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Background: There is evidence that macula densa nitric oxide (NO) inhibits tubuloglomerular feedback (TGF). However, TGF response is not altered in mice deficient in neuronal nitric oxide synthase (nNOS) (-/-). Furthermore, nNOS expression in the macula densa is inversely related to salt intake, yet micropuncture studies have shown that NOS inhibition potentiates TGF in rats on high sodium intake but not in rats on a low-salt diet. These inconsistencies may be due to confounding systemic factors, such as changes in circulating renin. To further clarify the role of macula densa nNOS in TGF response, independent of systemic factors, we tested the hypothesis that (1) TGF response is inversely related to sodium intake, and (2) during low sodium intake, NO produced by macula densa nNOS tonically controls the basal diameter of the afferent arteriole (Af-Art).Methods: Af-Arts and attached macula densas were simultaneously microperfused in vitro. TGF response was determined by measuring Af-Art diameter before and after increasing NaCl in the macula densa perfusate. TGF response was studied in wild-type (+/+) and nNOS knockout mice (-/-), as well as in juxtaglomerular apparatuses (JGAs) from rabbits fed a low-, normal-, or high-NaCl diet.Results: TGF responses were similar in nNOS +/+ and -/- mice. However, in nNOS +/+ mice, 7-nitroindazole (7-NI) perfused into the macula densa significantly potentiated the TGF response (P = 0.001), while in nNOS -/- mice, this potentiation was absent. In rabbits on three different sodium diets, TGF responses were similar and were potentiated equally by 7-NI. However, in JGAs from rabbits on a low-NaCl diet, adding 7-NI to the macula densa while perfusing it with low-NaCl fluid caused Af-Art vasoconstriction, decreasing the diameter by 14% (from 21.7 +/- 1.3 to 18.6 +/- 1.5 microm; P < 0.001). This effect was not observed in JGAs from rabbits fed a normal- (19.0 +/- 0.5 vs. 19.3 +/- 0.8 microm after 7-NI) or high-NaCl diet (18.6 +/- 0.7 vs. 18.4 +/- 0.7 microm).Conclusions: First, in this in vitro preparation, chronic changes in macula densa nNOS do not play a major role in the regulation of TGF. Compensatory mechanisms may develop during chronic alteration of nNOS that keep TGF relatively constant. Second, nNOS regulates TGF response acutely. Third, the results obtained in the +/+ and -/- mice also confirm that the effect of 7-NI is due to inhibition of macula densa nNOS. Finally, during low sodium intake (without induction of TGF), the regulation of basal Af-Art resistance by macula densa nNOS suggests that NO in the macula densa helps maintain renal blood flow during the high renin secretion caused by low sodium intake. [ABSTRACT FROM AUTHOR]

Background: Sulfonylurea receptors are classified as either high-affinity type 1 (SUR1) or low-affinity type 2 receptors (SUR2), and the gene expression of SURs has recently been demonstrated in kidney. However, functional data regarding a renal SUR are lacking. We previously demonstrated that mesangial cell (MC) gene and protein expression of extracellular matrix components were up-regulated by the sulfonylurea, tolazamide. After noting this biological response, we next sought to investigate the presence of a sulfonylurea receptor in rat MCs.Methods: Equilibrium binding studies employing [3H]glibenclamide as a ligand were performed on crude MC membrane preparations. Gene expression for SUR was explored by Northern analysis of cultured MCs and whole kidney tissue. The effect of sulfonylurea on intracellular Ca2+ in MCs was assayed by spectrofluorometry, and glibenclamide-induced changes in the contractility of MCs were assessed.Results: MCs bound [3H]glibenclamide with a KD of 2.6 microM and a Bmax of 30.4 pmol/mg protein as determined by Scatchard analysis. Three SUR2 transcripts were detected in MCs. A major transcript was detected at 5.5 kb and minor transcripts at 7.5 and 8.6 kb. Following sulfonylurea treatment of MCs, real-time videomicroscopy revealed intense MC contraction, coinciding with oscillatory increments of intracellular Ca2+ concentration. Further evidence of sulfonylurea-induced MC contraction was demonstrated by glibenclamide-induced deformation of a silicone rubber substrate.Conclusions: These results demonstrate that SUR2 resides on MCs. Functional activation of this receptor by sulfonylurea induces Ca2+ transients that result in MC contraction. [ABSTRACT FROM AUTHOR]

The macula densa expresses a luminal Na(+)-K(+)-2Cl(-) cotransporter and a basolateral Cl(-) conductance. Although it is known that cotransport of Na(+), K(+), and Cl(-) is the first step in tubuloglomerular feedback (TGF), subsequent steps are unclear. We hypothesized that Na(+)-K(+)-2Cl(-) entry via the luminal Na(+)-K(+)-2Cl(-) cotransporter elevates intracellular Cl(-), increases electrogenic Cl(-) efflux across the basolateral membrane, and depolarizes the macula densa, initiating TGF. We perfused afferent arterioles with macula densa attached. The macula densa was perfused with solutions containing either 5 mM Na(+) and 3 mM Cl(-) (low NaCl) or 80 mM Na(+) and 77 mM Cl(-) (high NaCl). When the macula densa perfusate was changed from low to high NaCl, afferent arteriole diameter decreased from 15.8 +/- 0.8 to 13.1 +/- 0.7 mm (P < 0.05). Adding 10 microM furosemide to the macula densa lumen blocked TGF. When nystatin, a group I cation ionophore, was added to the macula densa lumen together with furosemide in the presence of low NaCl, it induced TGF (from 18.0 +/- 1.5 to 15.6 +/- 1.6 mm; P = 0.003). When valinomycin, a K(+)-selective ionophore, was added to the macula densa lumen together with furosemide in the presence of low NaCl containing 5 mM K(+), it did not induce TGF. Subsequent addition of 50 mM KCl to the macula densa perfusate induced TGF (from 21.7 +/- 0.8 to 17.5 +/- 1.3 mm; P = 0.0047; n = 6). Adding 50 mM KCl without valinomycin did not induce TGF. When 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB; 1 microM), a Cl(-) channel blocker, was added to the bath, it blocked TGF induced by high NaCl, but did not block TGF induced by valinomycin plus 50 mM KCl. NPPB did not alter afferent arteriole constriction induced by norepinephrine. We concluded that increased NaCl in the lumen of the macula densa leads to influx of Cl(-) via the Na(+)-K(+)-2Cl(-) cotransporter. The accelerated transport increases intracellular Cl(-). The subsequent exit of Cl(-) across the basolateral membrane via Cl( -) channels in turn leads to depolarization of the macula densa and thereby induces TGF.

We have reported that nitric oxide (NO) inhibits thick ascending limb (THAL) chloride absorption (J(Cl(-))). NaCl transport in the THAL depends on apical Na(+)-K(+)-2Cl(-) cotransporters, apical K(+) channels, and basolateral Na(+)-K(+)-ATPase. However, the transporter inhibited by NO is unknown. We hypothesized that NO decreases THAL J(Cl(-)) by inhibiting the Na(+)-K(+)-2Cl(-) cotransporter. THALs from Sprague-Dawley rats were isolated and perfused. Intracellular sodium ([Na(+)](i)) and chloride concentrations ([Cl(-)](i)) were measured with sodium green and SPQ, respectively. The NO donor spermine NONOate (SPM) decreased [Na(+)](i) from 13.5 +/- 1.2 to 9.6 +/- 1.6 mM (P < 0.05) and also decreased [Cl(-)](i) (P < 0.01). We next tested whether NO decreases Na(+)-K(+)-2Cl(-) cotransporter activity by measuring the initial rate of Na(+) transport. In the presence of SPM in the bath, initial rates of Na(+) entry were 49.6 +/- 6.0% slower compared with control rates (P < 0.05). To determine whether NO inhibits apical K(+) channel activity, we measured the change in membrane potential caused by an increase in luminal K(+) from 1 to 25 mM using a potential-sensitive fluorescent dye. In the presence of SPM, increasing luminal K(+) concentration depolarized THALs to the same extent as it did in control tubules. We then tested whether a change in apical K(+) permeability could affect NO-induced inhibition of THAL J(Cl(-)). In the presence of luminal valinomycin, which increases K(+) permeability, addition of SPM decreased THAL J(Cl(-)) by 41.2 +/- 10.4%, not significantly different from the inhibition observed in control tubules. We finally tested whether NO alters the affinity or maximal rate of Na(+)-K(+)-ATPase by measuring oxygen consumption rate (QO(2)) in THAL suspensions in the presence of nystatin in varying concentrations of Na(+). In the presence of 10.5 mM Na(+), nystatin increased QO(2) to 119.1 +/- 19.2 and 125.6 +/- 23.4 nmol O(2). mg protein(-1). min(-1) in SPM- and furosemide-treated tubules, respectively. In the presence of 145 mM extracellular Na(+), nystatin increased QO(2) by 104 +/- 7 and 94 +/- 20% in NO- and furosemide-treated tubules, respectively. We concluded that NO decreases THAL J(Cl(-)) by inhibiting Na(+)-K(+)-2Cl(-) cotransport rather than inhibiting apical K(+) channels or the sodium pump.

Background: Recent studies have revealed angiotensin II subtype 1 (AT1) receptors on macula densa cells, raising the possibility that angiotensin II (Ang II) could enhance tubuloglomerular feedback (TGF) by affecting macula densa cell function. We hypothesized that Ang II enhances TGF via activation of AT1 receptors on the luminal membrane of the macula densa., Methods: Rabbit afferent arterioles and the attached macula densa were simultaneously microperfused in vitro, keeping pressure in the afferent arteriole at 60 mm Hg., Results: The afferent arteriole diameter was measured while the macula densa was perfused with low NaCl (Na+, 5 mmol/L; Cl-, 3 mmol/L) and then with high NaCl (Na+, 79 mmol/L; Cl-, 77 mmol/L) to induce a TGF response. When TGF was induced in the absence of Ang II, the afferent arteriole diameter decreased by 2.4 +/- 0.5 microm (from 17.3 +/- 1.0 to 14.9 +/- 1.2 microm). With Ang II (0.1 nmol/L) present in the lumen of the macula densa, the diameter decreased by 3.8 +/- 0.7 microm (from 17.3 +/- 1.0 to 13.5 +/- 1.2 microm, P < 0.05 vs. TGF with no Ang II, N = 8). To test whether Ang II enhances TGF via activation of AT1 receptors on the luminal membrane of the macula densa, Ang II plus losartan (1 micromol/L) was added to the lumen. Losartan itself did not alter TGF. When TGF was induced in the absence of Ang II and losartan, the afferent arteriole diameter decreased by 2.3 +/- 0.3 microm (from 15.9 +/- 1.0 to 13.6 +/- 1.2 microm). When Ang II and losartan were both present in the macula densa perfusate, the diameter decreased by 2.4 +/- 0.4 microm (from 15.8 +/- 0.9 to 13.4 +/- 0.7 microm, P> 0.8 vs. TGF with no Ang II and losartan, N = 7). We then examined whether AT2 receptors on the macula densa influence the effect of luminal Ang II on TGF. When TGF was induced in the absence of Ang II plus PD 0123319-0121B (1 micromol/L), the afferent arteriole diameter decreased by 2.4 +/- 0.2 microm (from 17.0 +/- 0.9 to 14.6 +/- 0.8 microm). When Ang II and PD 0123319-0121B were both present in the macula densa lumen, the diameter decreased by 3.9 +/- 0.2 microm (from 16.8 +/- 0.9 to 12.9 +/- 0.9 microm, P < 0.001 vs. TGF with no Ang II and PD 0123319-0121B, N = 8). PD 0123319-0121B itself had no effect on TGF. To assure that this effect of Ang II was not due to leakage into the bath, losartan was added to the bath. When TGF was induced in the absence of Ang II with losartan in the bath, the afferent arteriole diameter decreased by 2.8 +/- 0.5 microm (from 19.3 +/- 1.2 to 16.5 +/- 0.8 microm). After Ang II was added to the macula densa perfusate and losartan to the bath, the diameter decreased by 4.0 +/- 0.7 microm (from 18.9 +/- 1.1 to 14.9 +/- 0.5 microm, P < 0.01 vs. TGF with no Ang II in the lumen and losartan in the bath, N = 8)., Conclusions: These results demonstrate that Ang II enhances TGF via activation of AT1 receptors on the luminal membrane of the macula densa.

Stimulation of alpha(2)-adrenergic receptors inhibits transport in various nephron segments, and the thick ascending limb of the loop of Henle (THAL) expresses alpha(2)-receptors. We hypothesized that selective alpha(2)-receptor activation decreases NaCl absorption by cortical THALs through activation of NOS and increased production of NO. We found that the alpha(2)-receptor agonist clonidine (10 nM) decreased chloride flux (J(Cl)) from 119.5 +/- 15.9 to 67.4 +/- 13.8 pmol. mm(-1). min(-1) (43% reduction; P < 0.02), whereas removal of clonidine from the bath increased J(Cl) by 20%. When NOS activity was inhibited by pretreatment with 5 mM N(G)-nitro-L-arginine methyl ester, the inhibitory effects of clonidine on THAL J(Cl) were prevented (81.7 +/- 10.8 vs. 71.6 +/- 6.9 pmol. mm(-1). min(-1)). Similarly, when the NOS substrate L-arginine was deleted from the bath, addition of clonidine did not decrease THAL J(Cl) from control (106.9 +/- 11.6 vs. 132.2 +/- 21.3 pmol. mm(-1). min(-1)). When we blocked the alpha(2)-receptors with rauwolscine (1 microM), we found that the inhibitory effect of 10 nM clonidine on THAL J(Cl) was abolished, verifying that alpha(2), rather than I(1), receptors mediate the effects of clonidine in the THAL. We investigated the mechanism of NOS activation and found that intracellular calcium concentration did not increase in response to clonidine, whereas pretreatment with 150 nM wortmannin abolished the clonidine-mediated inhibition of THAL J(Cl), indicating activation of phosphatidylinositol 3-kinase and the Akt pathway. We found that pretreatment of THALs with 10 microM LY-83583, an inhibitor of soluble guanylate cyclase, blocked clonidine-mediated inhibition of THAL J(Cl). In conclusion, alpha(2)-receptor stimulation decreases THAL J(Cl) by increasing NO release and stimulating guanylate cyclase. These data suggest that alpha(2)-receptors act as physiological regulators of THAL NO synthesis, thus inhibiting chloride transport and participating in the natriuretic and diuretic effects of clonidine in vivo.

ANG II increases fluid absorption in proximal tubules from young rats more than those from adult rats. ANG II increases fluid absorption in the proximal nephron, in part, via activation of protein kinase C (PKC). However, it is unclear how age-related changes in ANG II-induced stimulation of the PKC cascade differ as an animal matures. We hypothesized that the response of the proximal nephron to ANG II decreases as rats mature due to a reduction in the amount and activation of PKC rather than a decrease in the number or affinity of ANG II receptors. Because PKC translocates from the cytosol to the membrane when activated, we first measured PKC activity in the soluble and particulate fractions of proximal tubule homogenates exposed to vehicle or 10(-10) M ANG II from young (26 +/- 1 days old) and adult rats (54 +/- 1 days old). ANG II increased PKC activity to the same extent in homogenates from young rats (from 0.119 +/- 0.017 to 0.146 +/- 0.015 U/mg protein) (P < 0.01) and adult rats (from 0.123 +/- 0.020 to 0.156 +/- 0.023 U/mg protein) (P < 0.01). Total PKC activity did not differ between groups (0.166 +/- 0.018 vs. 0.181 +/- 0.023). We next investigated whether activation of the alpha-, beta-, and gamma-PKC isoforms differed by Western blot. In homogenates from young rats, ANG II significantly increased activated PKC-alpha from 40.2 +/- 6.5 to 60.2 +/- 9.5 arbitrary units (AU) (P < 0.01) but had no effect in adult rats (46.1 +/- 5.1 vs. 48.5 +/- 8.2 AU). Similarly, ANG II increased activated PKC-gamma in proximal tubules from young rats from 47.9 +/- 13.2 to 65.6 +/- 16.7 AU (P < 0.01) but caused no change in adult rats. Activated PKC-beta, however, increased significantly in homogenates from both age groups. Specifically, activated PKC-beta increased from 8.6 +/- 1.4 to 12.2 +/- 2.1 AU (P < 0.01) in homogenates from nine young rats and from 19.0 +/- 5.5 to 25.1 +/- 7.1 AU (P < 0.01) in homogenates from 12 adult rats. ANG II did not alter the amount of soluble PKC-alpha, -beta, and -gamma significantly. The total amount of PKC-alpha and -gamma did not differ between homogenates from young and adult rats, whereas the total amount of PKC-beta was 59.7 +/- 10.7 and 144.9 +/- 41.8 AU taken from young and adult rats, respectively (P < 0.05). Maximum specific binding and affinity of ANG II receptors were not significantly different between young and adult rats. We concluded that the primary PKC isoform activated by ANG II changes during maturation.

Isolation and perfusion of single nephron segments was first described by Burg et al. (1) in 1966. This technique has allowed us to study both transepithelial and transmembrane transport in individual nephron segments, including the proximal tubule, under carefully controlled circumstances. This has obvious advantages for experiments that require the epithelium and its junctional complexes to remain intact, but removed from the influences of endogenous neural or humoral mediators, including angiotensins. In addition, control of bath and luminal perfusate compositions and flow rates allows peptides to be added independently to each side of the epithelium. This has particular relevance in the case of angiotensin, which binds to luminal and basolateral receptors and initiates intracellular signal transduction pathways that may differ between the two sites (2).

Background: Afferent and efferent arteriole resistance exerts critical and opposite actions in the regulation of glomerular capillary pressure (PGC) and glomerular filtration rate (GFR). Tubuloglomerular feedback (TGF) plays an important role in the regulation of afferent arteriole resistance; however, the role of TGF in the regulation of efferent arteriole resistance is less well established. We hypothesized that TGF caused by increased NaCl in the tubular fluid stimulates the macula densa to initiate a cascade of events resulting in efferent arteriole vasodilation, mediated by adenosine via its A2 receptor., Methods: Rabbit efferent arterioles and adherent tubular segments with macula densa were simultaneously microperfused in vitro while changing NaCl concentration at the macula densa. To study whether autacoids produced by the glomerulus participate in the effect of TGF on efferent arterioles, they were perfused orthograde or retrograde. To eliminate the hemodynamic influence of the afferent arteriole during orthograde perfusion, the perfusion pipette was advanced to the distal end of the afferent arteriole, and the tip of the pressure pipette was placed beyond the afferent arteriole; for retrograde perfusion, the efferent arteriole was perfused from its distal end., Results: In efferent arterioles perfused orthograde and preconstricted with norepinephrine (NE), increasing NaCl concentration at the macula densa increased the diameter by 33%. In preconstricted efferent arterioles perfused retrograde, increasing NaCl at the macula densa increased the diameter by 33%. Efferent arteriole vasodilation was completely blocked by a selective adenosine A2 receptor antagonist (3, 7-dimethyl-1-propargylxanthine) but not by an adenosine A1 receptor antagonist (FK838)., Conclusions: Our data show that in vitro, preconstricted efferent arterioles dilate in response to increased macula densa NaCl, and this process is mediated by activation of adenosine A2 receptors. Thus, TGF changes efferent arteriole resistance in the opposite direction from the afferent arteriole, possibly amplifying TGF regulation of PGC and GFR. In vivo efferent arteriole TGF may only buffer the signals that cause efferent arteriole resistance to parallel changes in afferent arteriole resistance. Effects of TGF on efferent arterioles perfused orthograde or retrograde were similar, suggesting that glomerular autacoids do not participate in this process.

Background: In vivo and in vitro studies have shown that nitric oxide (NO) is an important modulator of transport processes along the nephron. The thick ascending limb (TAL) plays a significant role in the urine-concentrating mechanism and in the maintenance of acid/base balance., Methods: TALs from male Sprague-Dawley rats were isolated and perfused, and net bicarbonate flux (J(HCO3)(-) was determined., Results: In perfused TALs, 0.5 mmol/L L-arginine (L-Arg), the substrate for NO synthase, significantly lowered J(HCO3)(-) from 35.4 +/- 4.6 to 23.2 +/- 2.9 pmol. mm(-1). min(-1), a decrease of 36.9 +/- 11.6% (P < 0.025). D-Arg (0.5 mmol/L) had no effect on J(HCO3)(-) (N = 7). In the presence of 5 mmol/L L-NAME, an NO synthase (NOS) inhibitor, the addition of L-Arg did not affect TAL J(HCO3)(-) (43.4 +/- 4.4 vs. 44.6 +/- 5.0 pmol. mm(-1). min(-1)). L-NAME alone (5 mmol/L) did not affect TAL J(HCO3)(-). After removing L-Arg from the bath, J(HCO3)(-) increased from 26.2 +/- 3.9 to 34.8 +/- 3.2 pmol. mm(-1). min(-1) (P < 0.01), indicating no cytotoxicity of NO. We next investigated the effect of cGMP analogues on TAL J(HCO3)(-). 8-Br-cGMP (50 micromol/L) and db-cGMP (50 micromol/L) significantly decreased J(HCO3)(-) by 26.3 +/- 9.1% and 35.1 +/- 11.6%, respectively. In the presence of cGMP (50 micromol/L), the addition of L-Arg had no effect on J(HCO3)(-). In the presence of KT-5823 (2 mircromol/L), a protein kinase G inhibitor, the addition of L-Arg did not change TAL J(HCO3)(-) (N = 5)., Conclusions: We conclude that (1) endogenously produced NO inhibits TAL J(HCO3)(-) in an autocrine manner, (2) cGMP mediates all the effects of NO, and (3) this effect is mediated by protein kinase G activation.

Background: Previous studies have suggested that nitric oxide (NO) produced within cells of the macula densa (MD) modulates tubuloglomerular feedback (TGF). We tested the hypothesis that NO produced in the MD acts locally as an autacoid to activate soluble guanylate cyclase and cGMP-dependent protein kinase in the MD itself., Methods: Rabbit afferent arterioles (Af-Arts) and attached MD were simultaneously microperfused in vitro. The TGF response was determined by measuring the Af-Art diameter before and after increasing NaCl in the MD perfusate (from 17 mmol/L of Na and 2 of Cl to 65 mmol/L of Na and 50 of Cl). TGF was studied before (control TGF) and after inhibiting components of the NO-cGMP-dependent cascade in the tubular or vascular compartment., Results: Increasing NaCl concentration in the MD perfusate decreased the Af-Art diameter by 3.2 +/- 0.5 microm (from 18.5 +/- 1.3 to 15.4 +/- 1.3 microm, P < 0.001). Adding a soluble guanylate cyclase inhibitor (LY83583) to the MD increased TGF response to 6.3 +/- 1.1 microm (P < 0.031 vs. control TGF). Similarly, when cGMP-dependent protein kinase was inhibited with KT5823, TGF was augmented from 2.6 +/- 0.3 to 4.0 +/- 0.7 microm (P < 0.023). An analogue of cGMP in the MD reversed the TGF-potentiating effect of both 7-nitroindazole (7NI; an nNOS inhibitor) and LY83583. Inhibition of MD guanylate cyclase did not alter the effect of acetylcholine (a NO-cGMP-dependent vasodilator) on the Af-Art. Perfusing the Af-Art with the guanylate cyclase inhibitor did not potentiate TGF, suggesting that the effect of NO occurred at the MD via a cGMP-dependent mechanism. To determine whether the effect of NO in the MD was entirely mediated by cGMP, TGF was studied after giving (1) LY83583 or (2) LY83583 plus 7NI. Adding the nNOS inhibitor to the MD did not potentiate the TGF response further., Conclusions: We concluded the following: (1) NO produced by the MD inhibits TGF via stimulation of soluble guanylate cyclase, generating cGMP and activating cGMP-dependent protein kinase; (2) NO acts on the MD itself rather than by diffusing to the Af-Art; and (3) most, if not all, of the effect of NO in the MD is due to a cGMP-dependent mechanism rather than to other NO mediators.

Endothelin-1 (ET-1) inhibits transport in various nephron segments, and the thick ascending limb of the loop of Henle (TALH) expresses ET-1 receptors. In many tissues, activation of ET(B) receptors stimulates release of NO, and we recently reported that endogenous NO inhibits TALH chloride flux (J(Cl)). However, the relationship between ET-1 and NO in the control of nephron transport has not been extensively studied. We hypothesized that ET-1 decreases NaCl transport by cortical TALHs through activation of ET(B) receptors and release of NO. Exogenous ET-1 (1 nM) decreased J(Cl) from 118.3 +/- 15.0 to 62.7 +/- 13.6 pmol. mm(-1). min(-1) (48.3 +/- 8.2% reduction), whereas removal of ET-1 increased J(Cl) in a separate group of tubules from 87.6 +/- 10.7 to 115.2 +/- 10.3 pmol. mm(-1). min(-1) (34.5 +/- 6.2% increase). To determine whether NO mediates the inhibitory effects of ET-1 on J(Cl), we examined the effect of inhibiting of NO synthase (NOS) with N(G)-nitro-L-arginine methyl ester (L-NAME) on ET-1-induced changes in J(Cl). L-NAME (5 mM) completely prevented the ET-1-induced reduction in J(Cl), whereas D-NAME did not. L-NAME alone had no effect on J(Cl). These data suggest that the effects of ET-1 are mediated by NO. Blockade of ET(B) receptors with BQ-788 prevented the inhibitory effects of 1 nM ET-1. Activation of ET(B) receptors with sarafotoxin S6c mimicked the inhibitory effect of ET-1 on J(Cl) (from 120.7 +/- 12.6 to 75.4 +/- 13.3 pmol. mm(-1). min(-1)). In contrast, ET(A) receptor antagonism with BQ-610 did not prevent ET-1-mediated inhibition of TALH J(Cl) (from 96.5 +/- 10.4 to 69.5 +/- 8.6 pmol. mm(-1). min(-1)). Endothelin increased intracellular calcium from 96.9 +/- 14.0 to 191.4 +/- 11.9 nM, an increase of 110.8 +/- 26.1%. We conclude that exogenous endothelin indirectly decreases TALH J(Cl) by activating ET(B) receptors, increasing intracellular calcium concentration, and stimulating NO release. These data suggest that endothelin acts as a physiological regulator of TALH NO synthesis, thus inhibiting chloride transport and contributing to the natriuretic effects of ET-1 observed in vivo.

Nitric oxide (NO) inhibits transport in various nephron segments, and the thick ascending limb (TAL) expresses nitric oxide synthase (NOS). However, the effects of NO on TAL transport have not been extensively studied. We tested the hypothesis that NO inhibits apical and basolateral Na(+)/H(+) exchange by the TAL by measuring intracellular pH (pH(i)) of isolated, perfused rat TALs using the fluorescent dye 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF). The NO donor spermine NONOate (SPM, 10 microM) decreased steady-state pHi in medullary TALs from 7.18 +/- 0.13 to 7.13 +/- 0.14 (P < 0.02), whereas controls did not decrease significantly. We next measured the buffering capacity of medullary TALs and the rate at which they recovered from acid loads to investigate the mechanism whereby NO reduces steady-state pHi. SPM decreased H+ flux (JH) from 2.41 +/- 0.66 to 0.97 +/- 0.19 pmol. min(-1). mm(-1), 55%. To assure that the decrease in JH was due to NO, another donor, nitroglycerin (NTG; 10 microM), was used. NTG decreased J(H) from 1.65 +/- 0.11 to 1.07 +/- 0.24 pmol. min(-1). mm(-1), 37%. To determine the relative contributions of the apical and basolateral Na+/H+ exchangers, 5-(N,N-dimethyl)amiloride (DMA; 100 microM) was added to either bath or lumen. With DMA added to the bath, SPM decreased J(H) from 4.78 +/- 1.08 to 2.74 +/- 0.54 pmol. min(-1). mm(-1), an inhibition of 41%; and with DMA added to the lumen, SPM decreased J(H) from 2.31 +/- 0.29 to 1.74 +/- 0.27 pmol. min(-1). mm(-1), a reduction of 26%. Addition of DMA alone to both bath and lumen resulted in an 87% inhibition of JH. We conclude that NO inhibits both apical and basolateral Na+/H+ exchangers and consequently may play an important role in regulating pHi and may alter acid/base balance by directly affecting bicarbonate absorption in the TAL.

The factor responsible for salt sensitivity of blood pressure in Dahl rats is unclear but presumably resides in the kidney. We tested the hypotheses that (1) thick ascending limbs of Dahl salt-sensitive rats (DS) absorb more NaCl than those of Dahl salt-resistant rats (DR) and (2) NO inhibits transport to a lesser extent in thick ascending limbs from DS. We found that basal chloride absorption (J(Cl)) by thick ascending limbs from DR was 105.8+/-10.0 pmol. mm(-1). min(-1) (n=6). Ten and 100 micromol/L spermine NONOate, an NO donor, decreased J(Cl) in DR to 65.8+/-8.5 and 46.8+/-7.0 pmol. mm(-1). min(-1), respectively. Basal J(Cl) in DS was 131.6+/-13.4 pmol. mm(-1). min(-1) (n=7). In DS, 10 and 100 micromol/L spermine NONOate decreased J(Cl) to 111.5+/-12.8 and 46.8+/-6.2 pmol. mm(-1). min(-1), respectively. No difference was observed in basal or NO-inhibited Na absorption by cortical collecting ducts or in basal or NO-inhibited oxygen consumption by inner medullary collecting ducts. Because NO acts via generation of cGMP, we measured cGMP production by thick ascending limbs from DS and DR to see whether a difference in cGMP production could account for the difference in basal or NO-inhibited transport. Basal rates of cGMP production were similar between the 2 strains. Although NO increased cGMP production by thick ascending limbs from both strains, no difference existed between DS and DR. We concluded that the reduced ability of NO to block transport in thick ascending limbs in DS may account for at least part of the salt sensitivity of blood pressure in this strain.

1. Nitric oxide (NO) is produced and/or regulates transport in many segments of the nephron, including the proximal convoluted tubule, proximal straight tubule, thick ascending limb, cortical collecting duct and inner medullary collecting duct. 2. Endothelin (ET) is produced and/or regulates nephron transport in many of the segments that produce NO or in which transport is regulated by NO. 3. Four potential interactions between NO and ET are: (i) NO and ET may be antagonistic; (ii) NO and ET may be complementary; (iii) the effects of ET may be mediated via NO; and (iv) the effects of NO may be mediated by ET. 4. In conclusion, direct studies examining the interactions between NO and ET are few. However, circumstantial evidence suggests there may be many interactions between NO and ET in the regulation of nephron transport. In particular, recent data obtained from the collecting duct and thick ascending limb indicate that the effects of ET may be mediated by the production of NO and stimulation of its second messenger cascade.

Background: Measurements of Cl- in nanoliter samples, such as those collected during isolated, perfused tubule experiments, have been difficult, somewhat insensitive, and/or require custom-made equipment. We developed a technique using a fluorescent Cl- indicator, 6-methoxy-N-(3-sulfopropyl) quinolinium (SPQ), to make these measurements simple and reliable., Methods: This is a simple procedure that relies on the selectivity of the dye and the fact that Cl-quenches its fluorescence. To measure millimolar quantities of Cl- in nanoliter samples, we prepared a solution of 0.25 mm SPQ and loaded it into the reservoir of a continuous-flow ultramicrofluorometer, which can be constructed from commercially available components. Samples were injected with a calibrated pipette via an injection port, and the resultant peak fluorescent deflections were recorded. The deflections represent a decrease in fluorescence caused by the quenching effect of the Cl- injected., Results: The method yielded a linear response with Cl- concentrations from 5 to 200 mm NaCl. The minimum detectable Cl- concentration was approximately 5 mm. The coefficient of variation between 5 and 200 mm was 1.7%. Resolution, defined as two times the standard error divided by the slope, between 10 and 50 mm and between 50 and 200 mm was 1 mm and 2.6 mm, respectively. Furosemide, diisothiocyanostilbene-2,2'-disulfonic acid and other nonchloride anions (HEPES, HCO3, SO4, and PO4) did not interfere with the assay, whereas 150 mm NaBr resulted in a peak height greater than 150 NaCl. In addition, the ability to measure Cl- did not vary with pH within the physiological range., Conclusion: We developed an easy, accurate, and sensitive method to measure Cl- concentration in small aqueous solution samples.

Nitric oxide (NO) inhibits transport in various nephron segments, and the thick ascending limb of the loop of Henle (TALH) expresses NO synthase (NOS). However, the effects of NO on TALH transport have not been extensively studied. We hypothesized that endogenously produced NO directly decreases NaCl transport by the TALH. We first determined the effect of exogenously added NO on net chloride flux (JCl). The NO donor spermine NONOate (SPM; 10 microM) decreased JCl from 101.2 +/- 9.6 to 65.0 +/- 7.7 pmol. mm-1. min-1, a reduction of 35.5 +/- 6.4%, whereas controls did not decrease over time. To determine whether endogenous NO affects cortical TALH transport, we measured the effect of adding the NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME), the substrate L-arginine (L-Arg), or its enantiomer D-arginine (D-Arg) on JCl. L-NAME and D-Arg did not alter JCl; in contrast, addition of 0.5 mM L-Arg decreased JCl by 40.2 +/- 10.4% from control. The inhibition of chloride flux by 0.5 mM L-Arg was abolished by pretreatment with L-NAME, indicating that cortical TALH NOS is active, but production of NO is substrate-limited in our preparation. Furthermore, cortical TALH chloride flux increased following removal of 0.5 mM L-Arg from the bath, indicating that the reductions in chloride flux observed in response to L-Arg are not the result of NO-mediated cytotoxicity. We conclude that 1) exogenous NO decreases cortical TALH JCl; 2) cortical TALHs produce NO in the presence of L-Arg, which decreases JCl; and 3) the response of cortical TALHs to L-Arg is reversible in vitro. These data suggest an important role for locally produced NO, which may act via an autocrine mechanism to directly affect TALH sodium chloride transport. Thus TALH NO synthesis and inhibition of chloride transport may contribute to the diuretic and natriuretic effects of NO observed in vivo.

Bradykinin plays an important role in the regulation of renal hemodynamics. However, there have been few studies of the effect of bradykinin on isolated afferent arterioles, vascular segments that are important for the regulation of renal blood flow and glomerular filtration rate. Our purpose was to study (1) the effects of bradykinin on isolated perfused rabbit afferent arterioles and (2) the mechanisms of actions. Afferent arterioles dissected from rabbits were perfused in vitro at 60 mm Hg. In afferent arterioles preconstricted with phenylephrine, 10(-12) to 10(-10) mol/L bradykinin increased luminal diameter from 9.0+/-1.0 to 14.3+/-1.2 microm (P<0.003). In contrast, 10(-9) and 10(-8) mol/L bradykinin decreased luminal diameter to 10.8+/-1.4 and 9.7+/-1.2 microm, respectively (P<0.001). Bradykinin added to the bath had no effect on preconstricted afferent arterioles. The addition of [des-Arg9]-bradykinin (10(-9) and 10(-8) mol/L), a B1 receptor agonist, to the lumen decreased diameter from 9.7+/-1.2 to 6.7+/-1.2 microm at 10(-8) mol/L (P<0.002). Icatibant (Hoe 140), a B2 receptor antagonist, blocked both the vasodilation and vasoconstriction induced by bradykinin as well as the vasoconstriction induced by [des-Arg9]-bradykinin. L-NAME had no effect on bradykinin-induced dilation or constriction. Indomethacin blocked both the dilation induced by 10(-12) to 10(-10) mol/L bradykinin and the constriction induced by 10(-9) to 10(-8) mol/L bradykinin. In fact, in the presence of indomethacin, 10(-9) and 10(-8) mol/L bradykinin increased luminal diameter from 6.2+/-0.7 to 10.7+/-0.6 microm at 10(-8) mol/L (P<0.001), which was attenuated by L-NAME. Finally, in the presence of SQ29548, a prostaglandin H2/thromboxane A2 receptor antagonist, bradykinin caused dilation at all concentrations tested. In conclusion, bradykinin has a biphasic effect on afferent arterioles. Both dilation and constriction may be mediated by bradykinin B2 receptors. The mechanisms of vasodilation and vasoconstriction are due to cyclooxygenase products, not nitric oxide.

Nitric oxide has a diuretic effect in vivo. We have shown that nitric oxide inhibits antidiuretic hormone-stimulated osmotic water permeability in the collecting duct; however, the mechanism by which this occurs is unknown. We hypothesized that inhibition of antidiuretic hormone-stimulated water permeability by nitric oxide in the collecting duct is the result of activation of cGMP-dependent protein kinase, which in turn decreases intracellular cAMP. To test this hypothesis, we microperfused cortical collecting ducts. Antidiuretic hormone-stimulated water permeability was 317 +/- 47 microm/s (P < .001). Addition of spermine NONOate, a nitric oxide donor, to the bath decreased water permeability to 74 +/- 38 microm/s (P < .002). In the presence of LY 83583, an inhibitor of soluble guanylate cyclase, spermine NONOate did not change water permeability. Addition of spermine NONOate increased cGMP production (P < .01). In the presence of the cGMP-dependent protein kinase inhibitor, spermine NONOate did not change water permeability. Since antidiuretic hormone increases water permeability by increasing cAMP, we hypothesized that nitric oxide inhibits water permeability by decreasing cAMP. In tubules pretreated with antidiuretic hormone, intracellular cAMP was 18.9 +/- 3.9 fmol/mm. In tubules treated with antidiuretic hormone and spermine NONOate, cAMP was 9.3 +/- 1.7 fmol/mm (P < .03). We also examined the effect of spermine NONOate on dibutyryl-cAMP-stimulated water permeability. In the presence of dibutyryl-cAMP, water permeability was 388 +/- 30 microm/s. Addition of spermine NONOate had no significant effect on water permeability. Time controls and inhibitors by themselves did not change antidiuretic hormone-stimulated water permeability. We concluded that nitric oxide decreases antidiuretic hormone-stimulated water permeability by increasing cGMP via soluble guanylate cyclase, activating cGMP-dependent protein kinase and decreasing cAMP.

Measurement of sodium in the nanoliter samples obtained from in vitro studies of isolated, perfused nephron segments has traditionally been associated with time-consuming sample preparation and/or expensive instrumentation. A new instrument is described which can measure picomole quantities of sodium without the need for special processing. The instrument is based on the continuous flow devices developed by Vurek and a sodium-sensitive glass detector. The response to varying concentrations of NaCl was logarithmic as predicted from the Nernst equation. The response to 50 mM NaCl was 22.1 +/- 0.2 mV; 100 mM, 29.9 +/- 0.2 mV; 150 mM, 35.2 +/- 0.2 mV; and 200 mM, 39.3 +/- 0.6 mV (mean +/- SD). The slope of the standard curve was 27.8 mV/log unit change in sodium concentration. Potassium, calcium and ammonium, possible interfering ions, had no effect on the response to 100 mM NaCl. When the response to 100 mM NaCl was measured in buffered solutions of varying pH, it was 29.1 +/- 0.2 mV at pH 6.21, 28.9 +/- 0.4 mV at pH 7.04 and 28.7 +/- 0.4 mV at pH 8.54, indicating that pH did not alter the response to 100 mM NaCl. By treating the detector with NaOH and HCl, the response to 50, 100 and 150 mM improved to the point that the slope of the standard curve was 51.2 mV/log unit change in sodium concentration. The calculated resolution after treatment was 1.0 mM in the 50 to 100 mM range and 1.3 mM in the 100 to 150 mM range using a 10.9 nl pipet.(ABSTRACT TRUNCATED AT 250 WORDS)