The rat kidney contains high levels of prouroguanylin (the uroguanylin precursor) but does not express GC-C (the enteric uroguanylin receptor)

The peptide uroguanylin (Ugn) regulates enteric and renal electrolyte transport. Previous studies have shown that Ugn and its receptor GC-C (a ligand-activated guanylate cyclase) are abundant in the intestine. Less is known about Ugn and GC-C expression in the kidney. Here, we identify a 9.4-kDa polypeptide in rat kidney extracts that appears, based on its biochemical and immunological properties, to be authentic prouroguanylin (proUgn). This propeptide is relatively plentiful in the kidney (∼16% of intestinal levels), whereas its mRNA is marginally present (<1% of intestinal levels), and free Ugn peptide levels are below detection limits (<0.4% of renal proUgn levels). The paucity of preproUgn-encoding mRNA and free Ugn peptide raises the possibility that the kidney might absorb intact proUgn from plasma, where the concentration of propeptide greatly exceeds that of Ugn. However, immunocytochemical analysis reveals that renal proUgn is found exclusively in distal tubular segments, sites previously shown not to accumulate radiolabeled proUgn after intravascular infusions. Thus proUgn appears to be synthesized within the kidney, but the factors that determine its abundance (rates of transcription, translation, processing, and secretion) must be balanced quite differently than in the gut. Surprisingly, we also find negligible expression of GC-C in the rat kidney, a result confirmed both by RT-PCR and by functional assays that measure Ugn-activated cGMP synthesis. Taken together, these data provide evidence for an intrarenal Ugn system that differs from the well-described intestinal system in its regulatory mechanisms and in the receptor targeted by the peptide.

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Structures of soluble guanylate cyclase: implications for regulatory mechanisms and drug development

Biochemical Society Transactions ◽

10.1042/bst20130228 ◽

2014 ◽

Vol 42 (1) ◽

pp. 108-113 ◽

Cited By ~ 14

Author(s):

Opher Gileadi

Keyword(s):

Nitric Oxide ◽

Heart Failure ◽

Pulmonary Hypertension ◽

Drug Discovery ◽

Drug Development ◽

Guanylate Cyclase ◽

Therapeutic Intervention ◽

Soluble Guanylate Cyclase ◽

Regulatory Mechanisms ◽

Cgmp Synthesis

Activation of cGMP synthesis leads to vasodilation, and is an important mechanism in clinical treatment of angina, heart failure, and severe peripheral and pulmonary hypertension. The nitric oxide-responsive sGC (soluble guanylate cyclase) has been the target of recent drug discovery efforts. The present review surveys recent data on the structure and regulation of sGC, and the prospects of new avenues for therapeutic intervention.

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REGULATION OF RECEPTOR-OPERATED Ca2− CHANNEL OPENING IN HUMAN PLATELETS

10.1055/s-0038-1644677 ◽

1987 ◽

Author(s):

Katrina J Moffat ◽

D Euan MacIntyre

Keyword(s):

Adenylate Cyclase ◽

Contrast Agents ◽

Guanylate Cyclase ◽

Receptor Occupancy ◽

Human Platelets ◽

Regulatory Mechanisms ◽

Receptor Interaction ◽

Thrombin Receptors ◽

Channel Opening ◽

Camp And Cgmp

Agonist-induced elevation of the platelet intracellular free Ca2+ concentration ([Ca2−]i), as monitored using quin2, is not electrically mediated and is attenuated by removal of extracellular Ca2− and by lanthanides (e.g Gd3−).Collectively these data suggest that elevation of [Ca2−]i in platelets derives in part via influx of external Ca2−presumably through a receptor-operated Ca2− channel (ROC). Hal lam & Rink (FEBS Lett. 186: 175: 1985) showed that Mn2−also enters platelets via these ROC. To investigate the possible regulatory mechanisms that govern ROC status, we utilized quin2-labelled human platelets suspended in a Ca2+-free Hepes buffered Tyrodes solution, and monitored agonist-induced Mn2+-mediated quenching of quin2 fluorescence as an index of ROC opening.Thrombin (Th, 0.01-1 U/ml), Vasopressin (VP, 10-1000 nM) and the TxA2-mitnetic, EP171 (1-100 nM) all induced ROC opening which occurred rapidly (<30s), was maximal within 30-60s and thereafter declined. Gd3+ (≤2 mM) markedly impaired this Mn2ࢤ-mediated quenching of quin2 fluorescence induced by all 3 agonists. The adenylate cyclase stimulant PGD2 (3-3000 nM) and the guanylate cyclase stimulant sodium nitroprusside (0.01-10 μM) impaired ROC opening induced by Th (0.5 U/ml), VP (100 nM) and EP171 (25 nM) whether added to platelets ≤120sbefore or 30s after the agonists. In contrast, agents that selectively antagonize, at the receptor level, the effects of VP (e.g. d(CH2)5Tyr Me AVP, 10 ¼H) or EP171 (e.g.EP092, 250nM), or that inhibit the action of Th(e.g. Hirudin 1 U/ml)only impaired ROC opening when added to platelets simultaneously with or before the agonist.These results indicate that, although initiated by agonist-receptor interaction, maintenance of the open state of ROC in human platelets does not require continued receptor occupancy or activation by agonist. Moreover, besides acting to impair the transduction processes initiated following occupancy by agonist of platelet Vi, TP and Thrombin receptors, cAMP-and cGMP-dependent reactions also can terminate or otherwise limit opening of ROC.

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Na-K-ATPase isoform (alpha 3, alpha 2, alpha 1) abundance in rat kidney estimated by competitive RT-PCR and ouabain binding

AJP Renal Physiology ◽

10.1152/ajprenal.1996.271.2.f253 ◽

1996 ◽

Vol 271 (2) ◽

pp. F253-F260 ◽

Cited By ~ 7

Author(s):

K. Lucking ◽

J. M. Nielsen ◽

P. A. Pedersen ◽

P. L. Jorgensen

Keyword(s):

Polymerase Chain Reaction ◽

Rat Kidney ◽

Sodium Reabsorption ◽

Rt Pcr ◽

Ouabain Binding ◽

Chain Reaction ◽

Competitive Polymerase Chain Reaction ◽

High Affinity ◽

Upper Limit ◽

Polymerase Chain

For understanding the regulation of sodium reabsorption, it is important to know whether the alpha 2- or alpha 3-isoform of Na-K-adenosinetriphosphatase (Na-K-ATPase) is expressed in mammalian kidney in addition to the predominant alpha 1 beta 1-isozyme. Here we applied competitive polymerase chain reaction (PCR) for estimation of mRNA in parenchymal zones of rat kidney for comparison to high-affinity [3H]ouabain binding. The alpha 3-isoform mRNA was demonstrated to form 0.04-0.05% of the amount of alpha 1-isoform mRNA in the cortex, medulla, and papilla of rat kidney. The alpha 2-mRNA was demonstrated in all three zones and constituted 0.03% of the amount of alpha 1-mRNA in cortex. The abundance of the alpha 1 truncated mRNA was 0.1-0.8% of that of the alpha 1-mRNA. The upper limit for expression of Na-K-ATPase isozyme with high ouabain affinity (dissociation constant, 69-141 nM) was 0.096-0.14% of the concentration of alpha 1 beta 1-Na-K-ATPase. Thus a small but well-defined pool of alpha 2- and alpha 3-isoforms constitutes < or = 0.1% of the amount of alpha 1-isoform at both the mRNA and protein level in rat kidney.

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Urodilatin: binding properties and stimulation of cGMP generation in rat kidney cells

AJP Renal Physiology ◽

10.1152/ajprenal.1993.264.2.f267 ◽

1993 ◽

Vol 264 (2) ◽

pp. F267-F273

Author(s):

H. Saxenhofer ◽

W. R. Fitzgibbon ◽

R. V. Paul

Keyword(s):

Guanylate Cyclase ◽

Mesangial Cells ◽

Collecting Duct ◽

Rat Kidney ◽

Binding Characteristics ◽

Papillary Collecting Duct ◽

Collecting Duct Cells ◽

Medullary Collecting Duct ◽

Anp Receptors

Urodilatin (URO) [ANP-(95-126)] is an analogue of atrial natriuretic peptide (alpha-ANP) [ANP-(99-126)] that was first isolated from human urine. In rat mesangial cells, URO competed with high affinity for non-guanylate cyclase-coupled ANPR-C receptors [concentration at which 50% labeled ligand is displaced (IC50) approximately 70 pM], but with lesser affinity to the guanylate cyclase-linked ANPR-A receptors (IC50 approximately 800 pM). alpha-ANP bound to both receptors with similar affinity [dissociation constant (Kd) approximately 150 pM]. In papillary collecting duct homogenates, which possess only ANPR-A receptors, the apparent Kd value averaged 229 pM for alpha-ANP and 2.7 nM for URO. Intravenous URO was at least as potent and effective as alpha-ANP in inducing diuresis and natriuresis in anesthetized rats, but URO was approximately 10-fold less potent in stimulating guanosine 3',5'-cyclic monophosphate generation in mesangial and inner medullary collecting duct cells. We conclude that URO has a lesser affinity than alpha-ANP for guanylate cyclase-coupled ANP receptors in the kidney and that the relative natriuretic potency of URO in vivo cannot be directly attributed to its binding characteristics with ANPR-A receptors.

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Localization of mRNAs coding for isozymes of plasma membrane Ca(2+)-ATPase pump in rat kidney

AJP Renal Physiology ◽

10.1152/ajprenal.1992.263.1.f7 ◽

1992 ◽

Vol 263 (1) ◽

pp. F7-F14 ◽

Cited By ~ 10

Author(s):

M. Magosci ◽

M. Yamaki ◽

J. T. Penniston ◽

T. P. Dousa

Keyword(s):

Plasma Membrane ◽

Rat Kidney ◽

Rat Plasma ◽

Rt Pcr ◽

Chain Reaction ◽

Outer Medulla ◽

Unique Distribution ◽

Polymerase Chain ◽

Convoluted Tubules ◽

Specific Mrna

We have studied localization of mRNAs coding isozymes of rat plasma membrane Ca(2+)-adenosinetriphosphatase pump (rPMCA) in the rat kidney, with use of reverse transcription (RT) with subsequent amplification by polymerase chain reaction (PCR). When zones of the kidney were separated by macrodissection, a large amount of mRNA coding isozyme rPMCA1 was found in all zones; mRNA for isozyme rPMCA2 was abundant in cortex and in outer medulla, and mRNA for isozyme rPMCA3 was prominent in outer medulla. The mRNAs were analyzed in microdissected cortical nephron segments by use of RT-PCR approach described previously [T. Moriyama, H. R. Murphy, B. M. Martin, and A. Garcia-Perez. Am. J. Physiol. 258 (Renal Fluid Electrolyte Physiol. 27): F1470-F1474, 1990]. We detected mRNA for isozyme rPMCA2 in microdissected distal convoluted tubules (DCT) and in cortical thick ascending limbs (CTAL) and, less consistently, also in proximal convoluted tubule and in glomeruli. The mRNA for isozyme rPMCA1 was abundant in glomeruli but was absent in all examined cortical tubular segments. Our results document that mRNAs for all three major isozymes of rPMCA are present and show a unique distribution in the three major zones of rat renal parenchyma. Specific mRNA coding for rPMCA2 was detected in cortical tubules, namely in CTAL and DCT, whereas mRNA coding isozyme rPMCA1 was found in glomeruli. We suggest that isozyme rPMCA2 might be specifically related to epithelial cells and their function, whereas rPMCA1 is probably a component of nonepithelial cells including these in glomeruli.

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RT-PCR analysis of Na+/H+ exchanger mRNAs in rat medullary thick ascending limb

AJP Renal Physiology ◽

10.1152/ajprenal.1995.268.6.f1224 ◽

1995 ◽

Vol 268 (6) ◽

pp. F1224-F1228 ◽

Cited By ~ 10

Author(s):

P. Borensztein ◽

M. Froissart ◽

K. Laghmani ◽

M. Bichara ◽

M. Paillard

Keyword(s):

Rat Kidney ◽

Pcr Amplification ◽

Restriction Enzymes ◽

Pcr Analysis ◽

Thick Ascending Limb ◽

Rt Pcr ◽

Medullary Thick Ascending Limb ◽

Pcr Products ◽

Polymerase Chain ◽

Nhe Isoforms

The thick ascending limb (TAL) of rat kidney absorbs bicarbonate secondary to proton secretion, but displays both basolateral and luminal Na+/H+ exchange (NHE) activity. Several NHE genes, including NHE-1, NHE-2, NHE-3, and NHE-4, are expressed in the kidney. To identify the NHE isoforms expressed in the rat medullary TAL (MTAL), we used the reverse transcription-polymerase chain reaction (RT-PCR) to detect the mRNAs for NHE in microdissected MTAL. RT-PCR amplification from total RNA was performed between two specific primers for each NHE isoform. In rat kidney homogenate, the four NHE isoform mRNAs were detected, and the identity of the PCR products was demonstrated by the sizes of the fragments, digestion with restriction enzymes, and Southern blot analysis. In microdissected rat MTAL, NHE-3 was strongly expressed and NHE-1 mRNA was also detected, whereas NHE-2 and NHE-4 mRNAs were not detected. Therefore, NHE-3 could be the apical Na+/H+ exchanger, and NHE-1 could be the basolateral isoform in the MTAL.

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Sensing of tubular flow and renal electrolyte transport

Nature Reviews Nephrology ◽

10.1038/s41581-020-0259-8 ◽

2020 ◽

Vol 16 (6) ◽

pp. 337-351 ◽

Cited By ~ 3

Author(s):

Eric H. J. Verschuren ◽

Charlotte Castenmiller ◽

Dorien J. M. Peters ◽

Francisco J. Arjona ◽

René J. M. Bindels ◽

...

Keyword(s):

Electrolyte Transport ◽

Renal Electrolyte Transport ◽

Tubular Flow

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Distribution and regulation of guanylyl cyclase type B in the rat nephron

AJP Renal Physiology ◽

10.1152/ajprenal.1996.270.2.f311 ◽

1996 ◽

Vol 270 (2) ◽

pp. F311-F318 ◽

Cited By ~ 1

Author(s):

A. D. Dean ◽

V. M. Vehaskari ◽

D. Ritter ◽

J. E. Greenwald

Keyword(s):

Guanylyl Cyclase ◽

Rat Kidney ◽

Immunofluorescent Staining ◽

Hydration Status ◽

Mrna Levels ◽

Type B ◽

Rt Pcr ◽

Data Link ◽

Renal Inner Medulla ◽

Polymerase Chain

C-type natriuretic peptide (CNP) has been localized to the proximal and distal nephron. In this study, we examined the distribution and regulation of the CNP receptor, guanylyl cyclase type B (GC-B), in the rat kidney. GC-B mRNA was detected most frequently in microdissected glomeruli, thin and thick limbs of the loop of Henle, and outer and inner medullary collecting ducts by reverse transcription-polymerase chain reaction (RT-PCR). This pattern of expression is supported by immunofluorescent staining, using anti-GC-B-specific antiserum. Nearly equivalent levels of GC-B and guanylyl cyclase type A (GC-A) mRNAs were found by quantitative RT-PCR (5,662 +/- 1,622 and 5,187 +/- 1,204 molecules of cDNA/microgram total RNA, respectively; means +/- SE, n = 6). Renal inner medulla GC-B mRNA levels, but not renal CNP mRNA levels, were 3.2-fold greater in hypervolemic and 2.3-fold less in hypovolemic rats compared with euvolemic controls. Immunohistochemical staining also supports a greater GC-B expression with increased volume status. These data link hydration status and GC-B expression and suggest an additional and novel mechanism for regulating intravascular volume.

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The Expression of Dentin Sialophosphoprotein Gene in Bone

Journal of Dental Research ◽

10.1177/154405910208100607 ◽

2002 ◽

Vol 81 (6) ◽

pp. 392-394 ◽

Cited By ~ 183

Author(s):

C. Qin ◽

J.C. Brunn ◽

E. Cadena ◽

A. Ridall ◽

H. Tsujigiwa ◽

...

Keyword(s):

Specific Protein ◽

Regulatory Mechanisms ◽

Long Bone ◽

Mrna Transcript ◽

Rt Pcr ◽

Chain Reaction ◽

Dentin Sialophosphoprotein ◽

Dentin Phosphoprotein ◽

Polymerase Chain ◽

Dentin Sialoprotein

Dentin sialoprotein (DSP) and dentin phosphoprotein (DPP) are expressed as a single mRNA transcript coding for a large precursor protein termed dentin sialophosphoprotein (DSPP). DSP, DPP, and DSPP have been considered to be tooth-specific. To test for the expression of the dspp gene in bone, we performed Western immunoblots and reverse-transcription polymerase chain-reaction (RT-PCR). With Western immunoblots, we detected DSP in the Gdm/EDTA extracts of rat long bone, at a level of about 1/400 of that in dentin. Using RT-PCR, we detected DSPP mRNA in mouse calvaria. Similar to Western immunoblots, the results of RT-PCR indicated that the dspp gene is expressed at a lower level in bone than in dentin and odontoblasts. Analysis of the data shows that DSPP is not a tooth-specific protein, and that dramatically different regulatory mechanisms governing DSPP expression are involved in the bone and dentin.

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