The α1G-subunit of a voltage-dependent Ca2+ channel is localized in rat distal nephron and collecting duct

2000 ◽  
Vol 279 (6) ◽  
pp. F997-F1005 ◽  
Author(s):  
Ditte Andreasen ◽  
Boye L. Jensen ◽  
Pernille B. Hansen ◽  
Tae-Hwan Kwon ◽  
Søren Nielsen ◽  
...  
Keyword(s):  
Collecting Duct ◽  
Rnase Protection Assay ◽  
Confocal Laser ◽  
Pcr Analysis ◽  
Apical Plasma Membrane ◽  
Mrna Levels ◽  
Double Labeling ◽  
Rnase Protection ◽  
Principal Cells ◽  
Voltage Dependent

The molecular type and localization of calcium channels along the nephron are not well understood. In the present study, we assessed the distribution of the recently identified α1G-subunit encoding a voltage-dependent calcium channel with T-type characteristics. Using a RNase protection assay, α1G-mRNA levels in kidney regions were determined as inner medulla ≫ outer medulla ≅ cortex. RT-PCR analysis of microdissected rat nephron segments revealed α1G expression in the distal convoluted tubule (DCT), in the connecting tubule and cortical collecting duct (CT+CCD), and inner medullary collecting duct (IMCD). α1GmRNA was expressed in the IMCD cell line mIMCD-3. Single- and double-labeling immunohistochemistry and confocal laser microscopy on semithin paraffin sections of rat kidneys by using an anti-α1G antibody demonstrated a distinct labeling at the apical plasma membrane domains of DCT cells, CT principal cells, and IMCD principal cells.

Localization of prostaglandin E2 EP2 and EP4 receptors in the rat kidney

2001 ◽  
Vol 280 (6) ◽  
pp. F1001-F1009 ◽  
Author(s):  
Boye L. Jensen ◽  
Jane Stubbe ◽  
Pernille B. Hansen ◽  
Ditte Andreasen ◽  
Ole Skøtt
Keyword(s):  
Collecting Duct ◽  
Rat Kidney ◽  
Rnase Protection Assay ◽  
Receptor Expression ◽  
Pcr Analysis ◽  
Prostaglandin E ◽  
Rnase Protection ◽  
Ep4 Receptor ◽  
Vasa Recta ◽  
Resistance Vessels

We investigated the localization of cAMP-coupled prostaglandin E2 EP2 and EP4 receptor expression in the rat kidney. EP2 mRNA was restricted to the outer and inner medulla in rat kidney, as determined by RNase protection assay. RT-PCR analysis of microdissected resistance vessels and nephron segments showed EP2 expression in descending thin limb of Henle's loop (DTL) and in vasa recta of the outer medulla. The EP4 receptor was expressed in distal convoluted tubule (DCT) and cortical collecting duct (CCD) in preglomerular vessels, and in outer medullary vasa recta. Butaprost, an EP2 receptor-selective agonist, dose dependently raised cAMP levels in microdissected DTL and outer medullary vasa recta specimens but had no effect in EP2-negative outer medullary collecting duct segments. Dietary salt intake did not alter EP2 expression in the kidney medulla. These results suggest that PGE2 may act in the resistance vessels and in the DTL and DCT-CCD segments as a paracrine, cAMP-dependent regulator of vascular resistance and tubular transport, respectively.

scholarly journals A cell cycle analysis of growth-related genes expressed during T lymphocyte maturation.

1990 ◽  
Vol 111 (6) ◽  
pp. 2693-2701 ◽  
Author(s):  
J N Feder ◽  
C J Guidos ◽  
B Kusler ◽  
C Carswell ◽  
D Lewis ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Transcriptional Regulation ◽  
Steady State ◽  
T Lymphocyte ◽  
Ribonucleotide Reductase ◽  
Cellular Proliferation ◽  
Rnase Protection Assay ◽  
Minor Component ◽  
Mrna Levels ◽  
Rnase Protection

Fetal liver or bone marrow-derived T lymphocyte precursors undergo extensive, developmentally regulated proliferation in response to inductive signals from the thymic microenvironment. We have used neonatal mouse thymocytes size-separated by centrifugal elutriation to study the cell cycle stage-specific expression of several genes associated with cell proliferation. These include genes involved in the biosynthesis of deoxyribonucleotide precursors, such as dihydrofolate reductase (DHFR), thymidylate synthase (TS), and the M1 and M2 subunits of ribonucleotide reductase, as well as c-myc, a cellular oncogene of unknown function. Using nuclear run-on assays, we observed that the transcription rates for these genes, with the exception of TS, are essentially invariant not only throughout the cell cycle in proliferating cells, but also in noncycling (G0) cells. The TS gene showed a transient increase in transcription rate in cells which bordered between a proliferating and nonproliferating status. Studies of an elutriated T cell line, S49.1, yielded similar results, indicating that the process of immortalization has not affected the transcriptional regulation of these genes. Analysis of steady-state mRNA levels using an RNase protection assay demonstrated that the levels of DHFR and TS mRNA accumulate as thymocytes progress through the cell cycle. In contrast, only the M2 subunit of ribonucleotide reductase showed cyclic regulation. Finally, in contrast to cultured cell models, we observed an abrupt fivefold increase in the steady-state level of c-myc mRNA in the transition from G1 to S-phase. We conclude from these studies that the transcriptional regulation of specific genes necessary for cellular proliferation is a minor component of the developmental modulation of the thymocyte cell cycle.

Integrin alpha subunit mRNAs are differentially expressed in early Xenopus embryos

Development ◽  
1993 ◽  
Vol 117 (4) ◽  
pp. 1239-1249 ◽  
Author(s):  
C.A. Whittaker ◽  
D.W. DeSimone
Keyword(s):  
In Situ Hybridization ◽  
Rnase Protection Assay ◽  
Early Embryo ◽  
Mrna Levels ◽  
Rnase Protection ◽  
Dorsal Midline ◽  
Transmembrane Receptors ◽  
Early Embryos ◽  
Whole Mount

Adhesion of cells to extracellular matrix proteins is mediated, in large part, by transmembrane receptors of the integrin family. The identification of specific integrins expressed in early embryos is an important first step to understanding the roles of these receptors in developmental processes. We have used polymerase chain reaction methods and degenerate oligodeoxynucleotide primers to identify and clone Xenopus integrin alpha subunits from neurula-stage (stage 17) cDNA. Partial cDNAs encoding integrin subunits alpha 2, alpha 3, alpha 4, alpha 5, alpha 6 and an alpha IIb-related subunit were cloned and used to investigate integrin mRNA expression in early embryos by RNase protection assay and whole-mount in situ hybridization methods. Considerable integrin diversity is apparent early in development with integrins alpha 2, alpha 3, alpha 4, alpha 5 and alpha 6 each expressed by the end of gastrulation. Both alpha 3 and alpha 5 are expressed as maternal mRNAs. Zygotic expression of alpha 2, alpha 3, alpha 4 and alpha 6 transcripts begins during gastrulation. Integrin alpha 5 is expressed at relatively high levels during cleavage, blastula and gastrula stages suggesting that it may represent the major integrin expressed in the early embryo. We demonstrated previously that integrin beta 1 protein synthesis remains constant following induction of stage 8 animal cap cells with activin (Smith, J. C., Symes, K., Hynes, R. O. and DeSimone, D. W. (1990) Development 108, 289–298.). Here we report that integrin alpha 3, alpha 4 and alpha 6 mRNA levels increase following induction with 10 U/ml activin-A whereas alpha 5, beta 1 and beta 3 mRNA levels remain unchanged. Whole-mount in situ hybridization reveals that alpha 3 mRNAs are expressed by cells of the involuting mesoderm in the dorsal lip region of early gastrulae. As gastrulation proceeds, alpha 3 expression is localized to a stripe of presumptive notochordal cells along the dorsal midline. In neurulae, alpha 3 mRNA is highly expressed in the notochord but becomes progressively more restricted to the caudalmost portion of this tissue as development proceeds from tailbud to tadpole stages. In addition, alpha 3 is expressed in the forebrain region of later stage embryos. These data suggest that integrin-mediated adhesion may be involved in the process of mesoderm involution at gastrulation and the organization of tissues during embryogenesis.

Ischemia-reperfusion rapidly increases COX-2 expression in piglet cerebral arteries

1999 ◽  
Vol 277 (3) ◽  
pp. H1207-H1214 ◽  
Author(s):  
Ferenc Domoki ◽  
Roland Veltkamp ◽  
Nishadi Thrikawala ◽  
Greg Robins ◽  
Ferenc Bari ◽  
...  
Keyword(s):  
Ischemia Reperfusion ◽  
Cerebral Arteries ◽  
Rnase Protection Assay ◽  
Immunoblot Analysis ◽  
Mrna Levels ◽  
Rnase Protection ◽  
Time Control ◽  
Cox 2 ◽  
Cox 1 ◽  
Ischemic Stress

In the newborn, cyclooxygenase (COX)-derived products play an important role in the cerebrovascular dysfunction after ischemia-reperfusion (I/R). We examined effects of I/R on expression of COX-1 and COX-2 isoforms in large cerebral arteries of anesthetized piglets. The circle of Willis, the basilar, and the middle cerebral arteries were collected from piglets at 0.5–12 h after global ischemia (2.5–10 min, n = 50), hypoxia ( n = 3), or hypercapnia ( n = 2) and from time-control ( n = 19) or untreated animals ( n = 7). Tissues were analyzed for COX-1 and COX-2 mRNA and protein using RNase protection assay and immunoblot analysis, respectively. Ischemia increased COX-2 mRNA by 30 min, and maximal levels were reached at 2 h. Hypoxia or hypercapnia had minimal effects on COX-2 mRNA. COX-2 protein levels were also consistently elevated by 8 h after I/R. Increases in COX-2 mRNA or protein were not influenced by pretreatment with either indomethacin (5 mg/kg iv, n = 5) or nitro-l-arginine methyl ester (15 mg/kg iv, n = 7). COX-1 mRNA levels were low in time controls, and ischemic stress had no significant effect on COX-1 expression. Thus ischemic stress leads to relatively rapid, selective induction of COX-2 in cerebral arteries.

scholarly journals Developmental expression of the epithelial Na+ channel in kidney and uroepithelia

1999 ◽  
Vol 276 (2) ◽  
pp. F304-F314 ◽  
Author(s):  
Shigeru Watanabe ◽  
Kazumichi Matsushita ◽  
Paul B. McCray ◽  
John B. Stokes
Keyword(s):  
Urinary Bladder ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Rnase Protection Assay ◽  
Adult Life ◽  
Developmental Expression ◽  
Na Channel ◽  
Rnase Protection ◽  
Collecting Ducts ◽  
Uroepithelial Cell

The epithelial Na+ channel (ENaC) plays an important role in regulating Na+ balance in neonatal and adult life. Using in situ hybridization, we localized α-, β-, and γ-rat ENaC (rENaC) mRNA in developing rat kidney and uroepithelia. rENaC mRNA was first detectable on fetal day 16, and by fetal day 17, mRNA was abundant in the terminal collecting duct and uroepithelia. After birth, the intensity of the signals for all three subunits increased in the cortical collecting ducts and by 9 days after birth had diminished in the inner medullary collecting ducts. Expression in uroepithelial cells was different. mRNA for β- and γ-rENaC, but not α-rENaC, was detected in pelvis, ureters, and bladder at all stages of development beyond fetal day 16. By RNase protection assay (RPA), the greatest increase in subunit abundance in the kidney occurred before birth. Between postnatal days 9 and 30, the abundance of β- and γ-rENaC decreased relative to α-rENaC in outer and inner medulla. The urinary bladder, in contrast, demonstrated the greatest increase in β- and γ-rENaC mRNA abundance after birth. We were generally unable to detect α-rENaC by RPA in urinary bladder. Feeding weaned rats a diet of high or low NaCl did not change the abundance of any of the subunit mRNAs in bladder. These results demonstrate additional heterogeneity of developmental expression and regulation of ENaC. The differences between the collecting duct and uroepithelial cell rENaC mRNA regulation raise the possibility of significant differences in function.

Immunocytochemical localization of Na+ channels in rat kidney medulla

1989 ◽  
Vol 256 (2) ◽  
pp. F366-F369 ◽  
Author(s):  
D. Brown ◽  
E. J. Sorscher ◽  
D. A. Ausiello ◽  
D. J. Benos
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Apical Plasma Membrane ◽  
Na Channel ◽  
Thick Ascending Limb ◽  
Na Channels ◽  
Kidney Medulla ◽  
Principal Cells

Amiloride-sensitive Na+ channels were localized in semithin frozen sections of rat renal medullary collecting ducts, using polyclonal antibodies directed against purified bovine kidney Na+ channel protein. The apical plasma membrane of collecting duct principal cells was heavily stained by indirect immunofluorescence, whereas intercalated cells were negative. Basolateral plasma membranes of both cell types were unstained, as were subapical vesicles in the cytoplasm of these cells. In the thick ascending limb of Henle, some scattered granular fluorescence was seen in the cytoplasm and close to the apical pole of epithelial cells, suggesting the presence of antigenic sites associated with some membrane domains in these cells. No staining was detected in thin limbs of Henle, or in proximal tubules in the outer medulla. These results show that amiloride-sensitive sodium channels are located predominantly on the apical plasma membrane of medullary collecting duct principal cells, the cells that are involved in Na+ homeostasis in this region of the kidney.

Structural-functional relationships along the distal nephron

1986 ◽  
Vol 250 (1) ◽  
pp. F1-F15 ◽  
Author(s):  
K. M. Madsen ◽  
C. C. Tisher
Keyword(s):  
Atpase Activity ◽  
Collecting Duct ◽  
Basolateral Membrane ◽  
Distal Tubule ◽  
Structural Heterogeneity ◽  
Apical Plasma Membrane ◽  
Thick Ascending Limb ◽  
Intercalated Cells ◽  
Principal Cells ◽  
Functional Relationships

The distal tubule, which includes the thick ascending limb (TAL), the macula densa, and the distal convoluted tubule (DCT), and the collecting duct are structurally heterogeneous, thus reflecting the functional heterogeneity that is also present. As the TAL ascends from medulla to cortex, the surface area of the apical plasma membrane increases while that of the basolateral membrane decreases. The structure of the DCT resembles that of the medullary TAL. An excellent correlation exists between structure, Na-K-ATPase activity, and NaCl reabsorptive capacity in the distal tubule. The collecting duct is subdivided into the initial collecting tubule (ICT), and cortical (CCD), outer medullary (OMCD), and inner medullary (IMCD) collecting ducts. Between the distal tubule and the collecting duct is a transition region termed the connecting segment or connecting tubule (CNT). Considerable structural heterogeneity exists along the collecting duct within the two major cell populations, the intercalated cells and the principal cells. In the CNT, the ICT, and the CCD, potassium loading and mineralocorticoids stimulate Na-K-ATPase activity and cause proliferation of the basolateral membrane of CNT cells and principal cells, thus identifying the cells responsible for mineralocorticoid-stimulated potassium secretion in these regions. Finally, at least two morphologically distinct populations of intercalated cells exist, types A and B. In the rat, type A predominates in the CNT and the OMCD and is believed to be responsible for H+ secretion, at least in the OMCD. Type B predominates in the CCD, where it may be involved in bicarbonate secretion.

scholarly journals Interactions between the promoter and first intron are involved in transcriptional control of alpha 1(I) collagen gene expression.

1988 ◽  
Vol 8 (11) ◽  
pp. 4851-4857 ◽  
Author(s):  
P Bornstein ◽  
J McKay ◽  
D J Liska ◽  
S Apone ◽  
S Devarayalu
Keyword(s):  
Growth Hormone ◽  
Human Growth Hormone ◽  
Transcriptional Control ◽  
Rnase Protection Assay ◽  
Human Growth ◽  
Collagen Gene ◽  
Mrna Levels ◽  
3T3 Cells ◽  
Rnase Protection ◽  
First Intron

The first intron of the human collagen alpha 1(I) gene contains several positively and negatively acting elements. We have studied the transcription of collagen-human growth hormone fusion genes, containing deletions and rearrangements of collagen intronic sequences, by transient transfection of chick tendon fibroblasts and NIH 3T3 cells. In chick tendon fibroblasts, but not in 3T3 cells, inversion of intronic sequences containing a previously studied 274-base-pair segment, A274, resulted in markedly reduced human growth hormone mRNA levels as determined by an RNase protection assay. This inhibitory effect was largely alleviated when deletions were introduced in the collagen promoter of plasmids containing negatively oriented intronic sequences. Evidence for interaction of the promoter with the intronic segment, A274, was obtained by gel mobility shift assays. We suggest that promoter-intron interactions, mediated by DNA-binding proteins, regulate collagen gene transcription. Inversion of intronic segments containing critical interactive elements might then lead to an altered geometry and reduced activity of a transcriptional complex in those cells with sufficiently high levels of appropriate transcription factors. We further suggest that the deleted promoter segment plays a key role in directing DNA interactions involved in transcriptional control.

scholarly journals Apical endosomes isolated from kidney collecting duct principal cells lack subunits of the proton pumping ATPase.

1992 ◽  
Vol 119 (1) ◽  
pp. 111-122 ◽  
Author(s):  
I Sabolic ◽  
F Wuarin ◽  
L B Shi ◽  
A S Verkman ◽  
D A Ausiello ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Proton Pump ◽  
Transmembrane Domain ◽  
Collecting Duct ◽  
Water Channels ◽  
Proton Pumping ◽  
Apical Plasma Membrane ◽  
Principal Cells ◽  
Kidney Collecting Duct ◽  
Early Endosomes

Endocytic vesicles that are involved in the vasopressin-stimulated recycling of water channels to and from the apical membrane of kidney collecting duct principal cells were isolated from rat renal papilla by differential and Percoll density gradient centrifugation. Fluorescence quenching measurements showed that the isolated vesicles maintained a high, HgCl2-sensitive water permeability, consistent with the presence of vasopressin-sensitive water channels. They did not, however, exhibit ATP-dependent luminal acidification, nor any N-ethylmaleimide-sensitive ATPase activity, properties that are characteristic of most acidic endosomal compartments. Western blotting with specific antibodies showed that the 31- and 70-kD cytoplasmically oriented subunits of the vacuolar proton pump were not detectable in these apical endosomes from the papilla, whereas they were present in endosomes prepared in parallel from the cortex. In contrast, the 56-kD subunit of the proton pump was abundant in papillary endosomes, and was localized at the apical pole of principal cells by immunocytochemistry. Finally, an antibody that recognizes the 16-kD transmembrane subunit of oat tonoplast ATPase cross-reacted with a distinct 16-kD band in cortical endosomes, but no 16-kD band was detectable in endosomes from the papilla. This antibody also recognized a 16-kD band in affinity-purified H+ ATPase preparations from bovine kidney medulla. Therefore, early endosomes derived from the apical plasma membrane of collecting duct principal cells fail to acidify because they lack functionally important subunits of a vacuolar-type proton pumping ATPase, including the 16-kD transmembrane domain that serves as the proton-conducting channel, and the 70-kD cytoplasmic subunit that contains the ATPase catalytic site. This specialized, non-acidic early endosomal compartment appears to be involved primarily in the hormonally induced recycling of water channels to and from the apical plasma membrane of vasopressin-sensitive cells in the kidney collecting duct.

scholarly journals Polarization of gelsolin and actin binding protein in kidney epithelial cells.

1990 ◽  
Vol 38 (8) ◽  
pp. 1145-1153 ◽  
Author(s):  
J H Hartwig ◽  
D Brown ◽  
D A Ausiello ◽  
T P Stossel ◽  
L Orci
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Water Permeability ◽  
Collecting Duct ◽  
Regulatory Proteins ◽  
Actin Binding ◽  
Apical Plasma Membrane ◽  
Actin Binding Protein ◽  
Principal Cells ◽  
Actin Regulatory Proteins

Vasopressin regulates transepithelial osmotic water permeability in the kidney collecting duct and in target cells in other tissues. In the presence of hormone, water channels are inserted into an otherwise impermeable apical plasma membrane and the apical surface of these cells is dramatically remodelled. Because cytochalasin B and D greatly reduce the response of these cells to vasopressin, actin filaments are believed to participate in the events leading to an increase in transepithelial water permeability. Modulation of the actin filamentous network requires the concerted action of specific actin regulatory proteins, and in the present study we used protein A-gold immunocytochemistry to localize two important molecules, gelsolin and actin binding protein (ABP), in epithelial cells of the kidney inner medulla. Gelsolin and, to a lesser extent, ABP were concentrated in clusters in the apical cell web of principal cells of the collecting duct. Aggregates of gold particles were often associated with the cytoplasmic side of plasma membrane regions forming surface extensions or microvilli. The basolateral plasma membrane was labeled to a much lesser extent than the apical plasma membrane. In the thin limbs of Henle, ABP was localized over the apical plasma membrane in ascending limbs, but gelsolin labeling was weak in these cells. In thin descending limbs, the pattern of labeling was completely reversed, with abundant apical gelsolin labeling but only weak ABP immunolabeling. Although the significance of the distribution of actin regulatory proteins in thin limbs is unknown, the abundance and the predominantly apical polarization of both ABP and gelsolin in principal cells of the collecting duct is consistent with a role of the actin cytoskeleton in the mechanism of vasopressin actin.

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