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W S Spielman

Publications and source records attributed to W S Spielman.

At least 19 recordsLinked to original sources
PubMed1992

Characterization of adenosine A1 receptor in a cell line (28A) derived from rabbit collecting tubule.

We have previously reported that in several renal cell types, adenosine receptor agonists inhibit adenylyl cyclase and activate phospholipase C via a pertussis toxin-sensitive G protein. In the present study, in 28A cells, both of these adenosine receptor-mediated responses were inhibited by 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), a highly selective A1 adenosine receptor antagonist. The binding characteristics of the adenosine A1 receptor in the 28A renal cell line were studied using the radiolabeled antagonist [3H]DPCPX to determine whether two separate binding sites could account for these responses. Saturation binding of [3H]DPCPX to 28A cell membranes revealed a single class of A1 binding sites with an apparent Kd value of 1.4 nM and maximal binding capacity of 64 fmol/mg protein. Competition experiments with a variety of adenosine agonists gave biphasic displacement curves with a pharmacological profile characteristic of A1 receptors. Comparison of [3H]DPCPX competition binding data from 28A cell membranes with rabbit brain membranes, a tissue with well-characterized A1 receptors, reveals that the A1 receptor population in 28A cells has similar agonist binding affinities to the receptor population in brain but has a considerably lower density. Addition of guanosine 5'-triphosphate (100 microM) to 28A cell membranes caused the competition curves to shift from biphasic to monophasic, indicating that the A1 receptors exist in two interconvertible affinity states because of their coupling to G proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine↗
PubMed1992

Functional localization of adenosine receptor-mediated pathways in the LLC-PK1 renal cell line.

The functional localization of three adenosine receptor-mediated signal transduction pathways in the LLC-PK1 renal cell line was investigated. LLC-PK1 cells were grown on Millicell-CM filter inserts, which allow for the independent exposure of the apical or basolateral side of a confluent cell monolayer to hormones. Adenosine stimulated inositol phosphate turnover, inhibition of adenosine 3',5'-cyclic monophosphate (cAMP) accumulation (A1 receptor), and stimulation of cAMP accumulation (A2 receptor). Adenosine (10 microM) selectively applied to the basolateral side induced a significant (P < 0.05) increase in inositol phosphates, whereas apical exposure did not. The adenosine receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (1 microM), blocked the stimulation of inositol phosphate production in LLC-PK1 cells, provided support for an adenosine receptor-mediated event. When adenosine (30 nM) was selectively applied to the apical side, forskolin-stimulated cAMP levels were not significantly decreased (approximately 8%, P > 0.05). However, adenosine (30 nM) presented to the basolateral side produced a significant decrease (approximately 23%, P < 0.05) in forskolin-stimulated cAMP levels. A high dose (100 microM) of adenosine elicited a significant increase (P < 0.05) in cAMP levels when presented to either the apical or the basolateral cell surface. Adenosine (100 microM) applied to the apical side elicited significantly higher cAMP levels (P < 0.05) than the same dose applied basolaterally. LLC-PK1 cells grown on permeable supports exhibit a polarity of functional responses following activation by adenosine. These data support a topographic separation of the multiple adenosine signaling systems in a renal epithelial cell line.

Adenosine↗
PubMed1992

Renal hemodynamic effects of exogenously administered adenosine and polyadenylic acid.

Steady-state intrarenal arterial infusion of adenosine (Ado) suggests that there may be both afferent and efferent arteriolar actions of Ado. This study attempts to further differentiate vascular sites of action of Ado during an intrarenal infusion of Ado. We measured the filtration fraction (FF) during intrarenal infusion of Ado (33.3 nmol.kg-1 x min-1) in anesthetized dogs to determine its transient actions on renal hemodynamics. FF remained unchanged from preinfusion levels (0.42 +/- 0.01 vs. 0.46 +/- 0.01, respectively) at a time when renal blood flow (RBF) was significantly decreased (52 +/- 6% of control). During steady state, RBF was 96 +/- 5% of control, while FF was significantly decreased from control (0.27 +/- 0.02). To determine whether vasoconstriction and dilation to Ado are mediated by receptors accessible from intra- or extravascular compartments, two Ado analogues [oligoadenylic acid (oligo[A]), mol wt 5,000, and polyadenylic acid (poly[A]), mol wt > 100,000] were injected into the renal artery, and RBF response was compared with that of Ado. Poly[A] produced a transient vasodilation (42 +/- 6% increase in RBF), whereas oligo[A] produced a transient vasoconstriction (25 +/- 5% decrease in RBF). Responses to steady-state infusion of poly[A] (10 nmol.kg-1 x min-1) were determined in 11 anesthetized sodium-depleted dogs. Poly[A] produced a sustained significant increase in RBF from 2.83 +/- 0.31 to 3.92 +/- 0.40 ml.g-1 x min-1. This decrease in renal vascular resistance was blocked by an intrarenal infusion of the Ado antagonist theophylline (0.5 mumol.kg-1 x min-1, 2.68 +/- 0.38 vs. 2.85 +/- 0.38 ml.g-1 x min-1).(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine↗
PubMed1992

Adenosine regulates a chloride channel via protein kinase C and a G protein in a rabbit cortical collecting duct cell line.

We examined the regulation by adenosine of a 305-pS chloride (Cl-) channel in the apical membrane of a continuous cell line derived from rabbit cortical collecting duct (RCCT-28A) using the patch clamp technique. Stimulation of A1 adenosine receptors by N6-cyclohexyladenosine (CHA) activated the channel in cell-attached patches. Phorbol 12,13-didecanoate and 1-oleoyl 2-acetylglycerol, activators of protein kinase C (PKC), mimicked the effect of CHA, whereas the PKC inhibitor H7 blocked the action of CHA. Stimulation of A1 adenosine receptors also increased the production of diacylglycerol, an activator of PKC. Exogenous PKC added to the cytoplasmic face of inside-out patches also stimulated the Cl- channel. Alkaline phosphatase reversed PKC activation. These results show that stimulation of A1 adenosine receptors activates a 305-pS Cl-channel in the apical membrane by a phosphorylation-dependent pathway involving PKC. In previous studies, we showed that the protein G alpha i-3 activated the 305-pS Cl- channel (Schwiebert et al. 1990. J. Biol. Chem. 265:7725-7728). We, therefore, tested the hypothesis that PKC activates the channel by a G protein-dependent pathway. In inside-out patches, pertussis toxin blocked PKC activation of the channel. In contrast, H7 did not prevent G protein activation of the channel. We conclude that adenosine activates a 305-pS Cl- channel in the apical membrane of RCCT-28A cells by a membrane-delimited pathway involving an A1 adenosine receptor, phospholipase C, diacylglycerol, PKC, and a G protein. Because we have shown, in previous studies, that this Cl- channel participates in the regulatory volume decrease subsequent to cell swelling, adenosine release during ischemic cell swelling may activate the Cl-channel and restore cell volume.

Adenosine↗
PubMed1991

Effects of adenosine on cAMP production and cytosolic Ca2+ in cultured rabbit medullary thick limb cells.

The cellular signaling mechanism of adenosine action has been studied in highly purified populations of cultured cells from the rabbit medullary thick ascending limb of Henle's loop (MTAL). The effects of specific adenosine-receptor agonists 5'(N-ethylcarboxamido)adenosine (NECA; A2) and N6-cyclohexyladenosine (CHA; A1) on basal and hormone-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) production, cytosolic free calcium concentration ([Ca2+]f), and formation of inositol phosphates were examined. Production of cAMP was stimulated by high doses of NECA and was inhibited by low doses of CHA. The inhibitory effect of CHA was observed in cells in which cAMP production was first stimulated with vasopressin, isoproterenol, prostaglandin E2 (10(-6) M), or calcitonin (100 ng/ml) and was abolished by pretreating the cells with pertussis toxin (PT) for 12-20 h. A highly selective adenosine A1 antagonist, 8-cyclopentyl-1,3-dipropylxanthine (CPX), also abolished the inhibitory effect of CHA. Both NECA and CHA induced a rapid (10 s) and transient increase in [Ca2+]f, and this was associated with an increased inositol trisphosphate (IP3) production. Single-cell [Ca2+]f measurements indicated that all MTAL cells responded to CHA. The removal of extracellular Ca2+ failed to inhibit these responses. Pretreatment with PT or administration of CPX abolished both the increase in [Ca2+]f and the formation of IP3 occurring in response to CHA and NECA. Our results suggest that both adenylate cyclase-coupled inhibitory (A1) and stimulatory (A2) adenosine receptors are present in pure populations of cultured MTAL cells. Moreover, activation of an adenosine receptor coupled to a PT substrate results in the increased production of inositol phosphate and elevation of [Ca2+]f.

Adenosine↗
PubMed1991

Adenosine receptors and signaling in the kidney.

It is now generally accepted that adenosine is capable of regulating a wide range of physiological functions. Nowhere is the diversity of this action better illustrated than in the kidney. When adenosine binds to plasma membrane receptors on a variety of cell types in the kidney, it stimulates functional responses that span the entire spectrum of renal physiology, including alterations in hemodynamics, hormone and neurotransmitter release, and tubular reabsorption. These responses to adenosine appear to represent a means by which the organ and its constituent cell types can regulate their metabolic demand such that it is maintained at an appropriate level for the prevailing metabolic supply. Extracellular adenosine, produced from the hydrolysis of adenosine 5'-monophosphate and stimulated by increased substrate availability and enzyme induction, acts on at least two types of cell surface receptors to stimulate or inhibit the production of cyclic adenosine-3',5'-monophosphate and also acts in some renal cells to stimulate the production of inositol phosphates and elevation of cytosolic calcium concentration. To understand when and why this complicated system becomes activated, how it interacts with other known extracellular effector systems, and ultimately how to manipulate the system to therapeutic advantage by selective agonists or antagonists, requires a detailed knowledge of renal adenosine receptors and their signaling mechanisms. The following discussion attempts to highlight our knowledge in this area, to present a modified hypothesis for adenosine as a feedback regulator of renal function, and to identify some important questions regarding the specific cellular mechanisms of adenosine in renal cell types.

Adenosine↗
PubMed1989

Successive histochemical differentiation steps during postnatal development of the collecting duct in rabbit kidney.

Immunohistochemical experiments with monoclonal antibodies (mabs) on the kidney of neonatal rabbits revealed that the primary expression of collecting duct typic structures does not occur in a continuous and parallel, but in a subsequent developmental process. Only mabs RCT-30 A, and CD 4-V revealed immunoreactivity at the ontogenetically oldest parts of the collecting duct, the ampullae, while the other used markers (CD 1-3, CD 5-V, RCT-30 and RMCX) did not. In contrast, all of the tested antibodies showed positive reactions at the medullary and cortical collecting duct of the neonatal kidney as well as of the adult kidney. Additional incubations with wheat-germ agglutinin (WGA) a marker of terminal-differentiated collecting duct cells demonstrated weak-labelled ampulla cells beside intensively labelled ampullary neck and medullary collecting duct cells. With peanut agglutinin (PNA) labelling a 3 step transition could be illuminated: weak-labelled ampulla cells were found beside continuously bright labelled ampullary neck cells and finally a punctuate pattern downwards to the papilla. If the ampullary neck is the zone of proliferation, our findings of WGA- and PNA-co-labelling in this zone indicate, that in early developmental stages characteristic structures of different mature cells, probably principal and intercalated cells, are co-expressed within one single cell type. Thus intercalated cells might derive from principal cells.

Animals↗
PubMed1989

Adenosine-sensitive phosphoinositide turnover in a newly established renal cell line.

To aid in characterizing adenosine receptors in renal cells, primary cultures of rabbit cortical collecting tubule (RCCT) cells were infected with an adenovirus 12-simian virus 40 hybrid, resulting in a continuous cell line. The cells, designated RCCT-28A, retained their epithelial morphology and reacted with a monoclonal antibody specific for rabbit collecting tubule. Adenosine 3',5'-cyclic monophosphate (cAMP) accumulation was stimulated by vasopressin (AVP), isoproterenol, prostaglandin E2 (PGE2), calcitonin, parathyroid hormone, and a potent adenosine A1- and A2-receptor agonist, 5'-N-ethylcarboxamidoadenosine (NECA). A more selective adenosine A1-receptor agonist, N6-cyclohexyl adenosine (CHA) inhibited basal and AVP-stimulated cAMP accumulation. Cytosolic free calcium was transiently elevated by bradykinin, PGE2, NECA, and CHA. To examine the mechanism by which adenosine analogues increase intracellular free calcium, phosphoinositide (PI) turnover was assessed in the 28A cells after labeling with myo-[3H]inositol. NECA and CHA increased [3H]inositol phosphate formation with an approximate half-maximal effective concentration of 0.1 microM for both analogues. The increase in PI turnover was blocked by the selective adenosine A1-receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine and pretreatment of the 28A cells with pertussis toxin. These results suggest that adenosine analogues increase cytosolic free calcium by stimulating PI turnover.

1-Methyl-3-isobutylxanthine↗
PubMed1989

Vasopressin V1 receptors on the principal cells of the rabbit cortical collecting tubule. Stimulation of cytosolic free calcium and inositol phosphate production via coupling to a pertussis toxin substrate.

The effects of arginine vasopressin (AVP) on the cytosolic free calcium concentration ([Ca2+]f) were examined in freshly immunodissected rabbit cortical collecting tubule cells using fluorescent Ca2+ indicators fura-2 and indo-1. The addition of AVP to a cell suspension resulted in a rapid and transient increase in the [Ca2+]f. The 1-deamino-8-D-AVP (dDVP), a V2 receptor agonist of AVP that stimulated adenosine 3',5' cAMP production in these cells, had no effect on [Ca2+]f and did not affect AVP-induced increase in [Ca2+]f. The AVP-induced increase in [Ca2+]f but not cAMP production was blocked by the V1 receptor antagonist, [1-(beta-mercapto-beta-beta-cyclopentamethylene propionic acid), 2-(O-methyl)tyrosine] Arg8-vasopressin. The AVP-stimulated increase in [Ca2+]f appeared to be largely due to Ca2+ release from intracellular stores as reduction of extracellular Ca2+ with EGTA had little if any effect on the AVP-induced increase in [Ca2+]f. This AVP-induced increase in [Ca2+]f was associated with an increase in inositol-1,4,5-trisphosphate production and appeared to involve a guanine nucleotide-binding protein (G), since the pretreatment of cells with pertussis toxin for 4-6 h inhibited this effect. Finally, measurements of [Ca2+]f in single cells suggest that only the principal cells of the collecting tubules respond to AVP with an increase in [Ca2+]f. In summary, these results demonstrate that the principal cells of the cortical collecting tubule possess two distinct receptor systems for vasopressin, the well-known V2 receptor coupled to adenylate cyclase, and a V1 receptor system that leads to the mobilization of cytosolic calcium, coupled through a pertussis toxin substrate (G protein) to a production of inositol phosphates.

Adenylate Cyclase Toxin↗
PubMed1988

Adenosine receptor-mediated calcium mobilization in cortical collecting tubule cells.

To investigate the cellular mechanisms underlying the epithelial actions of adenosine, we studied adenosine receptor-effector coupling in cultured rabbit cortical collecting tubule (RCCT) cells. We previously reported, in RCCT cells isolated by immunodissection, that a potent A2 adenosine analogue [5'-N-ethylcarboxamideadenosine (NECA)] stimulates cAMP production [effective concentration 50% (EC50) = 1 microM], and potent A1 analogues [N6-cyclohexyladenosine (CHA) and R-N6-phenylisopropyladenosine (PIA)] inhibit basal and AVP-stimulated cAMP production (EC50 = 5 nM). The present study was undertaken to determine whether adenosine receptors in RCCT cells are also coupled to a signal transduction system leading to the mobilization of intracellular free calcium. RCCT cells were loaded with the fluorescent calcium indicator, fura-2, and were treated with the adenosine analogues NECA, CHA, and PIA. All three adenosine analogues produced dose-dependent (1 nM-0.1 mM), transient increases in intracellular calcium concentration with equal potency (EC50 = 0.5 microM). Chelation of extracellular calcium with ethyleneglycol-bis(beta-aminoethyl ether)N,N,N',N' tetraacetic acid (EGTA) did not abolish the increase in calcium. The adenosine receptor antagonists, 1,3-diethyl-8-propylxanthine and 8-cyclopentyl-1,3-dipropylxanthine, and pretreatment of RCCT cells with pertussis toxin blocked the increase in calcium. These results demonstrate that RCCT cells have, in addition to adenosine receptors associated with the stimulation and inhibition of cAMP, a pertussis-toxin sensitive receptor system that leads to the mobilization of intracellular calcium.

Adenosine↗
PubMed1988

Immunodissection of mitochondria-rich cells from rabbit outer medullary collecting tubule.

Two types of mitochondria-rich (MR) cells have been identified in the rabbit collecting tubule based on differences in immuno- and lectin cytochemistry. We have produced a monoclonal antibody, immunoglobulin (Ig) G1 (mr-mct), that reacts specifically with the MR cells (identified by positive histochemical staining for succinate dehydrogenase) found predominantly in the outer medulla (OM) and cells of the proximal tubule. IgG1 (mr-mct) reacted with 18 +/- 2% of the cells of the outer medullary collecting tubule (OMCT) and did not colocalize with peanut lectin-binding MR cells in the cortex. To isolate MR-OMCT cells, collecting tubule cells from collagenase dispersions of the OM were first adsorbed onto plates treated with a monoclonal antibody reactive against all of the OMCT cells. Of the isolated OMCT cells, 17% reacted with IgG1 (mr-mct). Cells were then detached from the plate and transferred to plates coated with IgG1. Greater than 70% of the adsorbed cells were MR as determined by positive staining with IgG1 (mr-mct). This enrichment of MR-OMCT cells was associated with a severalfold increase in adenosine 3',5' cyclic monophosphate (cAMP) production in response to isoproterenol and an attenuated increase in cAMP production to vasopressin. In summary, we report the isolation of highly enriched populations of MR cells from the OM using two-stage solid-phase immunoadsorption. This approach should provide a useful and convenient method for further investigations of the physiological role of these poorly understood tubular cells.

Animals↗
PubMed1988

Immunodissection of cortical and medullary thick ascending limb cells from rabbit kidney.

A procedure was developed for isolating thick ascending limb cells from either the outer medulla or the inner cortex from rabbit kidneys. Dispersed cells derived from the medulla or cortex were incubated with goat anti-human uromucoid (Tamm-Horsfall glycoprotein) serum, washed, and applied to culture dishes coated with affinity-purified anti-goat immunoglobulin G. Nonadherent cells were removed by washing. Routinely, 10(6) or 7 X 10(4) adherent cells were obtained per gram of rabbit outer medulla or inner cortex, respectively. Greater than 97% of the adherent cells stained for Tamm-Horsfall antigen, and examination of freshly isolated cells by transmission electron microscopy established that they had morphological properties expected for thick limb cells. Freshly isolated medullary thick limb (MTALH) cells consistently accumulated cAMP in response to arginine vasopressin (AVP), thyrocalcitonin, prostaglandin E2 (PGE2), and glucagon. PGE2, thyrocalcitonin, parathyroid hormone, and AVP, but not isoproterenol or glucagon, reproducibly stimulated cAMP accumulation in freshly isolated cortical thick limb (CTALH) cells. MTALH cells produced immunoreactive PGE2 when incubated with 10 microM arachidonic acid. In summary, large numbers of highly purified and hormonally responsive rabbit MTALH and CTALH cells can be obtained by immunodissection using commercially available antibody preparations. Because the Tamm-Horsfall antigen is present as an extracellular determinant on thick ascending limb epithelia from many species, this general approach likely can be used to isolate CTALH and MTALH cells from most mammalian kidneys.

Animals↗
PubMed1987

Vasopressin increases cytosolic free calcium in LLC-PK1 cells through a V1-receptor.

We examined the effects of arginine vasopressin (AVP), parathyroid hormone (PTH), and bradykinin (BK) on the cytosolic free calcium concentration ([Ca]i) in cultured LLC-PK1 and MDCK kidney cell lines by use of the fluorescent Ca chelator fura-2. In LLC-PK1 cells, the addition of AVP but not [1-desamino-8-D-arginine]vasopressin (dDAVP, V2 agonist), PTH, or BK (10(-6) M) caused a significant increase in [Ca]i. The AVP-induced increase in [Ca]i from 61 +/- 6 to 225 +/- 44 nM (n = 7, P less than 0.01) was rapid and transient, returning to base line in 2 to 3 min. The effect of AVP was dose dependent and was present at 1 (61% increase) but not 5 min after extracellular Ca was removed. The effect of 10(-6) M AVP could be blocked with the pressor (V1) antagonist, d(CH2)5Tyr(Me)AVP, but not dDAVP. In MDCK cells, BK, but not AVP and PTH, increased [Ca]i from 146 +/- 11 to 281 +/- 31 nM (n = 9, P less than 0.001). The removal of extracellular Ca (5 min), reduced but did not abolish this effect. These results indicate that [Ca]i mobilized by activation of V1-receptors may mediate AVP-regulated function in some transporting epithelia.

Arginine Vasopressin↗
PubMed1987

A1 and A2 adenosine receptors in rabbit cortical collecting tubule cells. Modulation of hormone-stimulated cAMP.

Adenosine analogs were used to investigate the cellular mechanisms by which adenosine may alter renal tubular function. Cultured rabbit cortical collecting tubule (RCCT) cells, isolated by immunodissection, were treated with 5'-N-ethylcarboxamideadenosine (NECA), N6-cyclohexyladenosine (CHA), and R-N6-phenylisopropyladenosine (PIA). All three analogs produced both dose-dependent inhibition and stimulation of RCCT cell cyclic AMP (cAMP) production. Stimulation of cAMP accumulation occurred at analog concentrations of 0.1 microM to 100 microM with the rank order of potency NECA greater than PIA greater than CHA. Inhibition occurred at concentrations of 1 nM to 1 microM with the rank order of potency CHA greater than PIA greater than NECA. These effects on cAMP production were inhibited by 1,3-diethyl-8-phenylxanthine and isobutylmethylxanthine. CHA (50 nM) blunted AVP- and isoproterenol-stimulated cAMP accumulation. This modulation of hormone-induced cAMP production was abolished by pretreatment of RCCT cells with pertussis toxin. Prostaglandin E2 production was unaffected by 0.1 mM CHA. These findings indicate the presence of both inhibitory (A1) and stimulatory (A2) receptors for adenosine in RCCT cells. Moreover, occupancy of the A1 receptor causes inhibition of both basal and hormone-stimulated cAMP formation through an action on the inhibitory guanine nucleotide-binding regulatory component, Ni, of the adenylate cyclase system.

1-Methyl-3-isobutylxanthine↗
PubMed1987

Role for intrarenal adenosine in the renal hemodynamic response to contrast media.

The intrarenal injection of contrast media results in a transient fall in renal blood flow (RBF) and a decrease in glomerular filtration rate (GFR). These effects are enhanced by dietary sodium restriction and attenuated by sodium loading. A similar sodium-dependent response of RBF and GFR occurs with the intrarenal injection of adenosine. In view of these similarities, we sought to determine whether endogenous adenosine is involved in the renal hemodynamic response to contrast media. The intrarenal injection of contrast media (meglumine-Na diatrizoate, 76%) in six sodium-depleted, anesthetized dogs resulted in a 17% +/- 4% decrease in RBF and a 31% +/- 5% decrease in GFR. The infusion of the adenosine receptor antagonist, theophylline (5 mumol/min), reduced the decrease in RBF to 6% +/- 2% and in GFR to 12% +/- 3% of control values. During the intrarenal infusion of dipyridamole (24 micrograms/kg/min), a potentiator of adenosine through its action to inhibit the cellular uptake of nucleosides, the hemodynamic response to contrast media was greater; RBF fell 25% +/- 4% and GFR fell 44% +/- 7%. In addition, the urinary excretion of endogenous adenosine increased after the injection of contrast media (388 +/- 79 vs. 830 +/- 231 nmol/min). In summary, the contrast media-induced fall in RBF and GFR was attenuated by theophylline and augmented by dipyridamole, and the administration of contrast media resulted in an increase in the excretion of endogenous adenosine. These results support the hypothesis that endogenous adenosine is involved in the renal hemodynamic response to contrast media.

Adenosine↗
PubMed1986

Elevation of intrarenal adenosine by maleic acid decreases GFR and renin release.

Maleic acid administration produces a defect in tubular reabsorption resembling that seen in the Fanconi syndrome and also causes a decrease in glomerular filtration rate (GFR). The mechanism by which maleic acid alters renal function is uncertain, though the tubular defect is known to be associated with decreased ATP levels. Because of this alteration in nucleotide metabolism the present study was undertaken to determine the role of elevated endogenous adenosine in mediating the maleic acid-induced changes in renal function. Since the renal effects of exogenous adenosine are enhanced by sodium-depletion and attenuated by sodium-loading, the present study compared the time course of the effects of maleic acid on renal function in 10 dogs maintained on a low sodium diet, and 10 dogs maintained on a high sodium diet. In addition, we examined the effect of maleic acid on adenosine levels in renal venous plasma, on the urinary excretion of adenosine, and the effect of the adenosine antagonist, theophylline, on the maleic acid-induced changes in renal function. After 100 min of maleic acid, GFR was decreased significantly by 55 +/- 4% of control in the sodium-depleted dogs, and by 39 +/- 4% of control in the sodium-loaded dogs. In the sodium-depleted dogs, renin release was also significantly depressed (12 +/- 8% of control) during the infusion of maleic acid. The fractional excretion of sodium was significantly increased in both groups. The renal venous concentration of adenosine and the urinary excretion of adenosine were both significantly increased during maleic acid.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine↗
PubMed1986

Immunodissection and culture of rabbit cortical collecting tubule cells.

A mouse monoclonal antibody designated IgG3(rct-30) has been prepared that reacts specifically with an antigen on the surface of all cells comprising the cortical and medullary rabbit renal collecting tubule including the arcades. Plastic culture dishes coated with IgG3(rct-30) were used to isolate collecting tubule cells from collagenase dispersions of rabbit renal cortical cells by immunoadsorption. Typically, 10(6) rabbit cortical collecting tubule (RCCT) cells were obtained from 5 g of renal cortex (2 kidneys). Initial purity was greater than 96% based on immunocytofluorescent staining with three different anti-collecting tubule antibodies. Between 20 and 30% of the RCCT cells were reactive with peanut lectin suggesting that RCCT cells are a mixture of principal and intercalated cells. Approximately 10(7) RCCT cells were obtained after 4 to 5 days in primary culture. Moreover, RCCT cells continued to proliferate after passaging with a doubling time of approximately 32 h. RCCT cells passaged once and then cultured 4-5 days were found 1) to synthesize cAMP in response to arginine vasopressin (AVP), prostaglandin E2 (PGE2), isoproterenol, and parathyroid hormone, but not calcitonin, prostaglandin D2, or prostaglandin I, and 2) to release PGE2 in response to bradykinin but not arginine vasopressin or isoproterenol. Our results indicate that cultured RCCT cells retain many of the hormonal, histochemical, and morphological properties expected for a mixture of principal and intercalated rabbit cortical collecting tubule epithelia. RCCT cells should prove useful both for studying hormonal interactions in the cortical collecting tubule and as a starting population for isolating intercalated collecting tubule epithelia.

Animals↗