scholarly journals Expression of Tandem P Domain K+ Channel, TREK-1, in the Rat Carotid Body

2006 ◽  
Vol 54 (4) ◽  
pp. 467-472 ◽  
Author(s):  
Y. Yamamoto ◽  
K. Taniguchi
Keyword(s):  
In Situ Hybridization ◽  
Mrna Expression ◽  
Carotid Body ◽  
Nerve Fibers ◽  
K Channel ◽  
Membrane Excitability ◽  
Rt Pcr ◽  
Glomus Cells ◽  
Afferent Nerve Endings

TREK-1 is one of the important potassium channels for regulating membrane excitability. To examine the distribution of TREK-1 in the rat carotid body, we performed RT-PCR for mRNA expression and in situ hybridization and immunohistochemistry for tissue distribution of TREK-1. RT-PCR detected mRNA expression of TREK-1 in the carotid body. Furthermore, in situ hybridization revealed the localization of TREK-1 mRNA in the glomus cells. TREK-1 immunoreactivity was mainly distributed in the glomus cells and nerve fibers in the carotid body. TREK-1 may modulate potassium current of glomus cells and/or afferent nerve endings in the rat carotid body.

scholarly journals Quantification of TGF-β1 mRNA along rat nephron in obstructive nephropathy

2001 ◽  
Vol 281 (3) ◽  
pp. F513-F521 ◽  
Author(s):  
Kyoichi Fukuda ◽  
Koji Yoshitomi ◽  
Taihei Yanagida ◽  
Masanori Tokumoto ◽  
Hideki Hirakata
Keyword(s):  
In Situ Hybridization ◽  
Mrna Expression ◽  
Transforming Growth Factor ◽  
Interstitial Fibrosis ◽  
Proximal Tubules ◽  
Obstructive Nephropathy ◽  
Renal Tubules ◽  
Rt Pcr ◽  
Tgf Β1

Unilateral ureteral obstruction (UUO) leads to interstitial fibrosis of the obstructed kidney, and transforming growth factor-β1 (TGF-β1) is thought to play an important role in this process. Although increased TGF-β1 mRNA expression in the obstructed kidney has been demonstrated, the source of the increased TGF-β1 remains to be elucidated. To determine the precise localization of TGF-β1 in the obstructed kidney, we examined TGF-β1 mRNA expression using in situ hybridization and competitive RT-PCR in rats with UUO. In situ hybridization demonstrated that TGF-β1 mRNA expression was preferentially increased in tubular epithelial cells and to a lesser degree in infiltrating macrophages in obstructed kidneys. Quantitative analysis using competitive RT-PCR in microdissected nephron segments revealed that levels of TGF-β1 mRNA in obstructed kidneys relative to control kidneys increased significantly in proximal tubules, thick ascending limbs of Henle, and distal convoluted tubules, whereas those in glomeruli and collecting ducts did not change significantly. Of the tubular segments, the proximal tubules appeared to predominantly contribute to increased TGF-β1 mRNA. Our findings suggest that renal tubules, particularly proximal tubules, are the main contributors to increased TGF-β1 mRNA expression in obstructed kidneys and to the subsequent interstitial fibrosis.

Estrogen Receptor-β mRNA Expression in Rat Ovary: Down-Regulation by Gonadotropins

1997 ◽  
Vol 11 (2) ◽  
pp. 172-182 ◽  
Author(s):  
Michael Byers ◽  
George G. J. M. Kuiper ◽  
Jan-Åke Gustafsson ◽  
Ok-Kyong Park-Sarge
Keyword(s):  
Estrogen Receptor ◽  
In Situ Hybridization ◽  
Mrna Expression ◽  
Granulosa Cells ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Adult Rats ◽  
Mature Adult ◽  
Preovulatory Follicles

Abstract We have examined the expression and regulation of the two estrogen receptor (ERα and ERβ) genes in the rat ovary, using Northern blotting, RT-PCR, and in situ hybridization histochemistry. Northern blotting results show that the ovary expresses both ERα and ERβ genes as single (∼6.5-kb) and multiple (ranging from ∼1.0-kb to ∼10.0-kb) transcripts, respectively. ERα mRNA is expressed at a level lower than ERβ mRNA in immature rat ovaries. This relationship appears unchanged between sexually mature adult rats and immature rats. In sexually mature adult rats undergoing endogenous hormonal changes, whole ovarian content of ERβ mRNA, as determined by RT-PCR, remained more or less constant with the exception of the evening of proestrus when ERβ mRNA levels were decreased. Examination of ERβ mRNA expression at the cellular level, by in situ hybridization, showed that ERβ mRNA is expressed preferentially in granulosa cells of small, growing, and preovulatory follicles, although weak expression of ERβ mRNA was observed in a subset of corpora lutea, and that the decrease in ERβ mRNA during proestrous evening is attributable, at least in part, to down-regulation of ERβ mRNA in the preovulatory follicles. This type of expression and regulation was not typical for ERα mRNA in the ovary. Although whole ovarian content of ERα mRNA was clearly detected by RT-PCR, no apparent modulation of ERα mRNA levels was observed during the estrous cycle. Examination of ERα mRNA expression at the cellular level, by in situ hybridization, showed that ERα mRNA is expressed at a low level throughout the ovary with no particular cellular localization. To further examine the potential role of the preovulatory pituitary gonadotropins in regulating ERβ mRNA expression in the ovary, we used immature rats treated with gonadotropins. In rats undergoing exogenous hormonal challenges, whole ovarian content of ERβ mRNA, as determined by RT-PCR, remained more or less unchanged after an injection of PMSG. In contrast, a subsequent injection of human CG (hCG) resulted in a substantial decrease in whole ovarian content of ERβ mRNA. In situ hybridization for ERβ mRNA shows that small, growing, and preovulatory follicles express ERβ mRNA in the granulosa cells. The preovulatory follicles contain ERβ mRNA at a level lower than that observed for small and growing follicles. In addition, there is an abrupt decrease in ERβ mRNA expression in the preovulatory follicles after hCG injection. The inhibitory effect of hCG on ERβ mRNA expression was also observed in cultured granulosa cells. Moreover, agents stimulating LH/CG receptor-associated intracellular signaling pathways (forskolin and a phorbol ester) readily mimicked the effect of hCG in down-regulating ERβ mRNA in cultured granulosa cells. Taken together, our results demonstrate that 1) the ovary expresses both ERα and ERβ genes, although ERβ is the predominant form of estrogen receptor in the ovary, 2) ERβ mRNA is localized predominantly to the granulosa cells of small, growing, and preovulatory follicles, and 3) the preovulatory LH surge down-regulates ERβ mRNA. These results clearly implicate the physiological importance of ERβ in female reproductive functions.

scholarly journals Generation of Thyrotropin-Releasing Hormone Receptor 1-Deficient Mice as an Animal Model of Central Hypothyroidism

2004 ◽  
Vol 18 (6) ◽  
pp. 1450-1460 ◽  
Author(s):  
Roland Rabeler ◽  
Jens Mittag ◽  
Lars Geffers ◽  
Ulrich Rüther ◽  
Michael Leitges ◽  
...  
Keyword(s):  
Animal Model ◽  
In Situ Hybridization ◽  
Mrna Expression ◽  
Pcr Analysis ◽  
Target Cells ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Central Hypothyroidism ◽  
R1 Gene

Abstract To provide an animal model of central hypothyroidism, mice deficient in the TRH-receptor 1 (TRH-R1) gene were generated by homologous recombination. The pituitaries of TRH-R1−/− mice are devoid of any TRH-binding capacity, demonstrating that TRH-R1 is the only receptor localized on TRH target cells of the pituitary. With the exception of some retardation in growth rate, TRH-R1−/− mice appear normal, but compared with control animals they exhibit a considerable decrease in serum T3, T4, and prolactin (PRL) levels but not in serum TSH levels. In situ hybridization histochemistry and real-time RT-PCR analysis revealed that in adult TRH-R1−/− animals TSHβ-mRNA expression is not impaired whereas PRL mRNA and GH mRNA levels are considerably reduced compared with control mice. The numbers of thyrotropes, somatotropes, and lactotropes, however, are not affected by the deletion of the TRH-R1 gene. The mutant mice are fertile, and the dams nourish their pups well, indicating that TRH is not a decisive factor for suckling-induced PRL release. In situ hybridization and quantitative RT-PCR analysis, furthermore, revealed that, as in control animals, pituitary PRL-mRNA expression in TRH-R1−/− is considerably increased during lactation, albeit strongly reduced as compared with lactating control animals.

Acute O2 sensing through HIF2α-dependent expression of atypical cytochrome oxidase subunits in arterial chemoreceptors

2019 ◽  
Vol 13 (615) ◽  
pp. eaay9452 ◽  
Author(s):  
Alejandro Moreno-Domínguez ◽  
Patricia Ortega-Sáenz ◽  
Lin Gao ◽  
Olalla Colinas ◽  
Paula García-Flores ◽  
...  
Keyword(s):  
Ion Channels ◽  
Carotid Body ◽  
Acute Hypoxia ◽  
Mechanistic Explanation ◽  
Nerve Fibers ◽  
Adaptive Responses ◽  
Glomus Cells ◽  
Adult Mice ◽  
Genes Encoding ◽  
Regulation Of Breathing

Acute cardiorespiratory responses to O2 deficiency are essential for physiological homeostasis. The prototypical acute O2-sensing organ is the carotid body, which contains glomus cells expressing K+ channels whose inhibition by hypoxia leads to transmitter release and activation of nerve fibers terminating in the brainstem respiratory center. The mechanism by which changes in O2 tension modulate ion channels has remained elusive. Glomus cells express genes encoding HIF2α (Epas1) and atypical mitochondrial subunits at high levels, and mitochondrial NADH and reactive oxygen species (ROS) accumulation during hypoxia provides the signal that regulates ion channels. We report that inactivation of Epas1 in adult mice resulted in selective abolition of glomus cell responsiveness to acute hypoxia and the hypoxic ventilatory response. Epas1 deficiency led to the decreased expression of atypical mitochondrial subunits in the carotid body, and genetic deletion of Cox4i2 mimicked the defective hypoxic responses of Epas1-null mice. These findings provide a mechanistic explanation for the acute O2 regulation of breathing, reveal an unanticipated role of HIF2α, and link acute and chronic adaptive responses to hypoxia.

Expression of transforming growth factor alpha during development

1988 ◽  
Vol 66 (8) ◽  
pp. 1113-1121 ◽  
Author(s):  
V. K. M. Han ◽  
A. J. D'Ercole ◽  
D. C. Lee
Keyword(s):  
In Situ Hybridization ◽  
Growth Factor ◽  
Mrna Expression ◽  
Transforming Growth Factor ◽  
Transforming Growth Factor Alpha ◽  
Egf Receptors ◽  
Hybridization Histochemistry ◽  
In Situ Hybridization Histochemistry ◽  
Factor Alpha

Transforming growth factors (TGFs) are polypeptides that are produced by transformed and tumour cells, and that can confer phenotypic properties associated with transformation on normal cells in culture. One of these growth-regulating molecules, transforming growth factor alpha (TGF-α), is a 50 amino acid polypeptide that is related to epidermal growth factor (EGF) and binds to the EGF receptor. Previous studies have shown that TGF-α is expressed during rodent embryogenesis between 7 and 14 days gestation. To investigate the cellular sites of TGF-α mRNA expression during development, we have performed Northern analyses and in situ hybridization histochemistry on the conceptus and maternal tissues at various gestational ages. Contrary to previous reports, both Northern analyses and in situ hybridization histochemistry indicate that TGF-α mRNA is predominantly expressed in the maternal decidua and not in the embryo. Decidual expression is induced following implantation, peaks at day 8, and declines through day 15 when the decidua is being resorbed. In situ hybridization revealed that expression of TGF-α mRNA is highest in the region of decidua adjacent to the embryo and is low or nondetectable in the uterus, placenta, and embryo. In addition, we could not detect TGF-α mRNA expression in other maternal tissues, indicating that the induction of TGF-α transcripts in the decidua is tissue specific, and not a pleiotropic response to changes in hormonal milieu that occur during pregnancy. The developmentally regulated expression of TGF-α mRNA in the decidua, together with the presence of EGF receptors in this tissue, suggests that this peptide may stimulate mitosis and angiogenesis locally by an autocrine mechanism. Because EGF receptors are also present in the embryo and placenta, TGF-α may act on these tissues by a paracrine or endocrine mechanism.

scholarly journals Quantitative real-time RT-PCR and chromogenic in situ hybridization: precise methods to detect HER-2 status in breast carcinoma

BMC Cancer ◽  
2009 ◽  
Vol 9 (1) ◽  
Author(s):  
Fabíola E Rosa ◽  
Sara M Silveira ◽  
Cássia GT Silveira ◽  
Nádia A Bérgamo ◽  
Francisco A Moraes Neto ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Breast Carcinoma ◽  
Real Time ◽  
Rt Pcr ◽  
Her 2 ◽  
Chromogenic In Situ Hybridization

Comet and cup genes in Drosophila spermatogenesis: the first demonstration of post-meiotic transcription

2008 ◽  
Vol 36 (3) ◽  
pp. 540-542 ◽  
Author(s):  
Carine Barreau ◽  
Elizabeth Benson ◽  
Helen White-Cooper
Keyword(s):  
In Situ Hybridization ◽  
Expression Pattern ◽  
Reverse Transcription ◽  
Protein Product ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Single Cyst ◽  
Reverse Transcription Pcr

Post-meiotic transcription is widespread in mammalian spermatogenesis, but is generally believed to be absent from Drosophila spermatogenesis. Genes required during meiosis, in early spermatids or later in spermiogenesis are typically transcribed in primary spermatocytes in Drosophila. Their mRNAs are then stored in the cytoplasm until the protein product is needed. Recently, using in situ hybridization, we identified 17 Drosophila genes, collectively named ‘comets’ and ‘cups’, whose mRNAs are most abundant in, and localize to the distal ends of, elongating spermatids. Using a single-cyst quantitative RT–PCR (reverse transcription–PCR) assay, we confirmed this unusual expression pattern and conclusively demonstrate the existence of post-meiotic transcription in Drosophila spermatids. We found that transcription of comets and cups occurs just before protamines can be detected in spermatid nuclei.

Endothelin receptor mRNA expression in renal medulla identified by in situ RT-PCR

1995 ◽  
Vol 269 (3) ◽  
pp. F449-F457 ◽  
Author(s):  
L. H. Chow ◽  
S. Subramanian ◽  
G. J. Nuovo ◽  
F. Miller ◽  
E. P. Nord
Keyword(s):  
Mrna Expression ◽  
Collecting Duct ◽  
Renal Medulla ◽  
Receptor Subtypes ◽  
Rt Pcr ◽  
Receptor Mrna ◽  
Pcr Products ◽  
Medullary Collecting Duct ◽  
Labeled Probe

Three subtypes of endothelin (ET) receptors have been identified by cDNA cloning, namely ET-RA, ET-RB, and ET-RC. In the current study the precise cellular distribution of the ET receptor subtypes in the renal medulla was explored by detecting the corresponding polymerase chain reaction (PCR)-amplified cDNAs by in situ reverse transcription (RT)-PCR. The PCR-amplified cDNAs were detected either by direct incorporation using digoxigenin-dUTP (dig-dUTP) as a nucleotide substrate in the PCR reaction or by in situ hybridization with the dig-dUTP-labeled probe. ET-RB mRNA was detected exclusively in the epithelial cells of the inner and outer medullary collecting duct. In contrast, ET-RA message was observed primarily in interstitial cells and pericytes of the vasae rectae in the outer and inner medulla. Southern blot analysis of PCR-amplified cDNAs reverse transcribed from extracted RNA of rat renal medulla confirmed the specificity of the RT-PCR products. ET-RC mRNA was not detected. We conclude that ET-RB is the major ET receptor found in rat renal medulla and is expressed exclusively on inner medullary collecting duct cells. The pattern of ET receptor mRNA expression described suggests different physiological actions for ET on the diverse cellular structures of the renal medulla.

Expression of the mRNAs for the Kv3.1 potassium channel gene in the adult and developing rat brain

1992 ◽  
Vol 68 (3) ◽  
pp. 756-766 ◽  
Author(s):  
T. M. Perney ◽  
J. Marshall ◽  
K. A. Martin ◽  
S. Hockfield ◽  
L. K. Kaczmarek
Keyword(s):  
In Situ Hybridization ◽  
Rat Brain ◽  
K Channel ◽  
Rnase Protection ◽  
Hybridization Histochemistry ◽  
Adult Rat ◽  
In Situ Hybridization Histochemistry ◽  
Adult Rat Brain ◽  
Developing Rat

1. The gene for a mammalian Shaw K+ channel has recently been cloned and has been shown, by alternative splicing, to give rise to two different transcripts, Kv3.1 alpha and Kv3.1 beta. To determine whether these channels are associated with specific types of neurons and to determine whether or not the alternately spliced K+ channel variants are differentially expressed, we used ribonuclease (RNase) protection assays and in situ hybridization histochemistry to localize the specific subsets of neurons containing Kv3.1 alpha and Kv3.1 beta mRNAs in the adult and developing rat brain. 2. In situ hybridization histochemistry revealed a heterogeneous expression pattern of Kv3.1 alpha mRNA in the adult rat brain. Highest Kv3.1 alpha mRNA levels were expressed in the cerebellum. High levels of hybridization were also detected in the globus pallidus, subthalamus, and substantia nigra reticulata. Many thalamic nuclei, but in particular the reticular thalamic nucleus, hybridized well to Kv3.1 alpha-specific probes. A subpopulation of cells in the cortex and hippocampus, which by their distribution and number may represent interneurons, were also found to contain high levels of Kv3.1 alpha mRNA. In the brain stem, many nuclei, including the inferior colliculus and the cochlear and vestibular nuclei, also express Kv3.1 alpha mRNA. Low or undetectable levels of Kv3.1 alpha mRNA were found in the caudate-putamen, olfactory tubercle, amygdala, and hypothalamus. 3. Kv3.1 beta mRNA was also detected in the adult rat brain by both RNase protection assays and by in situ hybridization experiments. Although the beta splice variant is expressed at lower levels than the alpha species, the overall expression pattern for both mRNAs is similar, indicating that both splice variants co-expressed in the same neurons. 4. The expression of Kv3.1 alpha and Kv3.1 beta transcripts was examined throughout development. Kv3.1 alpha mRNA is detected as early as embryonic day 17 and then increases gradually until approximately postnatal day 10, when there is a large increase in the amount of Kv3.1 alpha mRNA. Interestingly, the expression of Kv3.1 beta mRNA only increases gradually during the developmental time frame examined. Densitometric measurements indicated that Kv3.1 alpha is the predominant splice variant found in neurons of the adult brain, whereas Kv3.1 beta appears to be the predominant species in embryonic and perinatal neurons. 5. Most of the neurons that express the Kv3.1 transcripts have been characterized electrophysiologically to have narrow action potentials and display high-frequency firing rates with little or no spike adaptation.(ABSTRACT TRUNCATED AT 400 WORDS)

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