductions of striatal prodynorphin and D1 dopamine receptor messenger RNAs during cocaine abstinence

It is well established that the opioid neuropeptide and dopamine systems are altered following the use of cocaine. However very little information is available about their possible involvement during cocaine abstinence. In the present study, the mRNA expression of the dopamine receptors, D1 and D2, and the opioid peptides, prodynorphin and proenkephalin, were analyzed in the rat striatum using in situ hybridization histochemistry. Saline or cocaine (30 mg/kg, i.p.) were administered to rats once daily for 1 or 10 days. To examine cocaine abstinence, animals were treated for 10 days as described followed by a 10-day drug free period. Acute and intermittent cocaine administration elevated the prodynorphin mRNA expression in the dorsal striatum, consistent with previous reports, while the abstinent phase resulted in a significant reduction of prodynorphin mRNA levels in the ventrorostral striatum. The D1-receptor mRNA was decreased in the caudorostral striatum during cocaine withdrawal, a finding opposite to the increase observed following a single injection of the drug. Proenkephalin and the D2-receptor mRNAs were not altered during cocaine abstinence, though proenkephalin was elevated following acute but not repeated cocaine administration. These results show long-term suppression on prodynorphin and D1-receptor systems in specific striatal populations localized mainly in rostral areas during withdrawal from cocaine.     

Expression of the avian VEGF receptor homologues Quek1 and Quek2 in blood-vascular and lymphatic endothelial and non-endothelial cells during quail embryonic development

Caricatures, which increase the distinctiveness of faces, are generally recognised at least as well as undistorted images of those faces. However, caricatures seem to facilitate recognition more for some faces than others. An investigation was made into whether the effectiveness of caricaturing depends on a face's initial distinctiveness. In experiments 1-3, subjects learned names for unfamiliar faces (photographs) that varied in distinctiveness, and were tested on recognition of caricatures, anticaricatures, and undistorted images of those faces. The test images were line drawing in experiment 1 and 2 photographic images in experiment 3. Experiments 1 and 2 were identical except that subjects had more exposure to the study photographs in experiment 1. In all three experiments, distinctive faces were recognised (named) more accurately than less-distinctive faces, and caricatures were recognised at least as accurately as undistorted images and better than anticaricatures. However, distinctiveness and caricature level did not interact. Nor did a face's initial distinctiveness correlate with the degree of recognition facilitation produced by caricaturing (experiments 1-3) or with the caricature level chosen as the best likeness (experiment 4). The effectiveness of caricatures varied across faces and experimental conditions, but these differences did not relate to differences in initial distinctiveness. These results prompted a more careful analysis of the expected relationship between initial distinctiveness and the power of caricatures, which indicated that the relationship may be curvilinear rather than linear. In addition, it was found that line-drawing caricatures functioned as superportraits (recognised better than undistorted images--experiment 1) but photographic caricatures did not (experiment 3) suggesting that the forensic potential of caricatures may be limited.     

Expression of mouse c-myb during embryonic development

Three members of the myb gene family, designated as A-myb, B-myb, and c-myb, have been described in many vertebrates. A large body of evidence indicates that the c-myb gene is essential for the development of most hematopoietic lineages. By contrast, the functions of A-myb and B-myb are less well understood. To further explore the relationship between the different myb family members we have compared the expression of A-myb and c-myb during mouse embryogenesis by Northern blotting and by in situ hybridization. In accordance with the important role of c-myb in the hematopoietic system, we detect high levels of c-myb expression in hematopoietic organs such as the fetal liver and the thymus. Surprisingly, we find that high levels of c-myb expression are not restricted to hematopoietic cells. We show that c-myb is strongly expressed in the neural retina and in epithelia of the respiratory tract. The side-by side analysis of c-myb and A-myb expression clearly shows that both genes are expressed in different, but overlapping sets of tissues. Our results suggest that the function of c-myb may not be restricted to hematopoietic cells.     

Expression of nerve growth factor and its high-affinity receptor Trk-A in the rat pancreas during embryonic and fetal life

We recently reported that nitric oxide (NO), which is produced by chondrocytes treated with interleukin-1beta (IL-1), releases basic fibroblast growth factor (bFGF) stored in the matrix of articular chondrocytes. To clarify the mechanism of the IL-1-induced bFGF release, we investigated the production and gene expression of bFGF, matrix metalloproteinases (MMPs), syndecan 3, and inducible NO synthase (iNOS) by IL-1-treated rabbit articular chondrocytes. IL-1 stimulated not only the release of bFGF but also the production of it. Gelatin and casein zymography revealed that IL-1 stimulated the production of not only MMP-9 but also MMP-3. The increase in the production of these MMPs preceded the IL-1-stimulated bFGF release. An MMP inhibitor partially suppressed the release of bFGF, indicating that matrix degradation is at least partially involved in the IL-1-stimulated bFGF release even if increased production of bFGF is related to the release. IL-1 sequentially stimulated mRNA expression of iNOS, membrane type 1-MMP, MMP-9 and -3, and bFGF, in that order. NG-Monomethyl-L-arginine, an inhibitor of NO production, inhibited gene expression of MMP-9 and bFGF. These findings suggest that elevation of the NO level via iNOS mRNA expression stimulated by IL-1 mediates gene expression and production of MMPs and bFGF, resulting in the release of bFGF, and also reveal molecular mechanisms implicating the degradation of articular cartilage followed by angiogenesis in the synovium in arthritic joints.     

The differential expression patterns of messenger RNAs encoding non-N-methyl-D-aspartate glutamate receptor subunits (GluR1-4) in the rat brain

The messenger RNA expression of non-N-methyl-D-aspartate glutamate receptor subunits (GluR1-4), considered alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid type, was investigated in rat brain by in situ hybridization histochemistry using oligonucleotide probes specific to each subunit sequence. GluR1-4 subunit messenger RNAs were expressed widely and abundantly throughout the CNS. However, the combination of expression pattern varied notably according to location. GluR2 messenger RNA was expressed most strongly and widely, with most areas except the Bergmann glia containing this messenger RNA. GluR4 messenger RNA was also present widely, although the expression level was low. However, we observed many areas which lacked or expressed very little GluR1 messenger RNA, such as some nuclei in the general motor system and auditory system. In addition, some nuclei in the hypothalamus and general somatosensory system lacked or expressed very little GluR3 messenger RNA. These results suggest that in the rat CNS non-N-methyl-D-aspartate receptors varied their composition according to the area where they were expressed, and that the combination pattern might be related to the functional role of neurons.     

Reduction of GABA and glutamate transporter messenger RNAs in the severe-seizure genetically epilepsy-prone rat

The genetically epilepsy-prone rat is an animal model of inherited generalised tonic-clonic epilepsy that shows abnormal susceptibility to audiogenic seizures and a lowered threshold to a variety of seizure-inducing stimuli. Recent studies suggest a crucial role for glutamate and GABA transporters in epileptogenesis and seizure propagation. The present study examines the levels of expression of the messenger RNAs encoding the glial and neuronal glutamate transporters, GLT-1 and EAAC-1, and the neuronal GABA transporter, GAT-1, in paired male genetically epileptic-prone rats and Sprague Dawley control rats using the technique of in situ hybridization. In a parallel study, semiquantitative immunoblotting was used to assess GLT-1 and EAAC-1 protein levels in similarly paired animals. Animals were assessed for susceptibility to audiogenic seizures on six occasions, and killed seven days following the last audiogenic stimulus exposure. Rat brains were processed for in situ hybridization with radioactive 35S-labelled oligonucleotide probes (EAAC-1 and GAT-1), 35S-labelled riboprobes (GLT-1), and Fluorescein-labelled riboprobes (GLT-1 and GAT-1) or processed for immunoblotting using subtype-specific antibodies for GLT-1 and EAAC-1. Semiquantitative analyses were carried out on X-ray film autoradiograms in several brain regions for both in situ hybridization and immunoblotting studies. Reductions in GAT-1 messenger RNA were found in genetically epileptic-prone rats in all brain regions examined (-8 to -24% compared to control). Similar reductions in GLT-1 messenger RNA expression levels were seen in cortex, striatum, and CA1 (-8 to -12%) of genetically epileptic-prone rats; the largest reduction observed was in the inferior colliculus (-20%). There was a tendency for a reduced expression of EAAC-1 messenger RNA in most regions of the genetically epileptic-prone rat brain although this reached statistical significance only in the striatum (-12%). In contrast, no significant differences in GLT-1 and EAAC-1 protein between genetically epileptic-prone rats and control animals were observed in any region examined, although there was a tendency to follow the changes seen with the corresponding messenger RNAs. These results show differences in the messenger RNA expression levels of three crucial amino acid transporters. For the two glutamate transporters, GLT-1 and EAAC-1, differences in messenger RNA levels are not reflected or are only partially reflected in the expression of the corresponding proteins.     

Exposure to prenatal nicotine transiently increases neuronal nicotinic receptor subunit alpha7, alpha4 and beta2 messenger RNAs in the postnatal rat brain

This study determined the effects of prenatal nicotine exposure (2 mg/kg/day) in Sprague Dawley CD rats via subcutaneously implanted osmotic minipumps, during gestational days 7-21, on postnatal levels of neuronal nicotinic receptor alpha4, alpha7 and beta2 subunit messenger RNAs. Northern analysis of postnatal day 1, 7, 14 and 28 hippocampal/septal and cortical total RNA using alpha-[32P]dCTP-labeled alpha4, alpha7 and beta2 complementary DNA probes identified a single (5.7-kb) alpha7 messenger RNA, three (2.4-, 3.8- and 8.0-kb) alpha4 messenger RNAs and four (3.7-, 5.0-, 7.5- and 10.0-kb) beta2 messenger RNAs. In comparison to prenatal saline, prenatal nicotine produced several significantly higher messenger RNA levels (cortical: 5.7-kb alpha7, 2.4-, 3.8- and 8.0-kb alpha4, 10.0-kb beta2; hippocampal/septal: 2.4- and 8.0-kb alpha4); these increases occurred predominantly on, but were not restricted to, postnatal day 14. Effects of nicotine were generally resolved by postnatal day 28. Collapsing the data across sex and age, a significant treatment effect indicated that hippocampal/septal and cortical 8.0-kb alpha4 messenger RNA levels and 10.0-kb beta2 messenger RNA levels were significantly higher following prenatal nicotine exposure. This is the first study indicating that prenatal nicotine produces alterations in developing postnatal rat neuronal nicotinic receptor messenger RNA levels, possibly by premature stimulation of neuronal nicotinic receptors. These results further implicate the teratogenic potential of nicotine in postnatal neuronal development.     

Changes in the content of progesterone receptor isoforms and estrogen receptor alpha in the chick brain during embryonic development

Progesterone and estradiol participate in the regulation of several reproductive functions through interaction with intracellular progesterone receptors (PR) and estrogen receptors (ER), respectively. In this work, we determined PR and ER-alpha isoforms content in the brain of chicks of both sexes on days 8 and 13 of embryonic development as well as on the day of hatching by Western blot analysis. PR isoforms protein content increased during embryonic development in both female and male chick brain. The highest PR isoforms content was observed on the day of hatching in both sexes. Interestingly, PR-A content was higher in the brain of chick males than in that of females on day 8 of embryonic development. PR-A/PR-B ratio was higher in the brain of males than in that of females at all ages. We found two ER-alpha isoforms of 66 and 52 kDa; the content of both isoforms was higher in the brain of females than in that of males on days 8 and 13 of embryonic development. An opposite pattern of ER-alpha isoforms content was observed. In males, ER-alpha content increased during embryonic development whereas in the females it decreased during this process. These results indicate that the content of PR and ER-alpha isoforms is related to the degree of brain development in chicks, and suggest that PR and ER-alpha isoforms should exhibit sexual dimorphism in the brain of chicks during embryonic development.     

Expression of non-classical islet hormone-like peptides during the embryonic development of the pancreas

The highly enriched anaerobic bacterium that couples the reductive dechlorination of tetrachloroethene to growth, previously referred to as PER-K23, was obtained in pure culture and characterized. The bacterium, which does not form spores, is a small, gram-negative rod with one lateral flagellum. It utilized only H2 as an electron donor and tetrachloroethene and trichloroethene as electron acceptors in an anaerobic respiration process; it could not grow fermentatively. Acetate served as a carbon source in a defined medium containing iron as the sole trace element, the two vitamins thiamine and cyanocobalamin, and the three amino acids arginine, histidine, and threonine. The cells contained menaquinones and b-type cytochromes. The G+C content of the DNA was 45.3 +/- 0.3 mol%. The cell wall consisted of type-A3gamma peptidoglycan with ll-diaminopimelic acid and one glycine as an interpeptide bridge. The cells are surrounded by an S-layer; an outer membrane was absent. Comparative sequence analysis of the 16S rRNA sequence showed that PER-K23 is related to gram-positive bacteria with a low G+C content of the DNA. Based on the cytological, physiological, and phylogenetic characterization, it is proposed to affiliate the isolate to a new genus, Dehalobacter, with PER-K23 as the type strain of the new species Dehalobacter restrictus.     

Cellular localization of dopamine D2 receptor messenger RNA in the rat trigeminal ganglion

The actions of dopamine are mediated by specific, high-affinity, G protein-coupled receptors. Multiple subtypes of dopamine receptors have been characterized, including the D2 subtype (D2R). Cells within the dorsal root and petrosal ganglia of the rat express D2R messenger RNA (mRNA) consistent with D2R expression by primary sensory neurons. We hypothesized that neurons of the trigeminal ganglion express D2R mRNA. Total cellular RNA from rat trigeminal ganglia was analyzed on Northern blots under high stringency conditions. Hybridization of trigeminal ganglion RNA resulted in a signal which comigrated with striatal, pituitary, and hypothalamic D2R mRNA. To determine the distribution of D2R expressing cells in the trigeminal ganglion, cryostat sections were analyzed by in situ hybridization followed by emulsion autoradiography. We identified a population of clustered cells labeled with dense grain concentrations over their cytoplasms. These findings demonstrate the expression of D2 dopamine receptor mRNA in discrete subpopulations of neurons in the rat trigeminal ganglion. Our observations suggest that drugs active at dopamine receptors of the D2 subtype are potential modulators of sensory activity of neurons whose cell bodies reside in the trigeminal ganglion. D2 dopamine receptors may thus have a role in clinical pain syndromes involving the head and neck.     

Increased expression of trkB and trkC messenger RNAs in the rat forebrain after focal mechanical injury

Tyrosine protein kinases trkA, trkB and trkC are signal transduction receptors for a family of neurotrophic factors known as the neurotrophins. Here we report on changes in the expression of messenger RNAs for trkA, trkB and trkC in the brain following an injury caused by insertion of a 30-gauge needle into adult rat hippocampus or neocortex. Quantitative in situ hybridization revealed no change in the level of trkA messenger RNAs in any brain region following this insult. In contrast, increased levels of trkB messenger RNA compared to untreated animals were seen in the granule cell layer of the dentate gyrus ipsilateral to the injury already 30 min after the injury. The increase reached maximal levels (four-fold) between 2 and 4 h, but returned to control levels 8 h after the injury. No change was seen in the contralateral dentate gyrus. The levels of trkC messenger RNA increased in the same brain regions as trkB messenger RNA, though with a delayed response, reaching a maximal increase of 3.3-fold 4 h after the injury. As for trkB messenger RNA, the level of trkC messenger RNA then tapered off and reached control levels 8 h after the injury. However, 4 h after the injury, a 1.7-fold increase of trkB and trkC messenger RNAs were seen in the ipsilateral piriform cortex. The increases of trkB and trkC messenger RNAs were confirmed using a nuclease protection assay. Increases of both trkB and trkC messenger RNAs were also seen in the piriform cortex, but not in the hippocampus, following needle insertion into the neocortex. Pretreatment of the animals with the non-competitive N-methyl-D-aspartate antagonist ketamine completely prevented the increases of trkB and trkC messenger RNAs, suggesting that the brain injury caused a release of glutamate with subsequent activation of N-methyl-D-aspartate receptors. In contrast, the anticonvulsive drug diazepam, the muscarinic antagonist atropine and the calcium-channel antagonist nimodipine had no effect on the increases of trkB and trkC messenger RNAs. Combined with previous data on the expression of neurotrophin messenger RNAs following similar injuries, our results support the hypothesis that increased levels of neurotrophins and their receptors could protect against neuronal damage following a brain insult.     

Expression of thyroid hormone receptors alpha and beta-1 messenger RNAs in human endothelial cells. The T3 hormone stimulates the synthesis of the messenger RNA of the intercellular adhesion molecule-1

A study of the effect of the L-3,5,3'-triiodothyronine hormone on the expression of the mRNA of the adhesion molecule ICAM-1 led to the observation that the mRNA level is slightly up-regulated in human umbilical-cord endothelial cells. To analyze this induction at a molecular level, the search of T3 hormone receptors was undertaken. In this paper, we show that ECV 304 endothelial cells express the mRNAs encoding two thyroid hormone receptor isoforms alpha(alpha1 and alpha2) and one beta(beta1). This is, to our knowledge, a first important step towards the demonstration of the involvement of these receptors in the induction of the expression of ICAM-1 by the T3 hormone.