INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

ONE STRATEGY ORGANISMS USE to limit the toxic effects of xenobiotics is excretory transport. Recent evidence indicates that aquatic organisms possess one or more multidrug resistance-like transport mechanisms that restrict xenobiotic uptake, drive xenobiotic excretion, and thus increase chances of survival in polluted environments (reviewed in Ref. 10). For example, Western blotting shows the presence of proteins immunologically related to p-glycoprotein in tissues from marine and freshwater invertebrates and vertebrates, and binding assays with membrane fractions show xenobiotic binding with the broad specificity characteristics of p-glycoprotein. Also, the ability of sponges, marine mussels, freshwater clams, and marine worms to limit accumulation of xenobiotics is reduced after exposure to p-glycoprotein modifiers, e.g., verapamil. Finally, several studies show induction of the multixenobiotic resistance phenotype in organisms exposed to polluted water (10). Together, these findings make a case for the presence of one or more xenobiotic excretion systems in these organisms. These transport systems appear to function at some basal level in animals living in unpolluted environments, and they are upregulated on exposure to foreign chemicals.

In recent years, we focused on the function and regulation of ATP-driven xenobiotic export pumps in killifish renal proximal tubules (12-14, 17), teleost analogs of p-glycoprotein, and the multidrug resistance-associated protein isoform 2 (MRP2). These transporters exhibit very wide but differing specificities. In general, p-glycoprotein handles cationic and uncharged compounds (3), whereas MRP2 transports anionic and uncharged compounds (9). Clearly, there is some overlap in the chemicals handled by these two multispecific transporters. In all vertebrates studied to date, such transporters are expressed at high levels in the luminal membranes of epithelial cells in the renal proximal tubule, intestine, and liver (9, 16, 21), which puts them in the right location to excrete xenobiotics into urine, bile, and gut lumen. We recently found MRP2, but not p-glycoprotein, expression in the dogfish shark rectal salt gland, an excretory tissue noted for its ability to excrete salt and fluid rather than xenobiotics (15). In this tissue, as in other excretory epithelia, MRP2 was localized to the luminal membrane of the epithelial cells. With the use of confocal microscopy, we showed that isolated rectal gland tubules exhibited MRP2-mediated transport of two large organic anions, a fluorescent methotrexate derivative and sulforhodamine 101-free acid.

The present study addresses the regulation of MRP2 function in shark rectal gland tubules. The starting point for these experiments was the observation that in teleost renal proximal tubule MRP2-mediated transport was regulated by endothelin (ET) acting through an ET_B receptor and protein kinase C (PKC) (13). Hormone binding to the basolateral receptor activated PKC, which signaled decreased transport at the luminal membrane. Similar to teleosts, sharks produce ET analogs, and shark tissues express ET receptors that are pharmacologically similar to those in other vertebrates (2, 11). We show here a similar regulatory mechanism for MRP2-mediated transport in shark rectal salt gland tubules. In addition, we found evidence that cAMP affects transporter function by a protein kinase A (PKA)-independent mechanism.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Chemicals. Phorbol 12-myristate 13-acetate (PMA), calphostin C, and H-89 were purchased from Molecular Probes (Eugene, OR). Sulforhodamine 101-free acid, forskolin, and RP-cAMP were purchased from Sigma Chemical (St. Louis, MO). ET-1, ET-2, ET-3, Big-ET-1, ET_A receptor antagonist JKC-301, and ET_B receptor antagonist RES-701-1 were obtained from Peninsula Laboratories (Belmont, CA). All other chemicals were obtained from commercial sources at the highest purity available.

Animals. Male and female dogfish sharks (2-4 kg) were collected outside of Frenchman's Bay, ME, and maintained at the Mount Desert Island Biological Laboratory in large circular tanks with running seawater. After sharks were pithed, glands were removed and suspended in ice-cold elasmobranch saline [containing (in mM) 270 NaCl, 4 KCl, 3 MgCl₂, 2.5 CaCl₂, 8 NaHCO₃, 1 KH₂PO₄, 0.5 Na₂SO₄, and 350 urea, pH adjusted to 7.8 by equilibration with 99% O₂-1% CO₂].

Tubule isolation. Tubules were isolated at 4°C using a modification of the procedure of Ecay and Valentich (1). The ends of each gland and the connective tissue were removed, and the gland was minced into 1-mm cubes using razor blades. The tissue was washed three times with 15 ml of elasmobranch saline to remove blood cells and digested in 25 ml of elasmobranch saline containing 0.05 g of collagenase (Boehringer Mannheim). During digestion, the tubes were agitated and gassed with 99% O₂-1% CO₂. After 25 min, the digest was drawn in and out of a 25-ml pipette 10 times to break up the tubules. The mixture was allowed to settle for 30 s, and the supernatant was then removed and put into a 50-ml centrifuge tube. The supernatant was centrifuged at 1,000 rpm for 45 s, and the resulting tubule pellet was resuspended in 10 ml of gassed elasmobranch saline. The tubules were stored at 4°C until used.

Confocal fluorescence microscopy. Rectal gland tubules were pipetted into a chamber (Bionique) containing 1 ml gassed elasmobranch saline, 5 µM sulforhodamine 101, and inhibitors. This fluorescent organic anion was chosen as substrate because it is relatively resistant to photobleaching and its fluorescence is insensitive to medium pH in the range 6.0-8.0 (12, 15). The chamber floor was a 4 × 4-cm glass coverslip to which the tubules adhered lightly and through which the tubules could be viewed by means of an inverted microscope; the chamber cover was a small petri dish. Tubule incubation and confocal microscopy were carried out at 18-20°C. The chamber containing renal tubules was mounted onto the stage of an Olympus FluoView inverted confocal laser-scanning microscope and viewed through a ×40 water immersion objective (numerical aperture 1.15). Excitation was provided by the 568-nm line of a krypton laser. A 570-nm dichroic filter and a 575-nm long-pass emission filter were used. Neutral density filters and low laser intensity were used to avoid photobleaching. With the photomultiplier gain set to give an average luminal fluorescence intensity of 1,500-3,000 (on a scale of 0-4,096), tissue autofluorescence was undetectable. To obtain an image, dye-loaded tubules in the chamber were viewed under reduced, transmitted light illumination, and a single tubule with well-defined lumen and undamaged epithelium was selected. The plane of focus was adjusted to cut through the center of the tubular lumen, and an image was acquired by averaging four scans. The confocal image was viewed on a high-resolution monitor and saved to an optical disk. With the use of this system, we found a linear relationship between fluorescence intensity and dye concentration (Miller et al., unpublished data). However, because of the many uncertainties in relating cellular fluorescence to actual compound concentration in cells and tissues with complex geometry, data are reported here as average measured pixel intensity rather than estimated dye concentration.

Immunostaining. Rectal glands were washed in elasmobranch saline and fixed for 10 min at room temperature in 2% (vol/vol) formaldehyde-0.1% (vol/vol) glutaraldehyde. After being washed in PBS, tubules were permeabilized in 1% (vol/vol) Triton X-100 in PBS, washed, and incubated for 90 min at 37°C in PBS with 20 mg/ml of a sheep-derived, anti-ET_B receptor polyclonal antibody. After the tubules were washed, antibody binding was detected using a FITC-labeled donkey anti-sheep IgG (1:100) for 60 min at 37°C. Tubules were viewed with the Olympus Fluoview confocal laser-scanning microscope (488-nm line of Argon laser, 510-nm dichroic filter, and 515-nm long-pass emission filter).

Statistics. Data are given as means ± SE. Means were considered to be statistically different when the probability value was P < 0.05 with the use of an unpaired t-test.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

We previously reported that sulforhodamine 101 crosses shark rectal gland tubules by a two-step process (15). Consistent with simple diffusion, uptake into cells is not concentrative and is not affected by any inhibitor of transport or metabolism. In contrast, transport from cell to lumen is concentrative and inhibited by KCN, leukotriene C4, estradiol glucuronide, and chlorodinitrobenzene. Cell-to-lumen transport appears to be mediated by the xenobiotic export pump MRP2, which has been immunolocalized to the luminal membrane of rectal gland epithelial cells (15). Figure 1 shows representative transmitted light and confocal images of a control tubule incubated in medium containing 5 µM sulforhodamine 101. Fluorescence in the lumen is clearly higher than in the cells, and cellular fluorescence is lower than in the bath (Fig. 1C). A similar pattern of fluorescence was reported previously for rectal gland tubules exposed to sulforhodamine 101 (15).

View larger version (52K): [in this window] [in a new window]   Fig. 1.   Representative transmitted light (A) and confocal (B) images of dogfish shark rectal salt gland tubules. Tissue was incubated in medium containing 5 µM sulforhodamine 101. After 30 min, images were acquired as described in MATERIALS AND METHODS. C: fluorescence intensity plot across the tubule and along the white line shown in B. Fluorescence intensity clearly falls in going from medium to cell and rises substantially in going from cell to lumen.

The two experiments shown in Fig. 2 indicate that the addition of 0.1-10 nM ET-1 to the bath reduced cell-to-lumen transport of 5 µM sulforhodamine 101 in a concentration-dependent manner. With 0.1 nM ET-1, luminal fluorescence was reduced by 33 ± 10% (P < 0.01), whereas with 1 nM ET-1, luminal fluorescence was reduced by 58 ± 4% (P < 0.01; Fig. 2A). Increasing the ET-1 concentration from 1 to 10 nM caused a further reduction in transport (Fig. 2B). Cellular fluorescence was not affected by any concentration of ET-1. Figure 3 shows the early time course of 0.5 µM sulforhodamine 101 transport in rectal gland tubules. In agreement with previous experiments (15), cellular and luminal fluorescence in control tubules rose rapidly and reached steady-state values after ~10 min. For tubules exposed to 10 nM ET-1 from time 0 on, cellular fluorescence approximated control values, but luminal fluorescence was significantly lower than controls at all times tested (Fig. 3). At the earliest time for which we have data, 5 min, ET-1 reduced luminal fluorescence by 75%. Together, these data demonstrate that ET-1 is a potent and rapid-acting effector of MRP2-mediated transport in isolated shark rectal gland tubules.

View larger version (14K): [in this window] [in a new window]   Fig. 2.   Inhibition of sulforhodamine 101 transport by endothelin-1 (ET-1). Tubules were incubated in medium containing 5 µM sulforhodamine 101 alone (control) or with 0.1-10 nM ET-1. After 30 min, images were acquired as described in MATERIALS AND METHODS. Results of 2 experiments are shown. Data are given as means ± SE for 12-20 tubules. All ET-1 concentrations tested reduced luminal fluorescence (**P < 0.01). Concn, concentration.

View larger version (12K): [in this window] [in a new window]   Fig. 3.   Time course of sulforhodamine 101 accumulation. Tubules were incubated for the indicated time in medium containing 5 µM sulforhodamine 101 [control (Con)] or 5 µM sulforhodamine 101 plus 10 nM ET-1. Images were acquired as described in MATERIALS AND METHODS. Data are given as means ± SE for 10 tubules. ET-1 significantly reduced luminal fluorescence at all times, **P < 0.01.

ETs are produced as larger inactive precursors, the prepro-ETs. These are enzymatically cleaved to form the pro- or Big-ETs. Big-ETs are, in turn, converted into biologically active ETs by specific ET-converting enzymes (ECE) (6, 20). When isolated rectal gland tubules were incubated in medium with 2 µM sulforhodamine 101 plus 10 nM big-ET-1, luminal fluorescence was reduced to 65 ± 8% of control values (Fig. 4), suggesting that the tubules contained ECE activity. To determine whether this was the case, we exposed ET-1-treated tubules to the protease inhibitor phosphoramidon and measured sulforhodamine 101 transport. Phosphoramidon by itself (10 µM) did not affect sulforhodamine 101 transport (not shown). However, phosphoramidon did prevent the reduction in luminal fluorescence caused by 1 nM ET-1 (Fig. 4).

View larger version (15K): [in this window] [in a new window]   Fig. 4.   Inhibition of sulforhodamine 101 transport by Big-ET-1. Tubules were incubated in medium containing 5 µM sulforhodamine 101 alone (control) or with 10 nM big-ET-1 or 10 µM phosphoramidon (phosphor). After 30 min, images were acquired as described in MATERIALS AND METHODS. Data are given as means ± SE for 18-20 tubules. Big-ET-1 significantly reduced luminal fluorescence (**P < 0.01).

ETs interact with two distinct receptor subtypes, ET_A and ET_B, which belong to the superfamily of G protein-coupled receptors (20). The receptor subtypes are pharmacologically distinct, i.e., the ET_A receptor displays preferential affinity for ET-1 and ET-2 over ET-3, whereas the ET_B receptor exhibits roughly equal affinity for all three polypeptides. On the basis of affinity profiles and the ability of specific receptor antagonists to block the action of ET-1, our data for killifish renal proximal tubules suggest ET action through a B receptor subtype (13). To identify the receptor subtype responsible for ET-1 action in rectal gland tubules, specific antagonists for each receptor subtype were tested on their ability to reverse the ET-1-induced reduction in sulforhodamine 101 secretion in rectal gland tubules. Preliminary experiments showed that (as in the renal proximal tubule; Ref. 13) neither receptor antagonist by itself affected transport (not shown). However, at 100 nM, the ET_B receptor antagonist RES-701-1 prevented the ET-1 reduction in sulforhodamine 101 transport, but the ET_A receptor antagonist JKC-301 did not (Fig. 5).

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Effect of ET-receptor antagonists on ET-1 action. Tubules were incubated in medium containing 5 µM sulforhodamine 101 alone (control) or with 10 nM ET-1 or ET-1 plus 100 nM RES-701-1 or JKC-301. After 30 min, images were acquired as described in MATERIALS AND METHODS. Data are given as means ± SE for 17-20 tubules. ET-1 significantly reduced luminal fluorescence (**P < 0.01), and this inhibition was reversed by RES-701-1 but not by JKC-301.

Taken together, the transport data indicate that an ET_B receptor regulates sulforhodamine 101 transport in rectal gland tubules. In an initial attempt to identify the location of the receptor, we immunostained tubules using mammalian antibodies to the A- and B-type ET receptors. Confocal micrographs clearly showed specific plasma membrane staining for the B-type receptor (Fig. 6). Antibody staining was predominantly associated with the basolateral membrane; no staining was found at the luminal membrane. These results place the shark form of the B-type ET receptor at the correct pole of the cells for rapid activation by hormone added to the bath.

View larger version (40K): [in this window] [in a new window]   Fig. 6.   Immunolocalization of ETB receptors in rectal gland tubules. Tubules were fixed, permeabilized, and exposed to antibodies as described in MATERIALS AND METHODS. A and B: corresponding transmitted light and confocal images for a tubule exposed to secondary but not primary antibody; no staining is evident. C: confocal image of tubules exposed to primary (sheep-derived anti-ETB receptor polyclonal antibody) and secondary (FITC-labeled donkey anti-sheep IgG) antibodies. It shows intense staining at the basolateral pole of the epithelial cells, but no apical staining. B: bar indicates 20 µm.

One general model for ET signaling involves activation of an ET receptor-coupled G protein, followed by activation of a phospholipase and PKC. Previous experiments have shown that MRP2-mediated transport in killifish renal proximal tubules is controlled by ET-1 acting through PKC (13). To determine whether the same pattern held for rectal gland tubules, we exposed tubules to phorbol esters or protein kinase inhibitors during 30-min sulforhodamine 101 transport experiments. As shown in Fig. 7, 10 and 100 nM PMA reduced luminal accumulation of sulforhodamine 101. A phorbol ester that does not activate PKC, 4-PDD, at 100 nM had no effects on transport. The PKC-selective inhibitor calphostin C (100 nM) by itself was without effect; however, when used in combination with 100 nM PMA, calphostin C prevented the PMA-induced reduction in sulforhodamine 101 transport into the tubular lumen (Fig. 7). Thus, as in killifish renal proximal tubules, activation of PKC reduced MRP2-mediated secretion in shark rectal salt gland tubules.

View larger version (18K): [in this window] [in a new window]   Fig. 7.   Effects of protein kinase C (PKC) activation and inhibition on sulforhodamine 101 transport. Tubules were incubated in medium containing 5 µM sulforhodamine 101 alone (control) or with the indicated additions. After 30 min, images were acquired as described in MATERIALS AND METHODS. Data are given as means ± SE for 10-19 tubules. Phorbol 12-myristate 13-acetate (PMA) significantly reduced luminal fluorescence (**P < 0.01).

To determine whether PKC was involved in ET regulation of MRP2, we exposed tubules to 1 nM ET-1 in the absence and presence of 100 nM calphostin C and measured sulforhodamine 101 transport. Figure 8A shows that the PKC-selective inhibitor prevented the reduction of transport by ET-1. In contrast, the PKA-selective inhibitor H-89 (10 µM) was without effect (Fig. 8B). These findings implicate PKC in the chain of cellular events that connect activation of an ET_B receptor at the basolateral membrane with reduced transport by MRP2 at the luminal membrane.

View larger version (21K): [in this window] [in a new window]   Fig. 8.   Effects of PKC (A) and protein kinase A (PKA; B) inhibition on ET-1 action. Tubules were incubated in medium containing 5 µM sulforhodamine 101 alone (control) or with the indicated additions. After 30 min, images were acquired as described in MATERIALS AND METHODS. Data are given as means ± SE for 16-20 tubules. ET-1 significantly reduced luminal fluorescence (**P < 0.01), and calphostin C prevented this reduction.

Salt secretion in rectal gland tubules is activated when gland cAMP levels rise as a result of hormonal stimulation of adenyl cyclase. Although the data in Fig. 8 indicate that ET-1 does not act through PKA to reduce MRP2-mediated transport, they do not rule out the possibility that other hormones could affect transport through PKA. To test this possibility, we exposed tubules to the phosphodiesterase inhibitor forskolin and measured transport of sulforhodamine 101. The concentration of the drug chosen was one that rapidly increases cAMP content of rectal gland tissue and that activates salt secretion in intact glands (18). Figure 9 shows that forskolin did indeed reduce luminal accumulation of sulforhodamine 101, but the reduction was not altered by addition of the PKA-selective inhibitor H-89 to the medium. This result suggests that the effects of forskolin were not mediated by PKA. One possibility was that increased cAMP levels reduced transport directly by competition for transport at MRP2. To test this, we incubated tubules with RP-cAMP, a cAMP analog that does not activate PKA. This compound caused a concentration-dependent decrease in luminal sulforhodamine 101 accumulation (Fig. 9), suggesting that cAMP could interact directly with the transporter, possibly competing for excretory transport.

View larger version (15K): [in this window] [in a new window]   Fig. 9.   Inhibition of sulforhodamine 101 transport by forskolin and a cAMP analog. Tubules were incubated in medium containing 5 µM sulforhodamine 101 alone (control) or with the indicated additions. After 30 min, images were acquired as described in MATERIALS AND METHODS. Data are given as means ± SE for 16-18 tubules. Forskolin and RP-cAMP significantly reduced luminal fluorescence (**P < 0.01).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The results of the present study with isolated shark rectal salt gland tubules show that the ATP-driven drug export pump MRP2 is under hormonal control. As in killifish renal proximal tubules (13), transport on MRP2 was reduced by subnanomolar to nanomolar concentrations of the polypeptide hormone ET-1. The hormone acted very rapidly with significant reductions in transport being evident after 5 min of exposure. Transport was also reduced by Big-ET-1, a prohormone. However, the effects of Big-ET-1 were attenuated by phosphoramidon, an inhibitor of ECE, suggesting that rectal gland tubules could convert the prohormone to ET-1. Presumably, these tubules possess a shark form of ECE on the basolateral surface, implying that they may also store and release ET. Experiments with specific ET receptor antagonists indicated that ET-1 was acting through a B-type receptor, and immunostaining with an antibody that reacts with mammalian and teleost ET_B receptors indicated a basolateral, not luminal, localization. This basolateral localization of the ET_B receptor explains the rapid time course of action of the hormone when it is added to the bath, i.e., applied to the basolateral surface of the tubules.

ET receptors are G protein coupled, acting through second messenger pathways, such as PKC or PKA. In killifish renal proximal tubules, ET-1 reduced MRP2-mediated transport by acting through PKC (13). The present data show that this pattern also holds for the shark rectal salt gland. In rectal gland tubules, ET-1 action was mimicked by PMA, a phorbol ester that activates PKC, but not by 4-PDD, a PMA analog that does not activate the protein kinase. PMA was without effect in tubules treated with the PKC-selective inhibitor calphostin C. The PKA-selective inhibitor H-89 did not block PMA action. Consistent with ET-1 acting through PKC, hormone action was blocked by calphostin C but not by H-89.

Figure 10 summarizes the chain of events that signal reductions in MRP2-mediated sulforhodamine 101 transport in shark rectal salt gland tubules. This scheme and the present results are very similar to those recently published for control of MRP2 and p-glycoprotein in killifish renal proximal tubules (13). In both tissues, ET-1 signaled through a basolateral ET_B receptor and through PKC to rapidly (minutes) reduce transport from cell to lumen. These are the only data available on rapid hormonal regulation of MRP2 function in any epithelium. The exact mechanism by which transporter function is modified is not known in either tissue. In both, ET-1 action was so rapid that it cannot be explained by decreased transporter synthesis. Rather, it is likely that PKC signaled either a change in transporter function, perhaps by phosphorylation, or rapid removal from the plasma membrane to a subapical compartment. In this regard, in a recent confocal imaging study, exposing killifish proximal tubules to ET-1 caused no detectable broadening of the apical MRP2 immunostaining pattern (20). Thus, if the transporter had moved from the apical plasma membrane to a subapical compartment, that movement could not be resolved at the light level.

View larger version (54K): [in this window] [in a new window]   Fig. 10.   Model showing the sequence of events in ET regulation of multidrug resistance-associated protein isoform 2 (MRP2)-mediated transport and potential consequences for gland salt secretion. Data presented in the present study indicate that ET acting at a basolateral ETB receptor activates PKC, which, in turn, reduces transport from cell to lumen on MRP2. The data also suggest that cyclic nucleotides are MRP2 substrates. Reduced cyclic nucleotide efflux on MRP2 could sustain PKA and protein kinase G (PKG) activation and thus salt secretion.

Salt secretion in the shark rectal gland is turned on by several hormones that elevate tissue cAMP levels and activate through PKA a shark homolog of the chloride channel, cystic fibrosis transmembrane conductance regulator (4, 18). Although we found no evidence that PKA was involved in the action of ET-1 on MRP2, forskolin did inhibit transport (present study). This inhibition was not attenuated by H-89, indicating an action of forskolin that did not involve PKA. Interestingly, a cAMP analog that does not activate PKA (RP-cAMP) reduced sulforhodamine 101 transport, and this reduction was not affected by H-89. Thus the most likely explanation for forskolin's effects on MRP2-mediated transport is that the drug raised tissue cAMP levels and the second messenger interacted directly with the transporter, competing with sulforhodamine 101 for binding and perhaps transport. This line of reasoning suggests that rectal gland stimulation of salt secretion by hormones that activate adenyl cyclase (and possibly guanyl cyclase) could also reduce excretory transport, not by PKA activation, but by competition for transport. It also suggests that cyclic nucleotides may be substrates for the transporter, a suggestion that has implications with regard to control of salt excretion (see Perspectives).

Perspectives

If shark MRP2 handled cyclic nucleotides, then the transporter could provide an additional mechanism for turning off cyclic nucleotide signaling, efflux of the second messenger. Such a link between MRP2 and control of salt secretion suggests chemicals that impact MRP2 function through signaling (ET and other hormones acting through PKC) or competition for transport (glutathione conjugates) could alter the balance of factors that control salt secretion. Consider, for example, the control of salt secretion by C-type natriuretic peptide (CNP), which can act directly on rectal gland epithelial cells to increase cGMP production and stimulate salt secretion (5, 19). Staurosporine reduces the stimulatory effect of CNP, suggesting involvement of PKC (19). Surprisingly, phorbol ester by itself did not stimulate secretion, nor did 8-bromo-cGMP. However, when given in concert, phorbol ester plus 8-bromo-cGMP did stimulate salt secretion. These results were interpreted by Silva et al. (19) to mean that CNP signaled through both PKC and protein kinase G, but that both systems had to be activated before salt secretion increased. The present results suggesting that MRP2 can transport cyclic nucleotides and that transport is inversely correlated with PKC activity suggest another interpretation: CNP directly stimulates salt secretion in the rectal gland by activating signaling through guanyl cyclase, but activation of PKC serves, at least in part, to reduce MRP2-mediated efflux of cGMP and thus sustain elevated intracellular cyclic nucleotide levels. Additional experiments are needed to directly test this hypothesis.