Role of PGE2 in alpha 2-induced inhibition of AVP- and cAMP-stimulated H2O, Na+, and urea transport in rat IMCD

Alexander J. Rouch1 and Lúcia H. Kudo2

1 Oklahoma State University College of Osteopathic Medicine, Tulsa, Oklahoma 74107; and 2 Faculdade de Medicina, Universidade de São Paulo, São Paulo, Brazil 01246


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

PGE2 inhibits osmotic water permeability (Pf) in the rat inner medullary collecting duct (IMCD) via cellular events occurring after the stimulation of cAMP, i.e., post-cAMP-dependent events. The alpha 2-agonists also inhibit Pf in the rat IMCD via post-cAMP-dependent events. The purpose of this study was to determine whether PGE2 plays a role in alpha 2-mediated inhibition of Pf, Na+, and urea transport in the rat IMCD. Isolated terminal IMCDs from Wistar rats were perfused to measure, in separate experiments, Pf, lumen-to-bath 22Na+ transport (Jlb), and urea permeability (Pu). Transport was stimulated with 220 pM arginine vasopressin (AVP) or 0.1 mM 8-(4-chlorophenylthio)-cAMP (CPT-cAMP). Indomethacin was used to inhibit endogenous prostaglandin synthesis, and the alpha 2-agonists clonidine, oxymetazoline, and dexmedetomidine were used to test the role of PGE2 in the alpha 2-mediated mechanism that inhibits transport. All agents were added to the bath. Indomethacin at 5 µM significantly elevated CPT-cAMP-stimulated Pf, Jlb, and Pu, and subsequent addition of 100 nM PGE2 reduced these transport parameters. Indomethacin reversed alpha 2 inhibition of CPT-cAMP-stimulated Pf, Jlb, and Pu, and subsequent addition of PGE2 reduced transport in each case. Indomethacin partially reversed alpha 2 inhibition of AVP-stimulated Pf, Jlb, and Pu, and PGE2 reduced transport back to the alpha 2-inhibited level. These results indicate that PGE2 is a second messenger involved in the mechanism of transport inhibition mediated by alpha 2-adrenoceptors via post-cAMP-dependent events in the rat IMCD.

signaling pathways; second messengers; inner medullary collecting duct; alpha 2-adrenoceptor; osmotic water permeability


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

SALT, WATER, AND UREA TRANSPORT in the inner medullary collecting duct (IMCD) play an important role in the renal regulation of salt and water excretion. Arginine vasopressin (AVP) stimulates these transport properties enhancing absorption in the IMCD, and alpha 2-agonists inhibit AVP-dependent transport (20, 29). This inhibitory mechanism has been associated with alpha 2-adrenoceptors coupling to an inhibitory G (Gi) protein that decreases adenylyl cyclase activity, reducing cellular levels of cAMP (8, 11, 20, 34, 36).

Evidence indicates that this inhibitory mechanism occurs in the presence of constant cellular cAMP levels. When water transport in collecting duct nephron segments is stimulated by nonhydrolyzable analogs of cAMP in lieu of AVP, alpha 2-agonists still produce significant inhibition (12, 29). The mechanism therefore appears to be more complex than just reducing adenylyl cyclase activity and must involve other second messengers.

PGE2 has been studied extensively as a potential regulator of renal salt and water excretion and has been shown to affect these transport properties in the collecting duct (2, 14, 15). Nadler et al. (22) reported that PGE2 reduced osmotic water permeability (Pf) stimulated by a nonhydrolyzable cAMP analog in the rat IMCD, indicating that PGE2 inhibits Pf via post-cAMP-dependent events, and inhibition of protein kinase C (PKC) by staurosporine prevented the PGE2-induced inhibition. They also reported that PGE2 increased intracellular calcium concentration ([Ca2+]i) levels. These findings suggest a role for the phospholipase C metabolites in controlling water permeability.

Because both alpha 2-agonists and PGE2 inhibit water permeability via post-cAMP-dependent events in the IMCD, we hypothesized that the alpha 2-inhibitory mechanism involves PGE2 as a second messenger. The purpose of the present study was to test this hypothesis on water, Na+, and urea transport in the isolated rat IMCD. Results indicate that PGE2 indeed plays a role in the alpha 2-inhibitory mechanism of these transport processes in the IMCD. The specific action of PGE2 in this mechanism remains to be determined.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

IMCD segments were isolated and perfused by techniques previously described (4, 26, 28). Wistar rats weighing 120-125 g and fed a standard chow (184 meq Na/kg ) were killed by decapitation, and the kidneys were rapidly removed and cut into small slices that were placed in chilled dissection solution of the same composition as the bathing solution or bath described below. IMCD segments were dissected and isolated from the terminal two-thirds of the inner medulla, i.e., the terminal IMCD (19, 30).

After isolation, the IMCD was transferred to a perfusion chamber on the stage of an inverted microscope and mounted on concentric pipettes that suspended the tubule in the bath. One end of the tubule was drawn by suction into the tip of one of the outer pipettes. The tip of the inner pipette containing the luminal perfusion solution, or perfusate, was advanced into the lumen of the tubule, and perfusion was initiated via air pressure.

The opposite end of the tubule was held in the tip of another glass micropipette where the perfusate accumulated. The tip of this pipette was coated with a viscous silicone liquid (Sylgard 184, Dow Corning) to isolate the perfusate from the bath. Samples of collected perfusate were taken periodically during an experiment with a constant-volume or volumetric pipette. The bath composition was as follows (in mM): 115 NaCl, 25 NaHCO3, 10 sodium acetate, 5 KCl, 1.0 CaCl2, 1.2 MgSO4, 1.2 NaH2PO4, and 5.5 glucose, pH = 7.4. The solution was continuously bubbled with 95% O2-5% CO2. All experiments were conducted at 37°C.

Pf was determined by measuring net fluid flux (Jv) in the presence of a lumen-to-bath osmotic gradient (80-90 to 295-300 mosmol/kgH2O). The perfusion solution was made hypotonic to the bath by reducing NaCl concentration to 50 mM, and rapid perfusion rates of 20-30 nl/min were used to avoid osmotic equilibrium. [14C]inulin at 50-100 counts/min (cpm)/nl in the luminal perfusate was used as the volume marker. Perfusion rate (Vi) was calculated as Vi = Vo(INo/INi), where INi and INo are the inulin cpm per nanoliter in the initial luminal perfusate and collected fluid, respectively, and Vo is the collection rate. Vo was determined directly by measuring the time to fill the volumetric pipette, and Jv (nl · mm-1 · min-1) was then calculated as Jv = (Vi - Vo)/l, where l is the tubule length measured with an eyepiece micrometer. Three timed fluid samples were collected in each experimental period. The Pf of each collection was calculated with established methods and equations (1), and the reported Pf for a given experimental period represents the average of the three determinations.

Lumen-to-bath 22Na+ transport (Jlb) was determined by measuring the disappearance rate of 22Na+ from the lumen. Perfusion and bath solutions were identical, and the composition was the same as the bath solution noted above. Three timed fluid samples were collected in each experimental period, and the reported Jlb for a given experimental period represents the average of the three determinations. Jlb (peq · cm2 · s-1) was calculated as
J<SUB>lb</SUB><IT>=</IT><FR><NU>[Na<SUP><IT>+</IT></SUP>]</NU><DE><IT>A·60</IT></DE></FR><IT> V</IT><SUB>i</SUB><FENCE><IT>1−</IT><FR><NU>C<SUB>o</SUB></NU><DE>C<SUB>i</SUB></DE></FR></FENCE>
where [Na+] is the [Na+] in the bath, A is the luminal area (cm2), and Co and Ci are the collected and perfused activities of 22Na+, respectively.

Pu was determined from the disappearance rate of [14C]urea (50-100 cpm/nl) from the luminal perfusate. As in the Jlb experiments, perfusion and bath solutions were identical. Because there was no net fluid absorption, Pu was calculated with the following equation
P<SUB>u</SUB><IT>=</IT><FR><NU><IT>V</IT><SUB>i</SUB></NU><DE><IT>A</IT></DE></FR> ln <FR><NU>C<SUB>i</SUB></NU><DE>C<SUB>o</SUB></DE></FR>
where A is the inner surface area of the tubule, and Ci and Co are the activities of [14C]urea (cpm/nl) in the initial luminal perfusate and collected fluid, respectively. The reported Pu values represent the averages of three individual samples taken in each experimental period.

Experimental protocols. Once the IMCD was mounted on concentric pipettes, perfusion was initiated and the bath temperature was raised to 37°C in 10-15 min. After an equilibration period of 30-35 min, the sampling procedure for the control period began. After three collections were taken, 220 pM AVP or 0.1 mM 8-(4-chlorophenylthio)-cAMP (CPT-cAMP) was added to the bath, followed by 15-20 min of equilibration and the sampling procedure. Other agents were added in subsequent experimental periods, followed by equilibration and the sampling procedure. The sequence of a given protocol is shown on the abscissa of Figs. 1-4 and described in their legends.

The alpha 2-agonists clonidine, oxymetazoline, and dexmedetomidine were used at 100 nM. In dose-response protocols, we found this to be the level that produced maximal or near- maximal inhibition (17, 29). Dexmedetomidine is the most potent of the three, with clonidine and oxymetazoline demonstrating equal potency.

Source of biochemicals. AVP, CPT-cAMP, oxymetazoline, and epinephrine were purchased from Sigma Chemical (St. Louis, MO). Clonidine and dexmedetomidine were kindly provided by Boehringer Ingelheim (Ridgefield, CT), and by Dr. Riku Aantaa, Chief of Research, Orion-Farmos Pharmaceutical, Turku, Finland, respectively. [14C]inulin was purchased from New England Nuclear (Boston, MA).

Statistical analysis. Data were analyzed with a single-factor ANOVA with repeated measures, and P values between treatments were determined by using the SuperAnova statistical package. P < 0.05 was considered significant.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Table 1 summarizes data showing that indomethacin increased CPT-cAMP-stimulated Pf, Jlb, and Pu and that subsequent addition of PGE2 reversibly decreased each form of transport.

                              
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Table 1.   Effect of indomethacin and PGE2 on CPT-cAMP-stimulated Pf, Jlb, and Pu

Figure 1 shows that clonidine, oxymetazoline, and dexmedetomidine inhibited CPT-cAMP-stimulated Pf by 25, 22, and 82%, respectively. In each protocol, addition of indomethacin increased Pf, and subsequent addition of PGE2 lowered Pf back to the alpha 2-inhibited level in the clonidine and oxymetazoline protocols. In the dexmedetomidine protocol, PGE2 reduced Pf significantly, although not completely back to the alpha 2-inhibited level.


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Fig. 1.   Effect of PGE2 on alpha 2-inhibition of cAMP-stimulated osmotic water permeability (Pf). After the control period, 8-(4-chlorophenylthio)-cAMP (CPT-cAMP) at 0.1 mM (cAMP in figure) increased Pf (P < 0.001). Addition of 100 nM clonidine (Clo; A), oxymetazoline (Oxy; B), or dexmedetomidine (Dex; C) decreased Pf (P < 0.001). Addition of 5 µM indomethacin (I) increased Pf in each protocol (P < 0.01), and addition of 100 nM PGE2 reduced Pf back to the alpha 2-inhibited level in A and B (P < 0.01) but not in C, although PGE2 significantly decreased Pf in C (P < 0.001). The sequence of experimental periods is shown on the x-axis. All agents were added to the bath. P values are with respect to the previous period. Each line represents 1 experiment of 1 inner medullary collecting duct (IMCD) from 1 rat. Values (means ± SE) are shown above each experimental period. * Significantly different from previous period.

Figure 2A shows that PGE2 prevented the indomethacin-induced increase in Pf reported in Table 1 and that the PKC inhibitor staurosporine failed to affect Pf with indomethacin and PGE2 in the bath. In another protocol, staurosporine slightly although significantly increased Pf with indomethacin, PGE2, and the alpha 2-agonist dexmedetomidine in the bath (Fig. 2B).


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Fig. 2.   Effect of staurosporine (St) on PGE2 and alpha 2-inhibition of Pf. A: addition of CPT-cAMP at 0.1 mM to the bath increased Pf (P < 0.001). Addition of 5 µM indomethacin and 100 nM PGE2 did not affect Pf, and Pf was not affected by subsequent addition of 10 nM staurosporine. B: addition of cAMP increased Pf (P < 0.001). Addition of 100 nM dexmedetomidine (D) with 5 µM indomethacin and 100 nM PGE2 decreased Pf (P < 0.001). Addition of 10 nM staurosporine reversibly increased Pf by 22% (P < 0.01). (See legend of Fig. 1 for format.) * Significantly different from previous period.

Figure 3 shows that clonidine, oxymetazoline, and dexmedetomidine inhibited CPT-cAMP-stimulated Jlb by 54, 56, and 77%, respectively. In each protocol indomethacin reversed the inhibition, and subsequent addition of PGE2 lowered Jlb back to the alpha 2-inhibited level.


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Fig. 3.   Effect of PGE2 on alpha 2-inhibition of cAMP-stimulated lumen-to-bath 22Na+ transport (Jlb). After the control period, CPT-cAMP at 0.1 mM increased Jlb (P < 0.001). Addition of 100 nM clonidine (A), oxymetazoline (B), or dexmedetomidine (C) decreased Jlb (P < 0.001). Addition of 5 µM indomethacin increased Jlb in each protocol (P < 0.001), and addition of 100 nM PGE2 reduced Pf back to the alpha 2-inhibited level (P < 0.001). (See legend of Fig. 1 for format.) * Significantly different from previous period.

Figure 4 shows that epinephrine at 100 nM and 1 µM (A and B, respectively) inhibited CPT-cAMP-stimulated Pu. In both protocols, indomethacin significantly increased Pu and subsequent addition of PGE2 lowered Pu. Clonidine and oxymetazoline slightly inhibited CPT-cAMP-stimulated Pu, as shown in Table 2.


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Fig. 4.   Effect of PGE2 on epinephrine-induced inhibition of cAMP-stimulated urea permeability (Pu). Addition of 0.1 mM CPT-cAMP increased Pu (P < 0.001). Epinephrine (Epi) added at 100 nM (A) or 1 µM (B) reduced Pu by 27 and 82%, respectively. Addition of 5 µM indomethacin raised Pu in both protocols (P < 0.01), and subsequent addition of 100 nM PGE2 decreased Pu (P < 0.05). (See legend of Fig. 1 for format.) * Significantly different from previous period.


                              
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Table 2.   Effect of PGE2 on alpha 2-mediated inhibition of cAMP-stimulated Pu

Table 3 summarizes data of the effect of PGE2 on alpha 2-mediated inhibition of AVP-stimulated Pf, Jlb, and Pu. The experimental protocol in these studies was the same as that in Figs. 1, 3, and 4, except that AVP not CPT-cAMP was used to stimulate transport. Clonidine and oxymetazoline inhibited AVP-stimulated Pf, and indomethacin reversed the inhibition by 51 and 25%, respectively. Subsequent addition of PGE2 lowered Pf back to the alpha 2-inhibited level in both protocols. Clonidine and oxymetazoline inhibited AVP-stimulated Jlb, and indomethacin reversed inhibition by 54 and 65%, respectively. Subsequent addition of PGE2 reduced Jlb back to the alpha 2-inhibited level in both protocols. Clonidine inhibited AVP-stimulated Pu by 27%. Indomethacin reversed this inhibition by 52%, and subsequent addition of PGE2 lowered Pu back to the clonidine-inhibited level. The same trend occurred with oxymetazoline, but statistical significance was not achieved.

                              
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Table 3.   Effect of PGE2 on alpha 2-mediated inhibition of AVP-stimulated Pf, Jlb, and Pu


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

PGE2 modulates transport in the collecting duct by affecting multiple cellular signaling pathways [see review by Hébert (13)]. With regard to this modulation, it is important to note species differences, segmental differences along the collecting duct, and experimental conditions. Sonnenburg and Smith (31) reported that in purified rabbit CCD cells PGE2 elevated basal cAMP content and reduced AVP-stimulated cAMP accumulation; however, PGE2 failed to affect AVP-stimulated cAMP in cultured cells. Using different techniques, Noland et al. (23) demonstrated that PGE2 can inhibit AVP-induced cAMP accumulation in cultured rabbit CCD cells. Chabardès et al. (5) reported that PGE2 reduced AVP-stimulated cAMP in dissected rabbit CCDs but not in rat CCDs, and Chen et al. (6) provided transport data consistent with these findings in that PGE2 inhibited AVP-stimulated Pf and Jlb in the rabbit but not rat CCD.

Although PGE2 modulates transport in the rabbit but not the rat CCD and the effect appears to be related to PGE2 regulation of cAMP, PGE2 plays a role in the rat IMCD by an apparent cAMP-independent mechanism. Maeda et al. (20) reported that PGE2 did not affect cAMP content in the rat IMCD with or without AVP. Nadler et al. (22) reported that 100 nM PGE2 reversibly inhibited CPT-cAMP-stimulated Pf in the isolated rat IMCD by ~40%, and the PKC inhibitor staurosporine prevented this inhibition. They concluded that in the rat IMCD PGE2 inhibits Pf via post-cAMP-dependent events that involve PKC.

Evidence regarding the effects of alpha 2-agonists on transport in the collecting duct also requires close attention to the species studied. Chen et al. (6) reported that the alpha 2-agonist clonidine inhibited AVP-stimulated Pf and Jlb in the rat but not the rabbit CCD. Chabardès et al. (5) reported that clonidine reduced AVP-stimulated cAMP accumulation in the rat but not the rabbit CCD. Maeda et al. (20) demonstrated alpha 2-inhibition of both AVP-stimulated cAMP accumulation and AVP-stimulated Pu in the rat IMCD. Edwards et al. (9) reported that alpha 2-agonists inhibited AVP-stimulated cAMP accumulation in rat but not in dog, pig, monkey, or human IMCD.

The classic mechanistic explanation related to alpha 2-adrenoceptors is that they couple to Gi proteins and inhibit adenylyl cyclase activity (8, 11, 25). However, evidence indicates that alpha 2 inhibition of AVP-stimulated transport in the IMCD occurs via post-cAMP-dependent events. We reported that the alpha 2-agonists dexmedetomidine, clonidine, and oxymetazoline reduced CPT-cAMP-stimulated Pf in the rat IMCD (17, 29). These findings indicate that the alpha 2-inhibitory mechanism in the IMCD involves unidentified second messengers. Because both PGE2 and alpha 2-agonists inhibit AVP-stimulated Pf via post-cAMP-dependent events, we hypothesized that PGE2 is one of those messengers associated with alpha 2-induced inhibition.

To examine this hypothesis, we tested the ability of the cyclooxygenase inhibitor indomethacin to reverse alpha 2-inhibition of AVP- and CPT-cAMP-stimulated Pf, Jlb, and Pu in the isolated rat IMCD. In addition, exogenous PGE2 was added to determine whether it would decrease the indomethacin-induced reversal of alpha 2-inhibition. We used the alpha 2-agonists dexmedetomidine, clonidine, and oxymetazoline, which inhibit AVP-stimulated Pf with dose-dependent profiles (17, 29). Dexmedetomidine is nonselective with respect to the alpha 2-subtypes (alpha 2A, alpha 2B, and alpha 2C) (24), clonidine is selective to both alpha 2- and imidazoline receptors (3, 10) and appears to bind to alpha 2B-adrenoceptors in the collecting duct (16, 36), and oxymetazoline is selective to the alpha 2A-adrenoceptor (35). We used these agonists because we knew of their inhibitory capability, and, because they demonstrate different pharmacological binding characteristics, there could be distinguishing characteristics with regard to cellular signaling.

We tested the effects of indomethacin and PGE2 on CPT-cAMP-stimulated Pf, Jlb, and Pu. Table 1 summarizes these data. Indomethacin increased CPT-cAMP-stimulated transport in all three protocols, and subsequent addition of PGE2 reversibly reduced the transport properties. These results expand on the findings of Nadler et al. (22) and demonstrate that endogenous PGE2 plays a role in regulating water, sodium, and urea transport via post-cAMP-dependent events.

Figure 1 contains results from three separate protocols showing that indomethacin reversed alpha 2-induced inhibition caused by clonidine, oxymetazoline, and dexmedetomidine (Fig. 1, A, B, and C, respectively) of CPT-cAMP-stimulated Pf. PGE2 added in the final period reduced Pf back to the alpha 2-inhibited level in (Fig. 1, A and B but not in C) although PGE2 still significantly reduced Pf. These results indicate a role for PGE2 in alpha 2-mediated inhibition of Pf.

In the Pf experiments we tested the effect of the PKC inhibitor staurosporine. Figure 2A shows that PGE2 added to the bath with indomethacin did not affect CPT-cAMP-stimulated Pf. PGE2 prevented the indomethacin-induced increase in CPT-cAMP-stimulated Pf (see Table 1). Subsequent addition of 10 nM staurosporine, the same concentration shown to block PGE2-induced inhibition of CPT-cAMP-stimulated Pf reported by Nadler et al., did not affect Pf. Figure 2B, however, shows that dexmedetomidine added with indomethacin and PGE2 reduced CPT-cAMP-stimulated Pf by 60% and the addition of staurosporine reversibly increased Pf by 22%. This suggests a role for PKC in the alpha 2-mediated inhibition of Pf.

The three alpha 2-agonists inhibited CPT-cAMP-stimulated Jlb (Fig. 3, A, B, and C). In all three cases, indomethacin reversed alpha 2-inhibition and the subsequent addition of PGE2 reduced Jlb back to the alpha 2-inhibited level. Dexmedetomidine inhibited Jlb (77%) more than clonidine (56%) or oxymetazoline (56%). PGE2 accounted for the major portion of the clonidine- and oxymetazoline-induced inhibition (70 and 79%, respectively), whereas it accounted for only 42% in the dexmedetomidine-induced inhibition. Rocha and Koda (27) reported that PGE2 did not affect bath-to-lumen Na+ flux.

In the Pu experiments we used the nonselective adrenergic agonist epinephrine because of our earlier study, which showed that dexmedetomidine is not an effective Pu inhibitor (29). Indomethacin reversed epinephrine-induced inhibition of CPT-cAMP-stimulated Pu, and PGE2 reduced this effect (Fig. 4). These results demonstrate that urea transport can be inhibited via post-cAMP-dependent events and PGE2 plays a role in modulating urea transport. Further data summarized in Table 2 show that clonidine and oxymetazoline significantly lowered CPT-cAMP-stimulated Pu. Indomethacin and PGE2 produced small effects that were significant in the oxymetazoline but not the clonidine experiments.

In addition to our results on CPT-cAMP-stimulated transport, we also tested the effect of PGE2 on alpha 2-mediated inhibition of AVP-stimulated Pf, Jlb, and Pu. These results are summarized in Table 3. Indomethacin reversed clonidine- and oxymetazoline-induced inhibition of AVP-stimulated Pf, and PGE2 reduced Pf back to the alpha 2-inhibited level. The same pattern was observed with clonidine- and oxymetazoline-induced inhibition of AVP-stimulated Jlb. Indomethacin reversed clonidine-induced inhibition of AVP-stimulated Pu, and PGE2 reduced Pu back to the clonidine-inhibited level. Oxymetazoline did not reduce AVP-stimulated Pu with statistical significance. Again, results with alpha 2-mediated inhibition of Pu are not as consistent as with Pf and Jlb, but it still appears that alpha 2-adrenoceptors are involved in the modulation of AVP-stimulated Pu.

Reversal of alpha 2-induced inhibition of transport by indomethacin was observed regardless of the method of transport stimulation (AVP or CPT-cAMP) and of the alpha 2-agonist used. No major distinguishing differences between clonidine- and oxymetazoline-induced inhibition were observed other than clonidine inhibited AVP-stimulated Pu whereas those results with oxymetazoline failed to produce statistical significance (Table 3). One observation worth noting is that indomethacin partially reversed clonidine- and oxymetazoline-induced inhibition of AVP-stimulated Pf and Jlb; i.e., the AVP period was significantly higher than the AVP+alpha 2-agonist+indomethacin period whereas it completely reversed clonidine- and oxymetazoline-induced inhibition of CPT-cAMP-stimulated Pf and Jlb and the CPT-cAMP period was not significantly different from the CPT-cAMP+alpha 2-agonist+indomethacin period. Endogenous production of cAMP via AVP likely provided a more effective transport stimulus that indomethacin at 5 µM did not block completely. AVP increases [Ca2+]i levels in the rat IMCD (32); thus it is also possible [Ca2+]i plays a role in alpha 2-inhibitory mechanism. We did not measure [Ca2+]i levels, but a zero-calcium bath did not reduce alpha 2-mediated inhibition of AVP-stimulated Pf in the rat IMCD (results not shown). We are unaware of any results with regard to the effect of alpha 2-agonists on phosphoinositide hydrolysis in the collecting duct; however, alpha 2-adrenoceptors have been shown to activate multiple signal transduction pathways, including those associated with arachidonic acid and the phosphoinositide system (7, 18, 21, 33).

Indomethacin partially reversed dexmedetomidine-induced inhibition of CPT-cAMP-stimulated Pf and Jlb (Figs. 1C and 3C). Dexmedetomidine, which as stated earlier is nonselective for the alpha 2-adrenoceptor subtypes, produces greater inhibition of AVP-stimulated transport than either clonidine or oxymetazoline. This could be due to higher potency, efficacy, or both. Evidence has been conflicting as to which alpha 2-adrenoceptor subtypes exist in the IMCD. Some results demonstrate the alpha 2B over the alpha 2A whereas other results suggest the opposite (34, 36). Our results could suggest that multiple adrenoceptors are responsible for the higher inhibition produced by dexmedetomidine. Because PGE2 accounts for a smaller portion of the inhibition produced by dexmedetomidine compared with clonidine and oxymetazoline, other second messengers could be involved in dexmedetomidine-induced inhibition. Future studies are required to determine these other messengers as well as alpha 2-adrenoceptor subtypes.

Finally, it is recognized that indomethacin can influence other cellular events besides cyclooxygenase inhibition, and thus it is conceivable that another indomethacin-induced event occurred in our experiments. We used indomethacin because it has been the most commonly used agent in these kinds of experiments. In addition to indomethacin we have tested naproxen and ketorolac, two other potent inhibitors of cyclooxygenase, on dexmedetomidine-induced inhibition of CPT-cAMP-stimulated Pf by using the same protocol as in Fig. 1C (i.e., replacing indomethacin with naproxen or ketorolac). Both agents reversed alpha 2-mediated inhibition to the same degree as indomethacin, and subsequent addition of PGE2 significantly reduced Pf (these data are not shown). Thus we think it is unlikely that endogenous prostaglandins were not the major cellular messengers being affected in this study.

In summary, results of this study indicate that alpha 2-adrenoceptors in the rat IMCD play a role in regulating water, sodium, and urea transport via a cellular mechanism that involves post-cAMP dependent events, which involve, among other second messengers, PGE2. PKC appears to be involved as well. This mechanism could involve multiple alpha 2-adrenoceptors and signaling pathways.


    ACKNOWLEDGEMENTS

This study was supported by National Science Foundation Career Award IBN-9507444 and CNPq-grant 303259 from Brazil (L. H. Kudo). Portions of this study have been published previously in abstract form (FASEB J 13: A728, 1999).


    FOOTNOTES

Address for reprint requests and other correspondence: A. J. Rouch, Oklahoma State Univ. College of Osteopathic Medicine, 1111 W. 17th St., Tulsa, OK 74107 (E-mail: rouch{at}osu-com.okstate.edu).

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. §1734 solely to indicate this fact.

Received 28 September 1999; accepted in final form 16 March 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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