C-peptide stimulates Na+,K+-ATPase activity via PKC alpha in rat medullary thick ascending limb.

Aims/hypothesis: C-peptide, the cleavage product of proinsulin processing exerts physiological effects including stimulation of Na(+),K(+)-ATPase in erythrocytes and renal proximal tubules. This study was undertaken to assess the physiological effects of connecting peptide on Na(+),K(+)-ATPase activity in the medullary thick ascending limb of Henle's loop.

Methods: Na(+),K(+)-ATPase activity was measured as the ouabain-sensitive generation of (32)Pi from gamma[(32)P]-ATP and (86)Rb uptake on isolated rat medullary thick ascending limbs.

Cellular localization of type 5 and type 6 ACs in collecting duct and regulation of cAMP synthesis.

The cellular distribution of Ca(2+)-inhibitable adenylyl cyclase (AC) type 5 and type 6 mRNAs in rat outer medullary collecting duct (OMCD) was performed by in situ hybridization. Kidney sections were also stained with specific antibodies against either collecting duct intercalated cells or principal cells. The localization of type 5 AC in H(+)-ATPase-, but not aquaporin-3-, positive cells demonstrated that type 5 AC mRNA is expressed only in intercalated cells.

Functional properties of Ca2+-inhibitable type 5 and type 6 adenylyl cyclases and role of Ca2+ increase in the inhibition of intracellular cAMP content.

Among the different adenylyl cyclase (AC) isoforms, type 5 and type 6 constitute a subfamily which has the remarkable property of being inhibited by submicromolar Ca2+ concentrations in addition to Galphai-mediated processes. These independent and cumulative negative regulations are associated to a low basal enzymatic activity which can be strongly activated by Galphas-mediated interactions or forskolin. These properties ensure possible wide changes of cAMP synthesis.

[Regulation of intracellular cAMP content in kidney tubules: role of adenylyl cyclases inhibitable by calcium].

Adenylyl cyclase (AC) isoforms 5 and 6 can be inhibited by submicromolar concentrations of Ca2+. Quantitative RT-PCR allowed to study the corresponding messengers (mRNA) along the rat nephron. The results demonstrate a significant expression of AC 6 mRNA all along the nephron and of AC 5 mRNA in the glomerulus and the collecting tubule located in the cortex and the outer medulla.

Cell-specific coupling of PGE2 to different transduction pathways in arginine vasopressin- and glucagon-sensitive segments of the rat renal tubule.

1. The aim of the present study was to investigate the transduction pathways elicited by prostaglandin E2 (PGE2) to inhibit hormone-stimulated adenosine 3':5'-cyclic monophosphate (cyclic AMP) accumulation in the outer medullary collecting duct (OMCD) and medullary thick ascending limb (MTAL) microdissected from the rat nephron. 2.

Co-expression of a Ca2+-inhibitable adenylyl cyclase and of a Ca2+-sensing receptor in the cortical thick ascending limb cell of the rat kidney. Inhibition of hormone-dependent cAMP accumulation by extracellular Ca2+.

The Ca2+-sensing receptor protein and the Ca2+-inhibitable type 6 adenylyl cyclase mRNA are present in a defined segment of the rat renal tubule leading to the hypothesis of their possible functional co-expression in a same cell and thus to a possible inhibition of cAMP content by extracellular Ca2+. By using microdissected segments, we compared the properties of regulation of extracellular Ca2+-mediated activation of Ca2+ receptor to those elicited by prostaglandin E2 and angiotensin II. The three agents inhibited a common pool of hormone-stimulated cAMP content by different mechanisms as follows.

Localization of mRNAs encoding Ca2+-inhibitable adenylyl cyclases along the renal tubule. Functional consequences for regulation of the cAMP content.

Expression of Ca2+-inhibitable types V and VI adenylyl cyclases was studied by reverse transcription-polymerase chain reaction in rat renal glomeruli and nephron segments isolated by microdissection. Quantitation of each mRNA was achieved using a mutant cRNA which differed from the wild type by substituting two bases to create a new restriction site in the corresponding cDNA. Type VI mRNA was present all along the nephron but was more abundant in distal than in proximal segments.

Arachidonic acid inhibits hormone-stimulated cAMP accumulation in the medullary thick ascending limb of the rat kidney by a mechanism sensitive to pertussis toxin.

The possible regulation of adenosine 3',5'-cyclic monophosphate (cAMP) accumulation by arachidonic acid (AA) was studied in segments, microdissected from the rat kidney, which are sensitive to arginine vasopressin (AVP). In the presence of 5 microM indomethacin, the addition of 5 microM AA did not impair AVP-dependent cAMP accumulation (measured during 4 min at 35 degrees C) in the cortical or outer medullary collecting tubule, but decreased this response in the thick ascending limb with an inhibition much more pronounced in the medullary portion (MTAL) than in the cortical portion. In MTAL, the response to 10 nM AVP was inhibited by 34.

The activation of protein kinase C prevents PGE2-induced inhibition of AVP-dependent cAMP accumulation in the rat outer medullary collecting tubule.

Previous studies have demonstrated that prostaglandin E2 (PGE2) inhibits arginine vasopressin-(AVP)dependent adenosine 3',5'-cyclic monophosphate (cAMP) accumulation in microdissected rat outer medullary collecting tubules (OMCD), by a mechanism unrelated to the inhibition of cAMP synthesis. The potential role of the activation of protein kinase C (PKC) to explain the negative regulation elicited by PGE2 was investigated in this study. Single OMCD samples were pre-incubated (10 min, 30 degrees C) in the presence or absence of either activators of PKC, phorbol 12-myristate 13-acetate (PMA), 1-oleoyl-2-acetyl-glycerol (OAG), dioctanoylglycerol (DOG) or an inhibitor of PKC, staurosporine (SSP).

Insulin stimulates Na+, Cl-, Ca2+, and Mg2+ transports in TAL of mouse nephron: cross-potentiation with AVP.

Insulin (Ins) decreases Na+ delivery in the final urine. To determine whether the loop of Henle participates in this reduction, the effects of Ins were tested on cortical (CTAL) and medullary thick ascending limbs (MTAL) of the mouse nephron, microperfused in vitro. In the MTAL, Ins increased the transepithelial potential difference (Vt) and the Na+ and Cl- net reabsorption fluxes (JNa and JCl, respectively) in a dose-dependent manner, the threshold being below 10(-9) M.

PGE2-induced inhibition of AVP-dependent cAMP accumulation in the OMCD of the rat kidney is cumulative with respect to the effects of alpha 2-adrenergic and alpha 1-adenosine agonists, insensitive to pertussis toxin and dependent on extracellular calcium.

The accumulation of cyclic adenosine 3',5'-phosphate (cAMP) elicited by antidiuretic hormone (arginine vasopressin, AVP) in the medullary collecting tubule (OMCD) microdissected from the rat kidney is inhibited by different factors: the A1 agonist of adenosine (-)-N6-(R-phenylisopropyl) adenosine (PIA), an alpha 2-adrenergic agonist clonidine (CLO), and prostaglandin E2 (PGE2). The negative regulation elicited by PGE2 was further characterized by measuring summation of inhibition with other inhibitors, by testing the effect of pertussis toxin and by studying the part played by extracellular calcium. Inhibitors were used at concentrations inducing maximum effects.

Effect of prostaglandin E2 on agonist-stimulated cAMP accumulation in the distal convoluted tubule isolated from the rabbit kidney.

The effects of calcitonin, vasoactive intestinal peptide (VIP), parathyroid hormone (PTH) and isoprenaline on intracellular cAMP accumulation were determined in the distal tubule (DCT) microdissected from collagenase-treated rabbit kidney. In DCTb (the initial "bright" portion) calcitonin (10 ng/ml) elicited a highly reproducible response 203.7 +/- 19.

Cholinergic stimulation of phosphoinositide metabolism in isolated rat glomeruli.

Cholinergic effects on kidney function have been observed in some mammals but the intrarenal localization and the cellular mechanisms of these effects are poorly defined to date. The aim of this work was to study the effects of carbachol on phosphoinositide metabolism in freshly isolated rat glomeruli labeled with myo-[3H]inositol. Carbachol rapidly and markedly stimulates phosphoinositide metabolism with a 50% effective concentration of 3 microM.

Establishment of renal cell lines derived from S2 segments of the proximal tubule.

The aim of this study was to establish epithelial cell lines derived from defined nephron segments. Primary cultures were prepared from dissected proximal S2 segments of the rabbit kidney, and grown in monolayers. Immortalization was observed after nuclear microinjection of the cells with simian virus 40 DNA and resulted in the development of cell lines of epithelial morphology.

Two mechanisms of inhibition by prostaglandin E2 of hormone-dependent cell cAMP in the rat collecting tubule.

The effect of exogenous prostaglandin E2 (PGE2) on hormone-dependent adenosine 3',5'-cyclic monophosphate (cAMP) accumulation was investigated by microradioimmunoassay in collecting tubules microdissected from the cortex (CCT) or outer medulla (MCT) of the rat kidney. Two phosphodiesterase inhibitors were used: either a xanthine derivative (isobutyl-methylxanthine (IBMX, 1 mM] active on all forms of phosphodiesterase or Ro 20-1724 (50 microM) active on the phosphodiesterase type III. A prostaglandin synthesis inhibitor was added to all media.

Developmental pattern of cyclic guanosine monophosphate production stimulated by atrial natriuretic peptide in glomeruli microdissected from kidneys of young rats.

Cyclic guanosine monophosphate (GMP) productions by alpha rat atrial natriuretic peptide 1-28 (alpha-rANP), carbamylcholine or sodium nitroprusside were assessed in isolated glomeruli microdissected from collagenase-treated kidneys of 2- to 34-day-old and adult rats. In both young and adult animals, alpha-rANP-stimulated cyclic GMP generation was proportional to the number of glomeruli and was enhanced in a dose-dependent and saturable fashion with increasing alpha-rANP concentrations. The apparent activation constant values were 6.

Enzyme immunoassays of adenosine cyclic 3',5'-monophosphate and guanosine cyclic 3',5'-monophosphate using acetylcholinesterase.

The pure tetrameric form of acetylcholinesterase (EC 3.1.1.

Distribution of vasoactive intestinal peptide-sensitive adenylate cyclase activity along the rabbit nephron.

The effect of vasoactive intestinal peptide (VIP) upon adenylate cyclase (AC) activity has been determined in defined microdissected renal tubules isolated from collagenase-treated rabbit kidneys. In the presence of 10 microM GTP, 1 microM VIP gave marked stimulations of AC over basal values in the bright portion of the distal convoluted tubule (DCTb) (10.1-fold), and in the collecting tubule isolated from the inner stripe of the outer medulla (OMCTi, 7.

Effect of PGE2 and alpha-adrenergic agonists on AVP-dependent cAMP levels in rabbit and rat CCT.

The effect of prostaglandins and alpha-adrenergic agonists on arginine vasopressin-induced adenosine 3',5'-cyclic monophosphate (cAMP) production was investigated in microdissected rat and rabbit cortical collecting tubules (CCT) incubated in vitro. In rabbit CCT, addition to all media of a prostaglandin synthesis inhibitor increased this production; exogenous prostaglandin E2 (PGE2) induced a reproducible dose-dependent inhibition of cAMP accumulation. Maximal inhibition (mean: 57.

Atrial natriuretic peptide effects on cGMP and cAMP contents in microdissected glomeruli and segments of the rat and rabbit nephrons.

A microradioimmunoassay has been developed in order to measure the changes in cGMP cell content induced in vitro by atrial natriuretic peptides (ANP) in either glomeruli or defined portions of tubules microdissected from collagenase treated rat and rabbit kidneys. When tested at 0.1 microM or 1 microM, all ANP analogues used produced in rat glomeruli a 20-25 fold increase in cGMP accumulation compared to basal values.

Inhibition of alpha 2-adrenergic agonists on AVP-induced cAMP accumulation in isolated collecting tubule of the rat kidney.

A microradioimmunoassay for cAMP was developed in order to analyse the effects of alpha-adrenergic agonists on vasopressin (AVP)-induced cAMP cell accumulation in single pieces of microdissected medullary (MCT) and cortical (CCT) rat collecting tubules. Under the experimental conditions chosen (4 min of incubation in the presence of a phosphodiesterase inhibitor), no cAMP could be detected either in the bathing solution or in non-stimulating samples of tubule. In MCT, 10(-6) M AVP stimulated cAMP generation up to 128.

Ontogenesis of hormone-dependent adenylate cyclase in isolated rat nephron segments.

Ontogenesis of hormone-dependent adenylate cyclase (AC) was investigated in rat kidney by single tubule microassay between two days postnatal and adulthood. This approach allowed us to analyze the kinetics of vasopressin-sensitive AC maturation in its tubular target sites, namely the thick ascending limb and collecting tubule. It was also possible to compare in a single segment--the thick ascending limb--the kinetics of AC ontogenesis for three hormones-- vasopressin, calcitonin, and parathyroid hormone.

Plasma antidiuretic hormone levels and kidney responsiveness to vasopressin in the jerboa, Jaculus orientalis.

The plasma antidiuretic hormone (ADH) concentration and the kidney medulla responsiveness to vasopressin were measured in adult jerboas ( Jaculus orientalis) in different states of hydration. In 15 jerboas adapted to 30 degrees and fed a dry diet, the average ADH concentration in blood plasma was 479 +/- 59 pg/ml, as measured by a radioimmunoassay. About 6 hr after receiving a 5% body wt water load by gavage, the plasma ADH concentration fell to 130 +/- 30 pg/ml in the 5 jerboas still producing hypertonic urine (1022 +/- 267 mosmol/liter) and to 41.