INTRODUCTION

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THE RENAL SYMPATHETIC NERVES are important modulators of renal sodium chloride excretion via the release of the neurotransmitter norepinephrine. Several segments of the nephron are closely associated with sympathetic neuronal varicosities, including the proximal convoluted tubule (2), the thick ascending limb (THAL) (3), and the distal nephron including distal convoluted tubule, connecting tubule, and collecting duct (5). Histological studies have revealed that the THAL possesses the greatest percentage of neural fibers per tubule of the nonvascular nephron components (4). Studies in the whole animal using low-level renal sympathetic efferent nerve stimulation have found increased sodium reabsorption in the absence of altered renal hemodynamics, indicating that the so-called "intrinsic" renal nerves are functional and exert direct tubular effects manifested as antinatriuresis (44).

Adrenergic receptors mediate the effects of norepinephrine, and each main type (e.g., -1, -2, -1, and -2) has been localized in the kidney (35, 46, 49). The antinatriuretic effects of renal sympathetic activity have been primarily ascribed to the action of -1 adrenergic receptors (39, 50) with -adrenergic (21) receptors mediating lesser effects depending on the nephron segment studied. In contrast, -2 adrenergic receptors have been suggested to mediate both inhibition and stimulation of transport along the nephron. For example, in proximal tubule cell suspensions, -2 adrenergic receptor activation increased Na⁺/H⁺ exchange activity (37), whereas -2 adrenergic receptor activation inhibits proximal convoluted tubular fluid absorption (42).

The THAL is critical to salt and water homeostasis (27, 34) and possesses both -2 (33) and -receptors (15). We recently reported that the selective -2 agonist clonidine inhibits THAL sodium chloride absorption (40), whereas others have demonstrated that the selective -adrenergic receptor agonist isoproterenol increases sodium chloride absorption by the THAL (1). The endogenous neurotransmitter norepinephrine activates each type of adrenergic receptor (30). Furthermore, renal -2 adrenergic receptors are expressed in a two- to fourfold greater number than -1 or -adrenergic receptors (43). However, the role of -2 adrenergic receptors in the tubular response to norepinephrine is still unclear. Taking our recent findings along with those of previous investigators suggests that the response of THAL sodium chloride absorption to norepinephrine may be differentially modulated via activation of -2 and -adrenergic receptors. However, we are currently unaware of any studies evaluating the response of isolated perfused THALs to the nonselective, native sympathetic neurotransmitter norepinephrine. Therefore, the current study tested the hypothesis that both -2 and -adrenergic receptors are responsive to norepinephrine in the THAL and that activation of -2 receptors inhibits, whereas activation of -receptors stimulates, THAL chloride flux (J_Cl).

	METHODS

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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 to 0.9 mm) were transferred to a temperature-regulated chamber and perfused between concentric glass pipettes at 37°C as described previously (41). The composition of the basolateral bath and perfusate (in mmol/l) was: 114 NaCl, 25 NaHCO, 2.5 NaH₂PO, 4 KCl, 1.2 MgSO, 6 alanine, 1 Na₃ citrate, 5.5 glucose, 2 Ca lactate₂, and 5 raffinose. In addition, we included 4 µM L-arginine in the tubular perfusate and bath solutions. 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 a rate of 5 to 10 nl/min. Norepinephrine bitartrate, the -selective adrenergic agonist isoproterenol HCl and antagonist propranolol HCl, the -2-selective agonist clonidine HCl and antagonist rauwolscine HCl, and the -1-selective agonist phenylephrine HCl were all purchased from Sigma (St. Louis, MO). Time-control studies were conducted for each protocol to determine the stability of tubular transport and any nonspecific effects of antagonists.

Net J_Cl. Chloride concentrations were determined in samples of perfusate and collected fluid using a previously described microfluorometric technique (16). Because chloride reabsorption was not accompanied by significant fluid reabsorption, net J_Cl was calculated according to the formula J_Cl = PR ([Cl₀]  [Cl₁]), where PR is the perfusion rate normalized for tubule length, Cl₀ is the chloride concentration in the perfusion fluid, and Cl₁ is the chloride concentration in the collected tubular fluid.

Experimental protocols. We initially tested the effects of incremental concentrations (10 pM-10 µM) of the native sympathetic neurotransmitter norepinephrine on THAL J_Cl. In this protocol, after a 20-min equilibration period, three basal measurements were made (control period). An initial concentration of norepinephrine was then added to the basolateral bath, and 15 min later, three additional collections were made (experimental period 1). After completion of the first experimental period, a greater concentration of norepinephrine was added to the basolateral bath, and 15 min later, three final collections were made (experimental period 2). To control for any nonspecific effects of norepinephrine concentration on J_Cl, the norepinephrine concentrations in experimental periods 1 and 2 were randomized, with the provision that the lower concentration was always presented first. In a separate series of experiments, a paired study was conducted to evaluate the effects of a high concentration (10 µM) of NE alone on THAL J_Cl. This protocol was similar to those described above, with the exception that the effects of only one concentration of norepinephrine were tested.

Statistics. Experimental results are expressed as means ± SE. Data for concentration-response experiments with norepinephrine were analyzed by one-way ANOVA for repeated measures with the same tubule being measured under different conditions. For all concentrations, a subset of all possible contrasts was specified a priori as being of primary importance. Bonferroni comparisons were used for significant differences between concentrations. Data for all other protocols were evaluated with Student's paired t-test. The criterion for statistical significance was P < 0.05 in all experiments. All experiments in which at least two measurements of J_Cl were made during each period were reported.

	RESULTS

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The current investigation sought to determine the response of isolated perfused THALs to incremental concentrations of the native sympathetic neurotransmitter norepinephrine and the receptor(s) mediating these responses. Figure 1 illustrates the effect of graded concentrations of norepinephrine (10 pM-10 µM) on J_Cl in isolated perfused THALs. During the control period, tubules absorbed chloride at a rate of 114.2 ± 8.1 pmol · mm¹ · min¹. After the addition of 10 pM norepinephrine, J_Cl began to decrease, with transport reduced to 93.7 ± 14.9 pmol · mm¹ · min¹ at 100 pM and reaching the nadir at 1 nM (67.7 ± 8.8 pmol · mm¹ · min¹). Additional increases in bath norepinephrine concentrations reversed the inhibition observed at low concentrations, with J_Cl exceeding the control rate at 1 µM (120.2 ± 17.9 pmol · mm¹ · min¹) and achieving maximal stimulation at 10 µM (131.0 ± 28.5 pmol · mm¹ · min¹). When analyzed as changes from the minimum transport rate, both 1 and 10 µM norepinephrine significantly increased J_Cl (P < 0.01). Thus incremental concentrations of norepinephrine produced a biphasic response in THAL J_Cl with low concentrations inhibiting and high concentrations stimulating transport.

View larger version (17K): [in this window] [in a new window]   Fig. 1.   The endogenous sympathetic neurotransmitter norepinephrine (NE) exerts biphasic effects on net chloride flux (JCl) in isolated perfused cortical thick ascending limbs of the loop of Henle (THALs). Low concentrations of NE decreased THAL JCl from control (114.2 ± 8.1 pmol · mm1 · min1) with the nadir occurring at 1 nM (67.7 ± 8.8 pmol · mm1 · min1), whereas high concentrations of NE increased JCl with the maximum rate (131.0 ± 18.5 pmol · mm1 · min1) occurring at 10 µM (*P < 0.05 vs. control; #P < 0.05 vs. minimum JCl value).

To determine the receptor type(s) mediating the descending and ascending limbs of the biphasic response, the strategy was to examine the J_Cl response of isolated THALs to norepinephrine in the presence of selective antagonists of -2 and -adrenergic receptors. Figure 2 depicts the effects of 1 µM norepinephrine on J_Cl in tubules pretreated with the selective -adrenergic receptor antagonist propranolol. This concentration of norepinephrine was chosen because we found that it did not significantly decrease J_Cl from the control rate (Fig. 1) but rather may have slight stimulatory effects on chloride absorption when administered alone. In the presence of 10 µM propranolol, THALs absorbed chloride at a rate of 148.9 ± 16.4 pmol · mm¹ · min¹. Twenty minutes after addition of 1 µM norepinephrine to the bath, J_Cl decreased significantly to a rate of 76.2 ± 13.6 pmol · mm¹ · min¹ (47.7 ± 7.1%; P < 0.01; n = 6). There were no differences in perfusion rates between experimental periods. In addition, time-control experiments determined that propranolol alone does not exert any effects on J_Cl (control: 84.9 ± 10.9 pmol · mm¹ · min¹ vs. propranolol: 85.8 ± 6.5 pmol · mm¹ · min¹; n = 6). These findings indicate that in the absence of -adrenergic receptor activation, 1 µM norepinephrine decreases THAL chloride absorption.

View larger version (20K): [in this window] [in a new window]   Fig. 2.   A concentration of NE (1 µM), which exerted no effects on JCl alone, decreased JCl from 148.9 ± 16.4 to 76.2 ± 13.6 pmol · mm1 · min1, a reduction of 48 ± 7% (*P < 0.01; n = 6) in isolated perfused THALs pretreated with the -adrenergic receptor antagonist propranolol.

To verify that the decrease in chloride absorption observed to norepinephrine in the previous protocol was due to selective activation of -2 adrenergic receptors, we examined the effect of a selective agonist of -2 adrenergic receptors on THAL chloride absorption. Figure 3 illustrates the effect of the selective -2 adrenergic agonist clonidine (10 nM) on J_Cl in six isolated THALs. During the control period, tubules absorbed chloride at a rate of 102.4 ± 9.9 pmol · mm¹ · min¹. After 10 nM clonidine were added to the bath, tubules absorbed chloride at a rate of 54.0 ± 15.7 pmol · mm¹ · min¹. Thus 10 nM clonidine decreased J_Cl by 49.1 ± 11.0% (P < 0.05), further suggesting that the reduction in transport observed to norepinephrine in the presence of -receptor inhibition was mediated by -2 adrenergic receptors.

View larger version (18K): [in this window] [in a new window]   Fig. 3.   -2 Adrenergic receptor agonist clonidine inhibits net JCl in isolated perfused cortical THALs. Addition of 10 nM clonidine to the basolateral bath resulted in a 49% reduction in JCl, from 102.4 ± 9.9 to 54.0 ± 15.7 pmol · mm1 · min1 (*P < 0.05; n = 6).

We next investigated the adrenergic receptor type mediating the ascending limb of the biphasic response of THAL J_Cl to norepinephrine. Figure 4 depicts the effects of 0.1 µM norepinephrine on J_Cl in tubules pretreated with the selective -2 adrenergic receptor antagonist rauwolscine. This concentration of norepinephrine was used because we found that it did not increase J_Cl from the control rate (Fig. 1) but rather may have slight inhibitory effects. In the presence of 1 µM rauwolscine, THALs absorbed chloride at a rate of 85.5 ± 20.1 pmol · mm¹ · min¹. Twenty minutes after addition of norepinephrine to the bath, J_Cl increased significantly to a rate of 111.8 ± 20.1 pmol · mm¹ · min¹ (49.8 ± 23.1%; P < 0.05; n = 7). There were no differences in the perfusion rates between experimental periods, and we have previously reported (41) that 1 µM rauwolscine alone does not exert any effects on THAL J_Cl. Thus these findings indicate that in the absence of -2 adrenergic receptor activation, 0.1 µM norepinephrine increases THAL chloride absorption.

View larger version (20K): [in this window] [in a new window]   Fig. 4.   A concentration of NE (0.1 µM), which exerted no effects on JCl alone, increased JCl from 85.5 ± 20.1 to 111.8 ± 20.1 pmol · mm1 · min1, an increase of 50 ± 23% (*P < 0.05; n = 7) in isolated perfused THALs pretreated with the -2 adrenergic receptor antagonist rauwolscine.

To determine whether the increase in chloride absorption observed to norepinephrine in the previous protocol was due to selective activation of -adrenergic receptors, we examined the effects of selective agonists of - and -1 adrenergic receptors on THAL chloride absorption. Figure 5 illustrates the effect of the selective -adrenergic agonist isoproterenol (10 nM) on J_Cl in six isolated THALs. During the control period, tubules absorbed chloride at a rate of 95.3 ± 11.6 pmol · mm¹ · min¹. After 10 nM isoproterenol were added to the bath, tubules absorbed chloride at a rate of 144.1 ± 15.0 pmol · mm¹ · min¹. Thus 10 nM isoproterenol increased THAL chloride absorption by 56.0 ± 13.9% (P < 0.005).

View larger version (19K): [in this window] [in a new window]   Fig. 5.   -Adrenergic receptor agonist isoproterenol stimulates net JCl in isolated perfused cortical THALs. Addition of 10 nM isoproterenol to the basolateral bath resulted in a 56% increase in JCl, from 95.3 ± 11.6 to 144.0 ± 15.0 pmol · mm1 · min1 (*P < 0.005; n = 6).

Because some investigators have suggested that -1 adrenergic receptors mediate the stimulatory effects of norepinephrine in the THAL (13), we next examined the effect of the selective -1 adrenergic receptor agonist phenylephrine on THAL J_Cl (Fig. 6). During the control period, tubules absorbed chloride at a rate of 107.2 ± 16.9 pmol · mm¹ · min¹. Twenty minutes after adding 10 nM phenylephrine to the bath, tubules absorbed chloride at a rate of 111.6 ± 23.0 pmol · mm¹ · min¹, a rate not different than control. These data indicate that selective activation of -1 adrenergic receptors in the isolated THAL do not increase absorption.

View larger version (20K): [in this window] [in a new window]   Fig. 6.   -1 Adrenergic receptor agonist phenylephrine had no effect on net JCl in isolated perfused cortical THALs. Addition of 10 nM phenylephrine to the basolateral bath resulted in no change in JCl, from 107.2 ± 16.9 to 111.6 ± 23.0 pmol · mm1 · min1 (n = 8).

Previous in vivo studies have reliably demonstrated increased tubular sodium excretion secondary to renal nerve stimulation and liberation of norepinephrine. However, our initial dose-response studies failed to demonstrate significant increases in J_Cl from baseline rates. Therefore, we performed a paired study to simply evaluate the effects of a high concentration (10 µM) of norepinephrine alone on THAL J_Cl (Fig. 7). During the control period, tubules absorbed chloride at a rate of 104.7 ± 6.5 pmol · mm¹ · min¹. Twenty minutes after adding 10 µM norepinephrine to the bath, tubules absorbed chloride at a rate of 148.4 ± 15.4 pmol · mm¹ · min¹, an increase of 41.3 ± 10.2% (n = 5; P < 0.05). These data indicate that high concentrations of norepinephrine increase THAL J_Cl from basal, previously unstimulated values.

View larger version (18K): [in this window] [in a new window]   Fig. 7.   High concentrations of the endogenous sympathetic neurotransmitter NE exert stimulatory effects on net JCl in isolated perfused cortical THALs. Addition of 10 µM NE to the basolateral bath resulted in a 41% increase in JCl, from 104.7 ± 6.5 to 148.4 ± 15.4 pmol · mm1 · min1 (*P < 0.05; n = 5).

	DISCUSSION

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In the present study, we evaluated the J_Cl response of the THAL to incremental concentrations of the native sympathetic neurotransmitter norepinephrine and used selective agonists and antagonists to determine the type of adrenergic receptor(s) mediating those responses. The confounding effects of altered renal hemodynamics or impinging parenchymal autocoids were thus avoided by studying this specific nephron segment in isolation. The current studies demonstrate that 1) norepinephrine produces biphasic effects on THAL J_Cl with low concentrations inhibiting and high concentrations stimulating transport; 2) selective activation of -2 receptors by either clonidine or norepinephrine in the presence of -antagonism inhibits THAL J_Cl; 3) selective activation of -adrenergic receptors by either isoproterenol or norepinephrine in the presence of -2 antagonism stimulates THAL J_Cl; and 4) the selective -1 adrenergic agonist phenylephrine exerted no effects on THAL transport. Taken together, these findings indicate that -2 and -adrenergic receptors differentially regulate the response of the isolated perfused THAL to norepinephrine. The current data suggest that rat THALs respond to adrenergic stimulation in vitro in a concentration-dependent manner, such that abluminal application of norepinephrine biphasically modulates J_Cl via differential activation of -2 and -adrenergic receptors.

We believe these are the first data showing that norepinphrine acts via -2 adrenergic receptors to modulate THAL transport. These data support in vivo data that suggest intrarenal administration of selective -2 agonists exerts direct effects on urinary sodium excretion. Gellai and Ruffolo (18) demonstrated that infusion of the -2 adrenergic receptor agonist clonidine increased osmotic and free water clearance in rats. Furthermore, other investigators have demonstrated that -2 adrenergic receptors mediate inhibition of nephron transport in vitro. Rouse et al. (42) demonstrated that direct -2 adrenergic receptor stimulation inhibits proximal convoluted tubular sodium absorption, whereas Krothapalli et al. (28) demonstrated -2 receptors mediate inhibition of vasopressin-stimulated hydroosmotic water permeability in isolated cortical collecting ducts. The current studies extend these previous findings to the THAL where selective -2 receptor activation with clonidine or by the native neurotransmitter norepinephrine in the presence of -receptor antagonism inhibited chloride absorption.

We found that -adrenergic receptor activation via selective agonist or the native neurotransmitter norepinephrine (in the presence of rauwolscine) stimulated THAL transport. These findings are in agreement with earlier reports suggesting direct stimulatory effects of -adrenergic agonists on tubular reabsorption (29,31,32). Bailly et al. (1) later directly demonstrated that selective activation of -adrenergic receptors increased reabsorption of multiple ions including sodium chloride in the isolated perfused THAL. The current studies demonstrate that the native sympathetic neurotransmitter norepinephrine is also capable of stimulating THAL transport through -receptor activation. Furthermore, the findings of significant increases in THAL J_Cl at high norepinephrine concentrations (Fig. 7) agree with previous reports demonstrating a stimulatory effect of renal sympathetic nerve stimulation on tubular reabsorption.

Other investigators (13) using whole animal preparations have implicated -1 receptors as mediating the increased THAL sodium chloride transport response to adrenergic stimulation. The current studies found no role for -1 adrenergic receptors in THAL transport. This contention is supported by three primary findings from the current studies. First, the -1-selective agonist phenylephrine did not alter THAL J_Cl from control rates (Fig. 6). The affinity of -1 receptors for phenylephrine is ~20-30 nM (25). Because we used 10 nM phenylephrine in the current studies, the lack of an increase in thick limb transport was not likely due to inadequate receptor activation. Second, addition of norepinephrine in the presence of -receptor antagonism inhibited THAL transport. The affinity of -1 receptors for norepinephrine is ~10 nM (23), whereas the concentration of norepinephrine used in these studies was 1 µM. At these concentrations, -1 receptors would likely be saturated by neurotransmitter. If -1 adrenergic receptors mediated a stimulatory effect on J_Cl, it would be expected to offset an -2-mediated reduction in THAL transport. However, this was not the case as THAL transport rates were consistently and significantly reduced by activation of the full complement of -adrenergic receptors (Fig. 2). Third, norepinephrine only modestly increased THAL J_Cl in the presence of rauwolscine (Fig. 4). If -1 adrenergic receptors participated in the response to norepinephrine, we may have observed synergy with the -receptor-mediated effect, in turn, producing a very robust increase in J_Cl similar to that observed with selective -activation alone (Fig. 5). Thus we must conclude that -adrenergic receptors rather than -1 adrenergic receptors mediate the stimulatory effects of adrenergic activation and/or catecholamines on isolated THAL chloride absorption.

The THAL occupies a pivotal role in the control of sodium excretion, absorbing ~25% of the filtered load of sodium chloride (27). Because the THAL is impermeable to water, the absorption of sodium chloride by this nephron segment serves to establish and maintain the hypertonic medullary solute gradient as well as generate dilute tubular fluid (20, 34). Therefore, adrenergic stimuli would be expected to directly alter THAL sodium chloride absorption and have potent effects on urinary sodium chloride excretion. Given the dense innervation and extremely tight juxtaposition of renal sympathetic varicosities with THALs (3), it is impossible to ascertain the exact norepinephrine concentration acting at THAL neuroeffector junctions. Others have reported that the local concentration of catecholamines in the kidney is in the range of 1 nM (26). In the current studies, THAL chloride absorption was at its nadir at identical concentrations (Fig. 1). Given the current findings, it would be expected that any maneuver that increased norepinephrine release and/or concentrations would move to the ascending portion of the concentration-response relationship and increase tubular reabsorption. Alternatively, if -adrenergic receptors are localized adjacent to the neurosecretory apparatus as has been demonstrated in the heart (14), and -2 receptors are extrajunctional as has been postulated by others (45), it may be the case that THAL -receptors are preferentially activated by neural derived norepinephrine released in vivo. The in vitro conditions of the current studies may have served to unmask an effect of -2 adrenergic receptors on THAL transport, and thus smaller increases in norepinephrine concentration may exert stimulatory effects in vivo.

The inability of other investigators to observe a role for -2 adrenergic receptors during activation of the renal nerves using in vivo models is difficult to reconcile with the current experimental design. There are several possibilities that may account for these differences. First, because -2 receptors mediate a myriad of effects in the kidney [e.g., presynaptic inhibition of neurotransmitter release, stimulation of endothelial nitric oxide (NO) production, and alterations in tubular transport], in vivo models may be unable to detect -2-mediated changes in THAL transport. Second, as stated above, some investigators have suggested that -2 receptors are located extrajunctionally and therefore are inaccessible to neurally released norepinephrine (45). Third, because of the mutually antagonistic effects of -2 and -adrenergic receptors on THAL transport, the integrated tubular response to norepinephrine may operate in the shallow portion of the dose-response curve producing effects that are undetectable in the whole animal. Finally, it may be the case that following acute surgical transection of the renal nerves, the surgical stress produces spontaneous discharge of neurotransmitter preventing norepinephrine concentrations from decreasing sufficiently to observe an -2 receptor-mediated tubular effect in vivo. Nonetheless, in the current studies, selective -2 receptor activation produced significant reductions in THAL sodium chloride absorption. Additional studies are required to address the location of -2 receptors and local norepinephrine concentrations under different experimental conditions.

The mechanism of biphasic adrenergic modulation of THAL J_Cl is currently unknown. -Adrenergic activation stimulates THAL transport through a well-established pathway of increased adenylyl cyclase activity with consequent production of cAMP (8). However, the mechanism of -2-mediated inhibition of THAL transport is less certain. We have recently reported that the -2 agonist clonidine inhibits THAL chloride absorption via stimulation of NO synthase and increased NO production (40). Other investigators have recently demonstrated that NO directly inhibits adenylyl cyclase isoform 6 (24), which is present in the THAL (7). cAMP is constitutively produced in the THAL (10) and stimulates THAL chloride absorption (22). Therefore, -2 adrenergic stimulation of endogenous NO production may act in an autocrine manner to inhibit cAMP production and, in turn, THAL J_Cl.

Alternatively, NO has been demonstrated to stimulate guanosine 3',5'-cyclic monophosphate (cGMP) production. We have reported that NO increases cGMP production in collecting duct cells by activating soluble guanylyl cyclase (47) and in the THAL (17), whereas others have shown that cGMP inhibits THAL chloride absorption (36). In addition, we have recently shown that NO-mediated increases in cGMP activate phosphodiesterase (PDE) II (38), which, in turn, enhances cAMP degradation (48). Because norepinephrine has greater affinity for -2 receptors than -receptors (23), it is reasonable to postulate that in the present experiments, -2 receptors were activated by lower concentrations of norepinephrine than -receptors. Thus low levels of adrenergic stimulation and -2-mediated NO production may inhibit THAL transport by directly inhibiting adenylyl cyclase and reducing cAMP production or indirectly by stimulating degradation of existing intracellular cAMP pools via soluble guanylate cyclase and cGMP-dependent activation of PDE II. At higher levels of adrenergic stimulation (Figs. 1 and 7), greater populations of -receptors may be activated with amplification of cAMP-mediated signaling (19), overwhelming the inhibitory effects of NO, and subsequently increasing transport.

In conclusion, the current studies directly demonstrate that norepinephrine exerts biphasic effects on chloride absorption in the isolated perfused THAL. In addition, these studies show that both -2 and -adrenergic receptors are activated in the THAL by sympathetic neurotransmitter. These findings suggest important roles for both stimulatory and inhibitory influences in the regulation of THAL sodium chloride transport. Thus the balance of -2 and -adrenoceptor activation may contribute to the ultimate response of the THAL to adrenergic stimulation.

Perspectives

Physiologically, the THAL is never presented with pure -2 receptor activation. However, the importance of a role for selective -2 receptor stimulation of the THAL can be expressed in the condition of human arterial hypertension. -2 Receptor agonists are frequently used in antihypertensive therapy, specifically to inhibit central sympathetic outflow. The results of the current studies as well as another recent report from our laboratory (40) indicate that there may be additional benefits to -2 agonist therapy via alterations in renal tubular function. These include the inhibition of sodium chloride absorption from the THAL that would promote natriuresis and, because of the primary role of the THAL in the generation of the corticomedullary solute gradient and urinary concentrating mechanism, water excretion as well. Both alterations in renal function would aid in the management of inappropriate salt and water retention in human hypertensives.