alpha 2-Adrenergic-mediated tubular NO production inhibits thick ascending limb chloride absorption

Craig F. Plato and Jeffrey L. Garvin

Hypertension and Vascular Research Division, Henry Ford Hospital, Detroit, Michigan 48202


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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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 (JCl) 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 JCl by 20%. When NOS activity was inhibited by pretreatment with 5 mM NG-nitro-L-arginine methyl ester, the inhibitory effects of clonidine on THAL JCl 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 JCl 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 µM), we found that the inhibitory effect of 10 nM clonidine on THAL JCl was abolished, verifying that alpha 2, rather than I1, 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 JCl, indicating activation of phosphatidylinositol 3-kinase and the Akt pathway. We found that pretreatment of THALs with 10 µM LY-83583, an inhibitor of soluble guanylate cyclase, blocked clonidine-mediated inhibition of THAL JCl. In conclusion, alpha 2-receptor stimulation decreases THAL JCl 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.

nitric oxide synthase; clonidine; kidney


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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DISCUSSION
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CLONIDINE IS AN ANTIHYPERTENSIVE agent that acts through stimulation of central alpha 2-adrenergic receptors (34), thereby inhibiting peripheral sympathetic tone and also markedly affecting renal function (44). In vivo, clonidine infusions have been associated with an increase in both sodium and water excretion (15, 42, 46). The effects on urinary output have been ascribed to activation of alpha 2-adrenergic receptors (22) both on the renal vasculature and along the nephron (3).

alpha 2-Adrenergic receptors have been shown to mediate inhibition of nephron transport in vitro. Rouse et al. (43) demonstrated that direct alpha 2-adrenergic receptor activation inhibits proximal convoluted tubular fluid absorption. Similarly, in the isolated cortical collecting duct, alpha 2-adrenergic receptor activation inhibits vasopressin-stimulated hydroosmotic water permeability (10, 24) and amiloride-sensitive sodium reabsorption (42). However, presently we know of no data directly evaluating the effect of alpha 2-adrenergic receptor activation on the thick ascending limb of the loop of Henle (THAL).

Previous studies have demonstrated that NO plays an important role in the control of renal sodium excretion both in vivo (24) and in vitro (40, 45). We recently reported that THAL chloride absorption is directly inhibited by endogenously produced NO (36), and that eNOS mediates this response (37). However, the physiological regulation of tubular NOS is poorly understood.

alpha 2-Adrenergic receptors stimulate NO release in the vascular endothelium. Blocking alpha 2-adrenergic receptors (2) enhances the vasoconstriction caused by the sympathetic neurotransmitter norepinephrine, and alpha 2-adrenergic receptor-induced vasodilatation is sensitive to NOS inhibition (48). Furthermore, inhibition of the vasodilator effects of clonidine by NG-monomethyl- L-arginine (L-NMMA) can be overcome by adding the substrate for NOS, L-arginine (39). Taken together, these findings suggest that endothelial alpha 2-adrenergic receptor activation stimulates NOS and increases NO production.

The THAL expresses alpha 2-adrenergic receptors (29, 55) and produces NO (36); however, it is not clear whether alpha 2-adrenergic receptor activation can inhibit transport via an NO-dependent mechanism. We hypothesized that the alpha 2-adrenergic receptor agonist clonidine decreases sodium chloride absorption in the THAL by activating alpha 2-adrenergic receptors, stimulating NOS, and increasing production of endogenous NO. Our findings indicate that clonidine inhibits chloride absorption in isolated perfused THALs by activation of alpha 2-adrenergic receptors, acting through a NOS-dependent mechanism via activation of phosphatidylinositol 3-kinase (PI3K). Thus alpha 2-adrenergic receptors may function as a physiological regulator of THAL NOS activity.


    MATERIALS AND METHODS
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INTRODUCTION
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Preparation of isolated nephron segments. Cortical THALs were obtained from male Sprague-Dawley rats, weighing 120-150 g (Charles River Breeding Laboratories, Wilmington, MA), which had been maintained on a diet containing 0.22% sodium and 1.1% potassium (Purina, Richmond, IN) with water ad libitum for at least 5 days. On the day of the experiment, rats were anesthetized with ketamine (100 mg/kg body wt ip) and xylazine (20 mg/kg body wt ip), and the abdominal cavity was opened to expose the kidney. The kidney was bathed in ice-cold saline and removed. Coronal slices were placed in oxygenated physiological saline at 12°C. Cortical THALs were dissected from medullary rays in the same solution under a stereomicroscope.

THAL perfusion. THALs (0.5-0.9 mm) were transferred to a temperature-regulated chamber and perfused between concentric glass pipettes at 37°C, as described previously (36). The composition of the basolateral bath and perfusate (in mmol/l) was 114 NaCl; 25 NaHCO3; 2.5 NaH2PO4; 4 KCl; 1.2 MgSO4; 6 alanine; 1 Na3 citrate; 5.5 glucose; 2 Ca lactate2 and 5 raffinose. In addition, a concentration of L-arginine (4 µM) approximating the Michaelis-Menten constant (Km) for eNOS (53) was included in the bath and perfusate solutions, unless otherwise indicated. Clonidine, the NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME), the NOS substrate L-arginine, the alpha 2-adrenergic receptor antagonist rauwolscine, and the PI3K inhibitor wortmannin were all purchased from Sigma (St. Louis, MO). The soluble guanylate cyclase inhibitor LY-83583 was purchased from Biomol (Plymouth Meeting, PA). The solution was bubbled with 5% CO2-95% O2 before and during the experiments. The pH of the bath was 7.4 and the osmolality of the bath solution was 290 ± 3 mosmol/kgH2O, as measured by freezing-point depression. The basolateral bath was exchanged at a rate of 0.5 ml/min, and tubules were perfused at 5 to 10 nl/min. Time-control studies were conducted for each protocol to determine the stability of tubular transport.

Net chloride flux. Chloride concentrations were determined in samples of perfusate and collected fluid using a previously described fluorometric technique (14). Because chloride reabsorption was not accompanied by significant fluid reabsorption, net chloride flux (JCl) was calculated according to the formula
<IT>J</IT><SUB>Cl</SUB><IT>=</IT>PR([Cl]<SUB>o</SUB><IT>−</IT>[Cl]<SUB>I</SUB>)
where PR is the perfusion rate normalized for tubule length, [Cl]o is the chloride concentration in the perfusion fluid, and [Cl]l is the chloride concentration in the collected tubular fluid. Because the ability of renal tubules to synthesize NO in vitro is substrate limited (36), as described above, we included 4 µM L-arginine in the tubular perfusate and bath to approximate the Km of NOS for L-arginine (53). To determine whether the clonidine response is dependent on NO production, a separate series of experiments measured THAL JCl in the absence of exogenous L-arginine. The dissociation constant (Kd) of alpha 2-receptors in the kidney for clonidine is well established and has been found to be ~2 nM (47). Therefore, to selectively activate alpha 2-receptors, we used 10 nM clonidine in all protocols evaluating the effects on THAL transport.

Experimental protocols. We first tested the effects of alpha 2-adrenergic receptor agonists and antagonists on THAL JCl. In these protocols, after a 20-min equilibration period, three basal measurements were performed (control period). Then, clonidine, an alpha 2-adrenergic receptor agonist, or rauwolscine, an alpha 2-receptor antagonist, was added to the bath. Twenty minutes later, three additional collections were made (experimental period).

To determine whether the inhibitory effects of clonidine on THAL JCl were secondary to cytotoxic effects of clonidine or endogenously generated NO, we next evaluated the reversibility of the response to clonidine. In this protocol, clonidine was present in the bath during the initial period. After 20 min, three measurements were made (basal period). The clonidine was then removed from the bath, and after a wash period, three additional collections were made (recovery period). Initial experiments demonstrated that JCl did not recover within 20 min. Thus the tubules in this protocol were washed 35-40 min before measurement of recovery.

We evaluated the role of NOS activity in the response of THALs to selective alpha 2-adrenergic receptor stimulation with clonidine, using two strategies. First, we measured the JCl response to clonidine in the presence of NOS inhibition. In this protocol, 5 mM L-NAME was present in the bath solution throughout the experiment. We have previously reported that 5 mM L-NAME alone does not significantly alter THAL JCl (36). After a 20-min equilibration period, three basal measurements were performed (control period). Clonidine was then added to the bath along with L-NAME. After 20 min, three additional collections were made (experimental period). Second, we evaluated the necessity of the substrate for NOS, L-arginine, in the response of THALs to clonidine. Thus in this protocol, L-arginine was omitted from the bath and perfusion solutions. After a 20-min equilibration period, three basal measurements were collected (control period). Clonidine was then added to the bath solution, and after 20 min, three additional collections were made (experimental period).

We evaluated the signaling cascade of NO in the response of THALs to selective alpha 2-adrenergic receptor stimulation. First, we measured the JCl response in the presence of an inhibitor of soluble guanylate cyclase, LY-83583 (10 µM). In this protocol, LY-83583 was present in the bath solution throughout the experiment. We have previously reported that 10 µM LY-83583 alone does not significantly alter THAL JCl (35). After a 20-min equilibration period, three basal measurements were performed (control period). Clonidine was then added to the bath along with LY-83583. After 20 min, three additional collections were made (experimental period).

We tested the specificity of clonidine's effects on THAL JCl by pretreating tubules with a selective alpha 2-adrenergic receptor antagonist (rauwolscine; 1 µM). After a 20-min equilibration period with rauwolscine in the bath, we took three basal measurements (control period). We then added clonidine (10 nM) to the bath along with the receptor antagonist. After a 20-min equilibration period, three additional collections were performed (experimental period).

Finally, we evaluated the mechanism of NOS activation in the response of THALs to selective alpha 2-adrenergic receptor stimulation with clonidine, using two strategies. First, we examined the effects of clonidine on intracellular calcium concentration using a ratiometric fluorescent indicator technique (20). Briefly, tubules were isolated, perfused, and superfused as described above and incubated with 5 µM fura 2-AM (Molecular Probes, Eugene, OR) for 1 h. After washing for 30 min, basal intracellular calcium concentration was determined. Excitation of fluorophores was performed below 400 nM, and fluorescent emission was detected at greater than 510 nM. After 5 min of stable basal recording, tubules were exposed to 10 nM clonidine, and the response was recorded. We calibrated the maximum and minimum calcium concentrations for each tubule by using 10 µM 4-Br-A23187 and 5 mM EGTA, respectively. Wavelength intensities and ratios were sampled every 20 s by use of the MetaFluor imaging software (Universal Imaging, West Chester, PA).

Second, we measured the JCl response in the presence of an inhibitor of the PI3K/Akt pathway, wortmannin. In this protocol, wortmannin (150 nM) was present in the bath solution throughout the experiment. After a 20-min equilibration period, three basal measurements were performed (control period). Clonidine was then added to the bath along with wortmannin. After 20 min, three additional collections were made (experimental period).

Statistics. Experimental results are expressed as means ± SE. Data were evaluated with Student's paired t-test. The criterion for statistical significance was P < 0.05 in all experiments.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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The THAL contains active NOS, and endogenously produced NO inhibits THAL transport (36). Others have reported that alpha 2-adrenergic receptors stimulate NO release (39). Thus, we first evaluated the response of isolated perfused THALs to clonidine, a selective alpha 2-adrenergic receptor agonist. Figure 1 illustrates the effect of clonidine (10 nM) on JCl in seven isolated THALs. During the control period, tubules absorbed chloride at a rate of 119.5 ± 15.9 pmol · mm-1 · min-1. After 10 nM clonidine was added to the bath, tubules absorbed chloride at a rate of 67.4 ± 13.8 pmol · mm-1 · min-1. Perfusion rates did not differ during the two periods, and time controls showed no reduction in chloride absorption over a 2-h period. Thus 10 nM clonidine inhibited THAL JCl by 43.3 ± 9.1% (P < 0.02).


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Fig. 1.   The alpha 2-adrenergic receptor agonist clonidine inhibits net chloride flux (JCl) in isolated perfused cortical thick ascending limbs of the loop of Henle (THALs). Addition of 10 nM clonidine to the basolateral bath resulted in a 43.3% reduction in JCl, from 119.5 ± 15.9 to 67.4 ± 13.8 pmol · mm-1 · min-1 (n = 7; *P < 0.05).

To verify that the reduction in transport was not secondary to any cytotoxic effects of clonidine, we evaluated the ability of cortical THALs to increase JCl after recovery from clonidine exposure. Figure 2 depicts the effects of removing 10 nM clonidine from the bath. In the presence of 10 nM clonidine, tubules absorbed chloride at a rate of 105.5 ± 11.8 pmol · mm-1 · min-1. Thirty minutes after we removed clonidine from the bath, JCl increased significantly to a rate of 125.4 ± 14.9 pmol · mm-1 · min-1 (20.3 ± 7.3%; P < 0.05; n = 7). These findings indicate that the reductions in JCl we observed in response to 10 nM basolateral clonidine administration were not secondary to cytotoxic effects.


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Fig. 2.   THAL JCl increases after recovery from treatment with clonidine. Removal of 10 nM clonidine from the bath solution increased JCl 20.3 ± 7.3%, from 105.5 ± 11.8 to 125.4 ± 14.9 pmol · mm-1 · min-1 (n = 7; *P < 0.05).

To test whether clonidine inhibits THAL JCl through a combination of NOS activation and increased NO production, we examined the effect of L-NAME on clonidine's ability to inhibit JCl (Fig. 3). In the presence of 5 mM L-NAME, tubules absorbed chloride at a rate of 81.7 ± 10.8 pmol · mm-1 · min-1 (n = 7). When we added 10 nM clonidine to the bath, THAL chloride absorption did not change significantly from the basal rate (71.6 ± 6.9 pmol · mm-1 · min-1). Because we have previously found that 5 mM L-NAME alone does not significantly alter JCl (36), these findings suggest that clonidine inhibits THAL transport via a NOS-dependent mechanism.


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Fig. 3.   NG-nitro-L-arginine methyl ester (L-NAME) inhibits the clonidine-induced decrease in JCl in isolated perfused THALs. Addition of 10 nM clonidine to the basolateral bath of cortical THALs pretreated with 5 mM L-NAME did not alter JCl (n = 7).

Because NOS inhibition abolished the ability of clonidine to inhibit THAL transport, we next evaluated the role of exogenous L-arginine, the substrate for NOS, in clonidine's effects by measuring the effect of clonidine on THAL JCl in the absence of exogenous L-arginine (Fig. 4). During the control period, tubules absorbed chloride at a rate of 106.9 ± 11.6 pmol · mm-1 · min-1. After 10 nM clonidine was added to the bath, chloride absorption was not significantly different from the basal rate (132.2 ± 21.3 pmol · mm-1 · min-1; n = 6). Thus removing the substrate for NOS prevented the reduction in THAL chloride absorption induced by 10 nM clonidine.


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Fig. 4.   Removal of the substrate for NOS, L-arginine (L-Arg), prevents the inhibitory effect of clonidine on THAL JCl. Addition of 10 nM clonidine to the basolateral bath in the absence of exogenous L-Arg did not alter the JCl of isolated perfused cortical THALs (n = 6).

The effects of NO in many tissues are mediated by the stimulation of guanylate cyclase and increased cGMP (32). To examine whether alpha 2-adrenergic receptor-mediated NO production follows a similar signaling cascade, we next examined the effects of an inhibitor of soluble guanylate cyclase, LY-83583, on clonidine's ability to inhibit THAL chloride absorption. In the presence of 10 µM LY-83583, tubules absorbed chloride at a rate of 66.1 ± 11.9 pmol · mm-1 · min-1 (n = 6). When we added 10 nM clonidine to the bath, THAL chloride absorption did not change significantly from the basal rate (53.5 ± 3.0 pmol · mm-1 · min-1; 6.8 ± 16.4%). Because we have previously reported that 10 µM LY-83583 alone does not significantly alter JCl (35), taken together, the present findings suggest that clonidine inhibits THAL transport via activation of NOS, increased NO production, and stimulation of soluble guanylate cyclase.

We have reported that the endothelial isoform of NOS mediates the inhibitory effects of L-arginine in the THAL (37). Other investigators have demonstrated that activation of endothelial NOS is calcium dependent (53). Therefore, we next examined the intracellular calcium response of isolated perfused THALs to activation of alpha 2-adrenergic receptors with clonidine. Intracellular calcium concentration increased only 22 ± 4% from the basal value of 114.5 ± 14.8 nM in response to 10 nM clonidine. These findings indicate that alpha 2-adrenergic receptors do not likely activate THAL NOS by increasing intracellular calcium concentration. Thus we explored alternative mechanisms for the activation of NOS by alpha 2-adrenergic receptors.

Previous studies have demonstrated that eNOS may also be activated through a calcium-independent pathway via stimulation of PI3K and phosphorylation of the serine/threonine kinase Akt (12, 13). Therefore, we next examined the possibility that alpha 2-adrenergic receptors stimulate THAL NOS and increase NO production through activation of PI3K. Figure 5 depicts the effects of 10 nM clonidine on five tubules pretreated with the PI3K inhibitor wortmannin (150 nM). During the control period, tubules absorbed chloride at a rate of 108.6 ± 13.1 pmol · mm-1 · min-1. After 10 nM clonidine was added to the bath, THAL chloride absorption did not change significantly from the basal rate (98.9 ± 10.7 pmol · mm-1 · min-1). In a separate series of experiments, addition of 150 nM wortmannin alone did not significantly alter JCl from control (82.6 ± 15.1 vs. 75.1 ± 7.9 pmol · mm-1 · min-1; n = 5). Taken together, these findings suggest that clonidine stimulates THAL NOS activity primarily through a PI3K-mediated pathway, whereas PI3K is not constitutively active under basal conditions.


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Fig. 5.   The phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin inhibits the clonidine-induced decrease in JCl in isolated perfused THALs. Addition of 10 nM clonidine to the basolateral bath of cortical THALs pretreated with 150 nM wortmannin did not alter JCl (108.6 ± 13.1 vs. 98.9 ± 10.7 pmol · mm-1 · min-1).

Other investigators have reported that clonidine stimulates I1-type imidazoline receptors (8) as well as alpha 2-adrenergic receptors. To verify that the effects of clonidine are specifically mediated by alpha 2-adrenergic receptors, we examined the effect of the alpha 2-adrenergic receptor antagonist rauwolscine on clonidine's ability to inhibit THAL chloride absorption (Fig. 6). In the presence of 1 µM rauwolscine, tubules absorbed chloride at a rate of 106.8 ± 24.4. pmol · mm-1 · min-1 (n = 6). After 10 nM clonidine was added to the bath, THAL chloride absorption did not change significantly from the basal rate (93.4 ± 18.7 pmol · mm-1 · min-1). Control experiments demonstrated that rauwolscine alone did not alter THAL JCl from control (101.1 ± 11.9 vs. 92.4 ± 14.3 pmol · mm-1 · min-1; n = 6). Thus clonidine inhibits THAL JCl specifically via alpha 2-adrenergic receptors.


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Fig. 6.   An alpha 2-adrenergic receptor antagonist abolishes the clonidine-induced decrease in JCl in isolated perfused THALs. Addition of 10 nM clonidine to the basolateral bath of cortical THALs pretreated with 1 µM rauwolscine, a selective alpha 2-adrenoceptor antagonist, did not alter JCl (n = 6).


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Our data show that clonidine reversibly inhibits JCl by isolated perfused THALs that can be blocked by a competitive inhibitor of NOS, the removal of the substrate for NOS, L-arginine, or the inhibition of soluble guanylate cyclase. Moreover, a selective alpha 2-receptor antagonist, rauwolscine, and wortmannin, an inhibitor of PI3K, were able to block clonidine-induced decreases in JCl. As a whole, these findings suggest that clonidine inhibits THAL transport by 1) enhancing endogenous NOS activity, 2) releasing NO, and 3) stimulating soluble guanylate cyclase via activation of alpha 2-adrenergic receptors and PI3K. This suggests that alpha 2-adrenergic receptors are physiological regulators of THAL NOS.

alpha 2-Receptors inhibit THAL transport. The present studies demonstrate that selective activation of alpha 2-adrenergic receptors inhibits THAL JCl, thus supporting the findings of other investigators who demonstrated alpha 2-mediated inhibition of transport in other nephron segments. Bello-Reuss (4) demonstrated that addition of the adrenergic neurotransmitter norepinephrine to the bath increased fluid absorption in microperfused proximal convoluted tubules. The stimulatory effects of norepinephrine were abolished by pretreatment with the beta -antagonist propranolol, which in turn unmasked significant inhibition of fluid absorption in response to norepinephrine, an effect that may have been mediated by alpha 2-adrenergic receptor activation. Rouse et al. (43) examined this phenomenon directly and demonstrated that selective alpha 2-adrenergic receptor stimulation with clonidine decreased fluid absorption in the isolated perfused proximal tubule.

alpha 2-Adrenergic receptors also inhibit fluid absorption, and depending on the species, sodium absorption, in the collecting duct. Krothapalli et al. (24) showed that alpha 2-adrenergic receptor activation inhibits arginine vasopressin (AVP)-induced osmotic water permeability in rabbit cortical collecting ducts, while Chen et al. (10) reported that clonidine reversibly inhibited both AVP-dependent osmotic water permeability and lumen-to-bath sodium flux in the rat cortical collecting duct. Chen et al. (10) were able to block these effects by pretreatment with a selective alpha 2-antagonist, indicating that clonidine's effects were indeed mediated by activation of alpha 2-adrenergic receptors. Thus our present findings support an inhibitory role for alpha 2-adrenergic receptors in the THAL, where clonidine decreases sodium chloride absorption through activation of alpha 2-adrenergic receptors.

We believe these are the first in vitro data showing that clonidine, acting via alpha 2-adrenergic receptors, stimulates NO production to inhibit transport in any nephron segment. Moreover, they support previous in vivo data suggesting that clonidine directly influences urinary sodium excretion. In previous studies, intrarenal administrations of clonidine increased free water clearance, and to a lesser extent, sodium excretion (3, 41, 46). The diuretic and natriuretic responses were observed independently of changes in glomerular filtration rate or renal perfusion pressure (5). Taken together, these data suggest that clonidine, acting through activation of alpha 2-adrenergic receptors, stimulates the production of NO, which in turn increases urinary sodium excretion by a direct tubular effect.

Evidence indicating that renal alpha 2-adrenergic receptors stimulate NO production has been previously assayed by measuring the conversion of the NOS substrate L-arginine to its byproduct, L-citrulline (49). That same study demonstrated that selective blockade of medullary alpha 2-adrenergic receptors with rauwolscine reduced renal NOS activity (and hence L-citrulline accumulation) and instigated arterial hypertension to otherwise subpressor intravenous infusions of norepinephrine (49). The renal hemodynamic response following rauwolscine was mimicked by renal interstitial infusion of L-NAME, suggesting that renal medullary alpha 2-adrenergic receptors act through stimulation of NO production. The THAL comprises a significant portion of the outer medulla (9), and the present study demonstrates that clonidine's ability to inhibit THAL transport is sensitive to both rauwolscine and L-NAME. Thus the alpha 2-receptor-mediated renal NO production observed in the previous whole-animal studies may have been derived in part from the THAL.

Some previous investigators have been unable to observe any effect of alpha 2-adrenergic receptors on transport in the THAL (3). The bulk of the earlier studies were concerned with the effects of efferent renal nerve stimulation or direct intrarenal infusions of alpha 2-adrenergic receptor agonists on renal function in the whole animal (11). alpha 2-Adrenergic receptor activation affects intrarenal neurotransmitter release (21), endothelial NO production (48), renal hemodynamics (15), and proximal tubular transport (16, 33, 43). Therefore, direct effects of alpha 2-adrenergic receptor-mediated effects of renal sympathetic nerve stimulation on THAL transport in the whole kidney may be obscured during intravenous or intrarenal infusions of antagonists. We believe the isolated perfused THAL preparation obviates many of the potentially confounding influences of simultaneous activation of alpha 2-adrenergic receptors in other renal cell types and thus clarifies their role, at least in this specific nephron segment.

alpha 2-Receptors and stimulation of NOS. The specific alpha 2-adrenergic receptor isoform(s) by which clonidine stimulates THAL NOS activity is presently unknown. However, we found that clonidine-mediated inhibition of THAL JCl was sensitive to the selective antagonist rauwolscine, suggesting that this response is dependent on alpha 2-adrenergic, rather than I1-imidazoline, receptor activation. Bockman et al. used differential receptor binding affinities to alpha 2-agonists and antagonists and implicated the alpha 2A-, alpha 2C- (6), and alpha 2D- (7) adrenergic receptor subtypes in stimulation of endothelial NO production. Furthermore, a recent study (55) using RT-PCR of microdissected nephron segments demonstrated expression of all known alpha 2-receptor subtypes in the rat THAL. Thus multiple isoforms of alpha 2-adrenergic receptors may be coupled to NO production in the THAL. Additional studies are necessary to determine which specific alpha 2-adrenergic receptor subtype mediates the stimulation of THAL NOS activity.

The mechanism by which alpha 2-adrenergic receptors stimulate NO production in the THAL is presently uncertain. However, our data showing abolition of clonidine-mediated inhibition of THAL JCl by L-NAME and substrate deprivation indicate that this response is dependent on activation of NOS and increased NO production. Each of the three isoforms of NOS has been localized to the THAL (1, 28, 30, 51). The constitutively expressed NOS isoforms [i.e., endothelial (eNOS) and neuronal (nNOS)] have traditionally been thought to require increased intracellular calcium for activation, whereas inducible NOS (iNOS) does not but is dependent on substrate availability (53). We have recently reported that eNOS mediates the inhibitory effects of exogenous L-arginine on THAL JCl (37), and others have found that alpha 2-adrenergic receptor activation increases intracellular calcium concentrations (17). Therefore we initially investigated the mechanism of alpha 2-adrenergic receptor activation of THAL NOS activity by examining the intracellular calcium response to clonidine. To our surprise, intracellular calcium concentration was unaffected by exposure to clonidine. Because the Km of NOS for calcium is 200 nM (19), increasing intracellular calcium from the basal concentration of 115 to 140 nM would be sufficient to increase NOS activity from 35 to 40% of its maximal rate. Given this very modest response of intracellular calcium, we evaluated an alternative signaling pathway for the activation of THAL NOS.

Recent findings have demonstrated that eNOS can be activated through calcium-independent mechanisms. Dimmeler et al. (12) have shown that activation of PI3K and stimulation of the serine/threonine protein kinase Akt can stimulate eNOS. This signaling pathway and NO production are sensitive to the fungal derivative wortmannin. In addition, others have reported that alpha 2-adrenergic receptor-mediated responses are sensitive to inhibition of PI3K (52). Wortmannin pretreatment abolished the inhibitory effects of clonidine on THAL JCl, indicating that alpha 2-adrenergic receptors are likely coupled to the activation of THAL NOS through stimulation of PI3K and increased Akt activity. To our knowledge, this is the first report of NOS activation being mediated through activation of the PI3K/Akt-signaling pathway in any tubular segment.

Possible physiological interactions and implications. The THAL is critical in the control of sodium excretion, absorbing ~25% of the filtered sodium chloride load (23), and the present studies demonstrate that clonidine inhibits THAL JCl. Because sodium is required for chloride transport across the apical membrane, and the Na-K-ATPase drives the Na-K-2Cl cotransporter, sodium reabsorption must accompany THAL chloride reabsorption (31). Therefore, clonidine may be expected to increase urinary sodium chloride excretion, with all other neurohumoral controllers of renal function remaining constant.

Because we have shown that clonidine stimulates THAL alpha 2-adrenergic receptors, and in turn, NOS activity, there may be additional effects on glomerular hemodynamics. Under normal conditions, the inhibition of THAL JCl would increase sodium chloride delivery to the macula densa and would result in tubuloglomerular feedback (TGF) that would reduce the delivery of sodium chloride from the THAL to a normal value. NO has been demonstrated to blunt the TGF response (50), whereas inhibitors of NOS augment the TGF response (54). NO has been predicted to have a half-life of 5 s (27) and has a diffusion constant of 3,300 µm2/s (26). Given these parameters, it is possible that NO produced by the THAL may be carried via the tubular fluid downstream to the macula densa. Alternatively, our laboratory has recently shown that NO produced by the macula densa directly inhibits TGF rather than via diffusion to the afferent arteriole (38). Thus clonidine-stimulated NO production by the THAL or the macula densa itself may reset the TGF mechanism, reducing its efficiency and promoting sodium chloride excretion.

The THAL is also impermeable to water, and absorption of salt by this nephron segment both establishes and maintains the hypertonic medullary solute gradient and also generates dilute tubular fluid (18, 31). To this end, acute clonidine-mediated reductions in sodium chloride absorption would be expected to reduce the diluting ability of the THAL. Exposure of the THAL to alpha 2-adrenergic stimulation would then decrease the corticomedullary solute gradient and decrease the kidney's ability to concentrate urine. The widely observed phenomena of dilute urine production and increased free water clearance due to clonidine in vivo have generally been attributed to alpha 2-receptors antagonizing the effects of vasopressin in the collecting duct (15, 24). However, the high transport rates and water-impermeant nature of the THAL are the cardinal attributes of the kidney that allow selective water abstraction in the downstream nephron. Therefore, the inhibition of sodium chloride absorption in the THAL may provide a mechanistic basis for the previous in vivo findings of clonidine having potent effects on urinary concentrating ability and hence promoting water excretion.

Conclusion. We found that clonidine inhibited chloride absorption by the isolated perfused THAL via activation of alpha 2-adrenergic receptors. This inhibition was abolished by L-NAME and required the substrate for NOS activity, L-arginine. The response was specifically mediated by alpha 2-adrenergic receptors, because pretreatment with the selective antagonist rauwolscine prevented the effects of clonidine. These findings indicate that the rat THAL responds to alpha 2-adrenergic receptor activation by increasing production of NO, which then inhibits transport via an autocrine mechanism. Thus alpha 2-adrenergic receptors may be physiological regulators of THAL NOS activity, and the inhibitory effects of clonidine on THAL chloride absorption may partially explain its ability to increase urinary sodium and water excretion in vivo.


    ACKNOWLEDGEMENTS

This work was conducted during the tenure of an American Heart Association Fellowship Grant awarded to C. F. Plato. It was supported by National Heart, Lung, and Blood Institute Grant HL-28982 awarded to J. L. Garvin.


    FOOTNOTES

Address for reprint requests and other correspondence: J. L. Garvin, Henry Ford Hospital, Hypertension and Vascular Research Division, 2799 W. Grand Blvd., Detroit, MI 48202.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 12 December 2000; accepted in final form 25 May 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Allcock, GH, Hukkanen M, Polak JM, Pollock JS, and Pollock DM. Increased nitric oxide synthase 3 in kidneys of DOCA-salt hypertensive rats. J Am Soc Nephrol 10: 2283-2289, 1999[Abstract/Free Full Text].

2.   Angus, JA, Cocks TM, and Satoh K. The alpha  adrenoceptors on endothelial cells. Fed Proc 45: 2355-2359, 1986[ISI][Medline].

3.   Barr, JG, and Kauker ML. Renal tubular site and mechanism of clonidine-induced diuresis in rats: clearance and micropuncture studies. J Pharmacol Exp Ther 209: 389-395, 1979[ISI][Medline].

4.   Bello-Reuss, E. Effect of catecholamines on fluid reabsorption by the isolated proximal convoluted tubule. Am J Physiol Renal Fluid Electrolyte Physiol 238: F347-F352, 1980[Abstract/Free Full Text].

5.   Blandford, DE, and Smyth DD. Dose selective dissociation of water and solute excretion after renal alpha-2 adrenoceptor stimulation. J Pharmacol Exp Ther 247: 1181-1186, 1988[Abstract].

6.   Bockman, CS, Jeffries WB, and Abel PW. Binding and functional characterization of alpha2-adrenergic receptor subtypes on pig vascular endothelium. J Pharmacol Exp Ther 267: 1126-1133, 1993[Abstract].

7.   Bockman, CS, Gonzalez-Cabrera I, and Abel PW. Alpha-2 adrenoceptor subtype causing nitric oxide-mediated vascular relaxation in rats. J Pharmacol Exp Ther 278: 1235-1243, 1996[Abstract].

8.   Bousquet, P, Feldman J, and Schwartz J. Central cardiovascular effects of the alpha-adrenergic drugs: differences between catecholamines and imidazolines. J Pharmacol Exp Ther 230: 232-236, 1984[Abstract].

9.   Chamberlin, ME, LeFurgey A, and Mandel LJ. Suspension of medullary thick ascending limb tubules from the rabbit kidney. Am J Physiol 247: F955-F964, 1984[ISI][Medline].

10.   Chen, L, Reif MC, and Schafer JA. Clonidine and PGE2 have different effects on Na+ and water transport in rat and rabbit CCD. Am J Physiol Renal Fluid Electrolyte Physiol 261: F126-F136, 1991[Abstract/Free Full Text].

11.   DiBona, GF, and Sawin LL. Role of renal alpha 2-adrenergic receptors in spontaneously hypertensive rats. Hypertension 9: 41-48, 1987[Abstract].

12.   Dimmeler, S, Fleming I, Fisslthaler B, Hermann C, Busse R, and Zeiher AM. Activation of nitric oxide synthase in endothelial cells by Akt-dependent phosphorylation. Nature 399: 601-605, 1999[ISI][Medline].

13.   Fulton, D, Gratton J-P, McCabe TJ, Fontana J, Fujio Y, Walsh K, Franke TF, Papapetropoulos A, and Sessa WC. Regulation of endothelium-derived nitric oxide production by the protein kinase Akt. Nature 399: 597-601, 1999[ISI][Medline].

14.   Garcia, NH, Plato CF, and Garvin JL. A fluorescent technique of chloride in nanoliter samples. Kidney Int 55: 321-325, 1999[ISI][Medline].

15.   Gellai, M, and Ruffolo RR. Renal effects of selective alpha-1 and alpha-2 adrenoceptor agonists in conscious normotensive dogs. J Pharmacol Exp Ther 240: 723-728, 1987[Abstract].

16.   Gesek, FA, and Strandhoy JW. Dual interactions between alpha 2-adrenoceptor agonists and the proximal Na+-H+ exchanger. Am J Physiol Renal Fluid Electrolyte Physiol 258: F636-F642, 1990[Abstract/Free Full Text].

17.   Godfraind, T, Miller RC, and Lima JS. Selective alpha 1- and alpha 2- adrenoceptor agonist induced contractions and 45Ca fluxes in the rat isolated aorta. Br J Pharmacol 77: 597-604, 1982[Abstract].

18.   Greger, R, and Velazquez H. The cortical thick ascending limb and early distal convoluted tubule in the urinary concentrating mechanism. Kidney Int 31: 590-596, 1987[ISI][Medline].

19.   Griffith, OW, and Steuhr DJ. Nitric oxide synthases: properties and catalytic mechanism. Annu Rev Physiol 57: 707-736, 1995[ISI][Medline].

20.   Grynkiewicz, G, Poenie M, and Tsien RY. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260: 3440-3450, 1985[Abstract].

21.   Hesse, IFA, and Johns EJ. The subtype of alpha -adrenoceptor involved in the neural control of renal tubular sodium reabsorption in the rabbit. J Physiol (Lond) 352: 527-538, 1984[Abstract].

22.   Hohage, H, Schlatter E, and Greven J. Effects of moxonidine and clonidine on renal function and blood pressure in anesthetized rats. Clin Nephrol 47: 316-324, 1997[ISI][Medline].

23.   Kirchner, KA, Crosby BA, Patel AR, and Granger JP. Segmental chloride transport in the Dahl-S rat kidney during L-arginine administration. J Am Soc Nephrol 5: 1567-1572, 1995[Abstract].

24.   Krothapalli, RK, Duffy WB, Senekjian HO, and Suki WN. Modulation of the hydro-osmotic effect of vasopressin on the rabbit cortical collecting tubule by adrenergic agents. J Clin Invest 72: 287-294, 1983[ISI][Medline].

25.   Lahera, V, Salom MG, Fiksen-Olsen MJ, Raij L, and Romero JC. Effects of NG-monomethyl-L-arginine and L-arginine on acetylcholine response. Hypertension 15: 659-663, 1990[Abstract].

26.   Lancaster, JR, Jr. A tutorial on the diffusibilty and reactivity of free nitric oxide. Nitric Oxide 1: 18-30, 1997[ISI][Medline].

27.   Liu, X, Miller MJ, Joshi MS, Sadowska-Krowicka H, Clark DA, and Lancaster JR, Jr. Diffusion-limited reaction of free nitric oxide with erythrocytes. J Biol Chem 273: 18709-18713, 1998[Abstract/Free Full Text].

28.   Mattson, DL, and Higgins DJ. Influence of dietary sodium intake on renal medullary nitric oxide synthase. Hypertension 27: 688-692, 1996[Abstract/Free Full Text].

29.   Meister, B, Dagerlind A, Nicholas AP, and Hokfelt T. Patterns of messenger RNA expression for adrenergic receptor subtypes in the kidney. J Pharmacol Exp Ther 268: 1605-1611, 1994[Abstract].

30.   Mohaupt, MG, Elzie JL, Ahn KY, Clapp WL, Wilcox CS, and Kone BC. Differential expression and induction of mRNAs encoding two inducible nitric oxide synthases in rat kidney. Kidney Int 46: 653-665, 1994[ISI][Medline].

31.   Molony, DA, Reeves WR, and Andreoli TA. Na+:K+:2Cl- contransport and the thick ascending limb. Kidney Int 36: 418-426, 1989[ISI][Medline].

32.   Moncada, S, Palmer RMJ, and Higgs EA. Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol Rev 43: 109-142, 1991[ISI][Medline].

33.   Nord, EP, Howard MJ, Hafezi A, Moradeshaji P, Vaystub S, and Insel PA. Alpha2-adrenergic agonists stimulate Na+-H+ antiport activity in the rabbit renal proximal tubule. J Clin Invest 80: 1755-1762, 1987[ISI][Medline].

34.   Onesti, G, Schwartz AB, Kim KE, Paz-Martinez V, and Swartz C. Antihypertensive effect of clonidine. Circ Res 28/29 (Suppl II): II-53-II-69, 1971.

35.   Ortiz, PA, and Garvin JL. NO inhibits NaCl absorption by rat thick ascending limb through activation of cGMP-stimulated phosphodiesterase. Hypertension 37: 467-471, 2001[Abstract/Free Full Text].

36.   Plato, CF, Stoos BA, Wang D, and Garvin JL. Endogenous nitric oxide inhibits chloride transport in the thick ascending limb. Am J Physiol Renal Physiol 276: F159-F163, 1999[Abstract/Free Full Text].

37.   Plato, CF, Sheseley EG, and Garvin JL. eNOS mediates L-arginine induced inhibition of thick ascending limb chloride flux. Hypertension 35: 319-323, 2000[Abstract/Free Full Text].

38.   Ren, YL, Garvin JL, and Carretero OA. Role of macula densa nitric oxide and cGMP in the regulation of tubuloglomerular feedback. Kidney Int 58: 2053-2060, 2000[ISI][Medline].

39.   Richard, V, Tanner FC, Tschudi M, and Luscher TF. Different activation of L-arginine by bradykinin, serotonin, and clonidine in coronary arteries. Am J Physiol Heart Circ Physiol 259: H1433-H1439, 1990[Abstract/Free Full Text].

40.   Roczniak, A, and Burns KD. Nitric oxide stimulates guanylate cyclase and regulates sodium transport in rabbit proximal tubule. Am J Physiol Renal Physiol 272: F106-F115, 1997.

41.   Roman, RJ, Cowley AW, Jr, and Lechene C. Water diuretic and natriuretic effect of clonidine in the rat. J Pharmacol Exp Ther 211: 385-393, 1979[ISI][Medline].

42.   Rouch, AJ, Chen L, Troutman SL, and Schafer JA. Na+ transport in isolated rat CCD: effects of bradykinin, ANP, clonidine, and hydrochlorothiazide. Am J Physiol Renal Fluid Electrolyte Physiol 260: F86-F95, 1991[Abstract/Free Full Text].

43.   Rouse, D, Williams S, and Suki WN. Clonidine inhibits fluid absorption in the rabbit proximal convoluted renal tubule. Kidney Int 38: 80-85, 1990[ISI][Medline].

44.   Schmitt, H. The pharmacology of clonidine and related products. In: Antihypertensive Agents. Handbook of Experimental Pharmacology, edited by Gross F.. New York: Springer-Verlag, 1977, vol. 39, p. 299-378.

45.   Stoos, BA, Carretero OA, Farhy RD, Scicli G, and Garvin JL. Endothelium-derived relaxing factor inhibits transport and increases cGMP content in cultured mouse cortical collecting duct. J Clin Invest 89: 761-765, 1992[ISI][Medline].

46.   Strandhoy, JW, Morris M, and Buckalew V. Renal effects of the antihypertensive, guanabenz, in the dog. J Pharmacol Exp Ther 256: 606-616, 1982[Abstract].

47.   Summers, RJ. Renal alpha  adrenoceptors. Fed Proc 43: 2917-2922, 1984[ISI][Medline].

48.   Sunano, S, Li-Bo Z, Matsuda K, Sekiguchi F, Watanabe H, and Shimamura K. Endothelium-dependent relaxation by alpha 2-adrenoceptor agonists in spontaneously hypertensive rat aorta. J Cardiovasc Pharmacol 27: 733-739, 1996[ISI][Medline].

49.   Szentivanyi, M, Zou AP, Maeda CY, Mattson DL, and Cowley AW, Jr. Increase in renal medullary nitric oxide synthase activity protects from norepinephrine-induced hypertension. Hypertension 35: 418-423, 2000[Abstract/Free Full Text].

50.   Thorup, C, Sundler F, Ekblad E, and Persson AEG Resetting of the tubuloglomerular feedback mechanism by blockade of NO-synthase. Acta Physiol Scand 148: 359-360, 1993[ISI][Medline].

51.   Tojo, A, Gross SS, Zhang L, Tisher CC, Schmidt HHHW, Wilcox CS, and Madsen KM. Immunocytochemical localization of distinct isoforms of nitric oxide synthase in the juxtaglomerular apparatus of normal rat kidney. J Am Soc Nephrol 4: 1438-1447, 1994[Abstract].

52.   Waen-Safranchik, VI, and Deth RC. Effects of wortmannin on alpha-1/alpha-2-adrenergic receptor-mediated contractile responses in rabbit vascular tissues. Pharmacology 48: 349-359, 1994[ISI][Medline].

53.   White, KA, Pufahl RA, Olken NM, Hevel JM, Richard MK, and Marletta MA. Nitric oxide synthase: mechanisms and relationship to cytochrome P450. In: Cytochrome P450. 8th International Congress, edited by Lechner MC.. Paris: Libbey Eurotext, 1994, p. 43-48.

54.   Wilcox, CS, Welch WJ, Murad F, Gross SS, Taylor G, Levi R, and Schmidt HHHW Nitric oxide synthase in macula densa regulates glomerular capillary pressure. Proc Natl Acad Sci USA 89: 11993-11997, 1992[Abstract].

55.   Zou, AP, and Cowley AW, Jr. alpha 2-Adrenergic receptor-mediated increase in NO production buffers renal medullary vasoconstriction. Am J Physiol Regulatory Integrative Comp Physiol 279: R769-R777, 2000[Abstract/Free Full Text].


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