V1 Receptor Stimulation Research Articles

Electrolyte handling in the isolated perfused rat kidney: demonstration of vasopressin V2-receptor-dependent calcium reabsorption

Background The most profound effect of vasopressin on the kidney is to increase water reabsorption through V2-receptor (V2R) stimulation, but there are also data suggesting effects on calcium transport. To address this issue, we have established an isolated perfused kidney model with accurate pressure control, to directly study the effects of V2R stimulation on kidney function, isolated from systemic effects. Methods The role of V2R in renal calcium handling was studied in isolated rat kidneys using a new pressure control system that uses a calibration curve to compensate for the internal pressure drop up to the tip of the perfusion cannula. Results Kidneys subjected to V2R stimulation using desmopressin (DDAVP) displayed stable osmolality and calcium reabsorption throughout the experiment, whereas kidneys not administered DDAVP exhibited a simultaneous fall in urine osmolality and calcium reabsorption. Epithelial sodium channel (ENaC) inhibition using amiloride resulted in a marked increase in potassium reabsorption along with decreased sodium reabsorption. Conclusions A stable isolated perfused kidney model with computer-controlled pressure regulation was developed, which retained key physiological functions. The preparation responds to pharmacological inhibition of ENaC channels and activation of V2R. Using the model, the dynamic effects of V2R stimulation on calcium handling and urine osmolality could be visualised. The study thereby provides evidence for a stimulatory role of V2R in renal calcium reabsorption.

Vasopressin receptor subtypes and renal sodium transport.

In mammals, three subtypes of V-receptors have been identified in the kidney. The effects of vasopressin, a hormone synthesized in the hypothalamus, are triggered by three distinct receptor isoforms: V2, V1a, and V1b. Stimulation of V2-receptors regulates urine osmotic concentration by increasing sodium reabsorption in the thick ascending limb of the loop of Henle and enhancing osmotic permeability of the epithelium cells in the collecting duct. Stimulation of V1a-receptors inhibits renal sodium reabsorption and induces natriuresis, comparable to the effect of the diuretic furosemide, in the thick ascending limb of the loop of Henle. Stimulation of V1b-receptors induces potassium secretion in the final parts of the distal segments and initial parts of the collecting ducts. In this review, we discuss the role of vasopressin and its interaction with V-receptor subtypes in natriuresis and for stabilizing the physicochemical parameters of the internal environment and water-salt homeostasis in humans. A better understanding of these systems and their regulation is necessary to facilitate identification of additional system components and mechanisms, clarify their contribution during various normal and pathological functional states, and suggest novel strategies for the development of therapeutic interventions.

Role of vasopressin in current anesthetic practice.

Arginine vasopressin (AVP), also known as antidiuretic hormone, is a peptide endogenously secreted by the posterior pituitary in response to hyperosmolar plasma or systemic hypoperfusion states. When administered intravenously, it causes an intense peripheral vasoconstriction through stimulation of V1 receptors on the vascular smooth muscle. Patients in refractory shock associated with severe sepsis, cardiogenic or vasodilatory shock, or cardiopulmonary bypass have inappropriately low plasma levels of AVP (‘relative vasopressin deficiency’) and supersensitivity to exogenously-administered AVP. Low doses of AVP and its synthetic analog terlipressin can restore vasomotor tone in conditions that are resistant to catecholamines, with preservation of renal blood flow and urine output. They are also useful in the treatment of refractory arterial hypotension in patients chronically treated with renin-angiotensin system inhibitors, cardiac arrest, or bleeding esophageal varices. In the perioperative setting, they represent attractive adjunct vasopressors in advanced shock states that are unresponsive to conventional therapeutic strategies.

Stimulation of V1a receptor increases renal uric acid clearance via urate transporters: insight into pathogenesis of hypouricemia in SIADH.

Hypouricemia is pathognomonic in syndrome of inappropriate secretion of antidiuretic hormone (SIADH) but the underlying mechanism remains unclear. Based on the previous studies, we hypothesized that V1a receptor may play a principal role in inducing hypouricemia in SIADH and examined uric acid metabolism using a rat model. Terlipressin (25ng/h), a selective V1a agonist, was subcutaneously infused to 7-week-old male Wistar rats (n=9). Control rats were infused with normal saline (n=9). The rats were sacrificed to obtain kidney tissues 3days after treatment. In addition to electrolyte metabolism, changes in expressions of the urate transporters including URAT1 (SLC22A12), GLUT9 (SLC2A9), ABCG2 and NPT1 (SLC17A1) were examined by western blotting and immunohistochemistry. In the terlipressin-treated rats, serum uric acid (UA) significantly decreased and the excretion of urinary UA significantly increased, resulting in marked increase in fractional excretion of UA. Although no change in the expression of URAT1, GLUT9 expression significantly decreased whereas the expressions of ABCG2 and NPT1 significantly increased in the terlipressin group. The results of immunohistochemistry corroborated with those of the western blotting. Aquaporin 2 expression did not change in the medulla, suggesting the independence of V2 receptor stimulation. Stimulation of V1a receptor induces the downregulation of GLUT9, reabsorption urate transporter, together with the upregulation of ABCG2 and NPT1, secretion urate transporters, all changes of which clearly lead to increase in renal UA clearance. Hypouricemia seen in SIADH is attributable to V1a receptor stimulation.

Factor VIII Is Synthesized in Human Endothelial Cells, Packaged in Weibel-Palade Bodies and Secreted Bound to ULVWF Strings.

The cellular synthesis site and ensuing storage location for human factor VIII (FVIII), the coagulation protein deficient in hemophilia A, has been elusive. FVIII stability and half-life is dependent on non-covalent complex formation with von Willebrand factor (VWF) to avoid proteolysis and clearance. VWF is synthesized in megakaryocytes and endothelial cells, and is stored and secreted from platelet alpha granules and Weibel-Palade bodies of endothelial cells. In this paper we provide direct evidence for FVIII synthesis in 2 types of primary human endothelial cells: glomerular microvascular endothelial cells (GMVECs) and umbilical vein endothelial cells (HUVECs). Gene expression quantified by real time PCR revealed that levels of F8 and VWF are similar in GMVECs and HUVECs. Previous clinical studies have shown that stimulation of vasopressin V2 receptors causes parallel secretion of both proteins. In this study, we found that both endothelial cell types express AVPR2 (vasopressin V2 receptor gene) and that AVPR2 mRNA levels are 5-fold higher in GMVECs than HUVECs. FVIII and VWF proteins were detected by fluorescent microscopy in Weibel-Palade bodies within GMVECs and HUVECs using antibodies proven to be target specific. Visual presence of FVIII and VWF in Weibel-Palade bodies was confirmed by correlation measurements. The high extent of correlation was compared with negative correlation values obtained from FVIII detection with cytoplasmic proteins, β-actin and Factor H. FVIII activity was positive in GMVEC and HUVEC cell lysates. Stimulated GMVECs and HUVECs were found to secrete cell-anchored ultra-large VWF strings covered with bound FVIII.

Vasopressin and Terlipressin in Neonates and Children with Refractory Septic Shock

Vasopressin and its analogue terlipressin are potent vasopressors which have been recently proposed in the treatment of catecholamine-resistant septic shock. We review the physiology, metabolism and pharmacology of vasopressin and terlipressin, as well as the available data on their efficacy and safety in neonates and children with septic shock. In adults, vasopressin deficiency can contribute to refractory shock states associated with sepsis. Differently, in children with septic shock vasopressin levels may be normal or even augmented. Nevertheless, low doses of vasopressin and terlipressin seem to have the potential to restore vasomotor tone in conditions refractory to catecholamines, improving organ perfusion with preservation of renal blood flow, while decreasing catecholamine requirements. Vasopressin and terlipressin produce vasoconstriction via stimulation of V1-receptors. In particular, terlipressin has a higher selectivity for V1-receptors and a longer half-life when compared to vasopressin, allowing for intermittent bolus doses. However, the pharmacology of vasopressin/terlipressin in newborns and children has not been sufficiently investigated and data on potential short and long-term adverse effects are still lacking. Further clinical, pharmacokinetic and pharmacodynamic studies are needed to better define the role of vasopressin and terlipressin in septic shock, as well as to prove their effectiveness and safety in infants and children.

Selective V1aagonism attenuates vascular dysfunction and fluid accumulation in ovine severe sepsis

Vasopressin analogs are used as a supplement to norepinephrine in septic shock. The isolated effects of vasopressin agonists on sepsis-induced vascular dysfunction, however, remain controversial. Because V(2)-receptor stimulation induces vasodilation and procoagulant effects, a higher V(1a)- versus V(2)-receptor selectivity might be advantageous. We therefore hypothesized that a sole, titrated infusion of the selective V(1a)-agonist Phe(2)-Orn(8)-Vasotocin (POV) is more effective than the mixed V(1a)-/V(2)-agonist AVP for the treatment of vascular and cardiopulmonary dysfunction in methicillin resistant staphylococcus aureus pneumonia-induced, ovine sepsis. After the onset of hemodynamic instability, awake, chronically instrumented, mechanically ventilated, and fluid resuscitated sheep were randomly assigned to receive continuous infusions of either POV, AVP, or saline solution (control; each n = 6). AVP and POV were titrated to maintain mean arterial pressure above baseline - 10 mmHg. When compared with that of control animals, AVP and POV reduced neutrophil migration (myeloperoxidase activity, alveolar neutrophils) and plasma levels of nitric oxide, resulting in higher mean arterial pressures and a reduced vascular leakage (net fluid balance, chest and abdominal fluid, pulmonary bloodless wet-to-dry-weight ratio, alveolar and septal edema). Notably, POV stabilized hemodynamics at lower doses than AVP. In addition, POV, but not AVP, reduced myocardial and pulmonary tissue concentrations of 3-nitrotyrosine, VEGF, and angiopoietin-2, thereby leading to an abolishment of cumulative fluid accumulation (POV, 9 ± 15 ml/kg vs. AVP, 110 ± 13 ml/kg vs. control, 213 ± 16 ml/kg; P < 0.001 each) and an attenuated cardiopulmonary dysfunction (left ventricular stroke work index, PaO(2)-to-FiO(2) ratio) versus control animals. Highly selective V(1a)-agonism appears to be superior to unselective vasopressin analogs for the treatment of sepsis-induced vascular dysfunction.

Terlipressin: An Asset for Hepatologists!

Terlipressin: An Asset for Hepatologists!

Effects of the vasopressin agonist terlipressin on plasma cAMP and ENaC excretion in the urine in patients with cirrhosis and water retention

Background. Terlipressin is a vasopressin analogue used for its potent V1a effects in cirrhotic patients. Recent data suggest that terlipressin has affinity to renal V2 receptors and modulates Aquaporin 2 (AQP2) expression and free water clearance. Stimulation of renal V2 receptors may also affect sodium transport via the Epithelial Sodium Channel (ENaC). Furthermore, endothelial V2 receptors may indirectly affect proximal sodium handling by increasing plasma cAMP. Methods. We investigated 18 patients with cirrhosis and ascites before and after infusion of 2 mg of terlipressin. Plasma cAMP and urine AQP2 were measured and a newly developed radioimmunoassay was used to quantify ENaC in the urine. Results. Mean arterial blood pressure increased from 87 ± 15 to 105 ± 19 mmHg, p < 0.001 after terlipressin infusion and GFR increased from 52 ± 6 to 69 ± 9 mL/min, p < 0.01. Urine-ENaC in ng/mmol creatinine increased from 42 ± 6 to 50 ± 7 ng/mmol creatinine, p = 0.05. Urine sodium increased from 43 ± 8 to 62 ± 9 mmol/L, p < 0.01. Plasma cAMP was not affected by terlipressin, 106 (63–673) vs. 103.5 (69–774) pmol/mL, NS. The rise in ENaC excretion correlated with the rise in AQP2 excretion, r = 0.63, p < 0.01. There was a weak correlation between the change in MAP and the rise in AQP2 excretion (p < 0.05). Conclusions. Increased ENaC excretion suggests increased abundance of ENaC and resultant increased distal sodium reabsorption. The V2 effects of terlipressin are insufficient to stimulate the endothelial V2 receptors since plasma cAMP is unaltered. Despite pronounced V1a and some V2 effects of terlipressin, additional effects on proximal sodium handling are therefore not likely.

Vasopressin V2 receptors, ENaC, and sodium reabsorption: a risk factor for hypertension?

Excessive sodium reabsorption by the kidney has long been known to participate in the pathogenesis of some forms of hypertension. In the kidney, the final control of NaCl reabsorption takes place in the distal nephron through the amiloride-sensitive epithelial sodium channel (ENaC). Liddle's syndrome, an inherited form of hypertension due to gain-of-function mutations in the genes coding for ENaC subunits, has demonstrated the key role of this channel in the sodium balance. Although aldosterone is classically thought to be the main hormone regulating ENaC activity, several studies in animal models and in humans highlight the important effect of vasopressin on ENaC regulation and sodium transport. This review summarizes the effect of vasopressin V2 receptor stimulation on ENaC activity and sodium excretion in vivo. Moreover, we report the experimental and clinical data demonstrating the role of renal ENaC in water conservation at the expense of a reduced ability to excrete sodium. Acute administration of the selective V2 receptor agonist dDAVP not only increases urine osmolality and reduces urine flow rate but also reduces sodium excretion in rats and humans. Chronic V2 receptor stimulation increases blood pressure in rats, and a significant correlation was found between blood pressure and urine concentration in healthy humans. This led us to discuss how excessive vasopressin-dependent ENaC stimulation could be a risk factor for sodium retention and resulting increase in blood pressure.

Chronic hyponatremia impairs memory in rats: effects of vasopressin antagonist tolvaptan

The effects of stable chronic hyponatremia on the central nervous system are largely unknown, clinically, or in experimental animals. The aim of this study was to identify and characterize these effects in rats. Tolvaptan, a vasopressin V(2) receptor antagonist, was used to correct hyponatremia and determine any potential benefits of such treatment in this condition. Stable chronic hyponatremia was induced by combination of the continuous vasopressin V(2) receptor stimulation and liquid food intake. The hyponatremic rats did not exhibit significant changes in general symptoms or neurological functions assessed by modified Irwin's method, or in motor function assessed by the rotarod test. In passive avoidance test, however, rats with moderate and severe hyponatremia had significantly reduced step-through latency, indicating impairment in memory. This reduced step-through latency was improved by the treatment of tolvaptan (0.25-8 mg/kg daily doses), a vasopressin V(2) receptor antagonist. This improvement is associated with normalization of plasma sodium concentrations in hyponatremic rats. In conclusion, these data suggest that chronic hyponatremia may impair memory, and treatments that normalize sodium level, such as vasopressin V(2) receptor antagonists, may be beneficial to patients with hyponatremia.

Role of the cAMP/PKA signaling cascade in vasopressin‐induced trafficking of TRPC3 channels in principal cells of the collecting duct

The transient receptor potential channels TRPC3 and TRPC6 are abundantly expressed in principal cells of the collecting duct (CD) along with the water channel, aquaporin‐2 (AQP2). All three channels localize to intracellular vesicles, but upon stimulation with arginine‐vasopressin (AVP), TRPC3 and AQP2 translocate to the apical membrane. In the present study, the effect of various activators and inhibitors of the adenylyl cyclase (AC)/cAMP/PKA signaling cascade on channel trafficking was examined in principal cells of the CD. Translocation of AQP2 and TRPC3 (but not TRPC6) was stimulated by [deamino‐Cys1, D‐Arg8]‐vasopressin (dDAVP), a specific V2‐receptor agonist, and blocked by [adamantaneacetyl1, O‐Et‐D‐Tyr2, Val4, Aminobutyryl6, Arg8,9]‐vasopressin (AEAVP), a specific V2 receptor antagonist. Translocation was also activated by forskolin, a direct activator of AC, or by di‐butyryl‐cAMP, a membrane‐permeable cAMP analog. AVP‐, dDAVP‐, and forskolin‐induced translocation was blocked by SQ22536 and H89, specific inhibitors of AC and PKA, respectively. Translocation stimulated by di‐butyryl‐cAMP was unaffected by AEAVP, but could be blocked by H89. Taken together, these results demonstrate that AVP stimulation of V2‐receptors in principal cells of the CD causes translocation of TRPC3 to the apical membrane via stimulation of the AC/cAMP/PKA signaling cascade. Supported in part by HL097355.

Role of cAMP/PKA signaling cascade in vasopressin-induced trafficking of TRPC3 channels in principal cells of the collecting duct

Transient receptor potential channels TRPC3 and TRPC6 are expressed in principal cells of the collecting duct (CD) along with the water channel aquaporin-2 (AQP2) both in vivo and in the cultured mouse CD cell line IMCD-3. The channels are primarily localized to intracellular vesicles, but upon stimulation with the antidiuretic hormone arginine vasopressin (AVP), TRPC3 and AQP2 translocate to the apical membrane. In the present study, the effect of various activators and inhibitors of the adenylyl cyclase (AC)/cAMP/PKA signaling cascade on channel trafficking was examined using immunohistochemical techniques and by biotinylation of surface membrane proteins. Both in vivo in rat kidney and in IMCD-3 cells, translocation of AQP2 and TRPC3 (but not TRPC6) was stimulated by [deamino-Cys(1), d-Arg(8)]-vasopressin (dDAVP), a specific V2-receptor agonist, and blocked by [adamantaneacetyl(1), O-Et-d-Tyr(2), Val(4), aminobutyryl(6), Arg(8,9)]-vasopressin (AEAVP), a specific V2-receptor antagonist. In IMCD-3 cells, translocation of TRPC3 and AQP2 was activated by forskolin, a direct activator of AC, or by dibutyryl-cAMP, a membrane-permeable cAMP analog. AVP-, dDAVP-, and forskolin-induced translocation in IMCD-3 cells was blocked by SQ22536 and H89, specific inhibitors of AC and PKA, respectively. Translocation stimulated by dibutyryl-cAMP was unaffected by AEAVP but could be blocked by H89. AVP- and forskolin-induced translocation of TRPC3 in IMCD-3 cells was also blocked by two additional inhibitors of PKA, specifically Rp-cAMPS and the myristoylated inhibitor of PKA (m-PKI). Quantification of TRPC3 membrane insertion in IMCD-3 cells under each assay condition using a surface membrane biotinylation assay, confirmed the translocation results observed by immunofluorescence. Importantly, AVP-induced translocation of TRPC3 as estimated by biotinylation was blocked on average 95.2 +/- 1.0% by H89, Rp-cAMPS, or m-PKI. Taken together, these results demonstrate that AVP stimulation of V2 receptors in principal cells of the CD causes translocation of TRPC3 to the apical membrane via stimulation of the AC/cAMP/PKA signaling cascade.

Copeptin and the Transplanted Kidney: Friends or Foes?

Copeptin and the Transplanted Kidney: Friends or Foes?

Arginine vasopressin increases plasma levels of von Willebrand factor in sheep

The V1a/V2 receptor dual-agonist arginine vasopressin (AVP) is increasingly used in catecholamine-resistant septic shock [1]. While V1a receptor stimulation results in vasoconstriction, V2 receptor stimulation promotes coagulation, at least in part through an increase in plasma von Willebrand factor antigen (vWF:Ag) activity. We hypothesized that, at an intravenous infusion rate representative of the requirements for the treatment of sepsis-induced vasodilatory hypotension in sheep [2], AVP provides procoagulant activities. We tested this hypothesis by measuring vWF:Ag activity in unanesthetized healthy sheep during administration of AVP in comparison with the vWF:Ag activity increase induced by the selective V2 receptor agonist desmo-pressin (dDAVP).

Effects of terlipressin on the aquaretic system: evidence of antidiuretic effects

The vasopressin analog terlipressin is believed to cause vasoconstriction selectively by V1 receptor stimulation. However, a possible antidiuretic effect by V2 receptor stimulation has never been ruled out. Twenty-two patients with ascites, including seven with refractory ascites, were included. The subjects were studied during a 400 ml/h oral water load before and after infusion of 2 mg of terlipressin (18 patients) or placebo infusion (4 patients). Effects on the V2 receptors were assessed by evaluating aquaporin (AQP)2 excretion, free water clearance (C(H2O)), urine osmolality (Uosm), and fractional distal water excretion (DFeH2O). After terlipressin the excretion of AQP2 increased by 89% [144 ng/mmol creatinine, 95% confidence interval (CI) 73-214 ng/mmol creatinine, P = 0.001]. C(H2O) decreased 1.05 ml/min (from 0.17 to -0.89 ml/min, P = 0.001), and DFeH2O decreased 37% (19 vs. 12; 95% CI 2-11, P = 0.01). Uosm increased by 27% (93 mosmol/kgH2O, 95% CI 23-164 mosmol/kgH2O, P = 0.02). Plasma sodium decreased 1.1 mmol/l (P < 0.01). An increase in AQP2 excretion and a decrease in C(H2O) and distal water excretion after terlipressin despite water loading is a clear indication of activation of the antidiuretic system (V2 receptor effect).

Effects of Vasopressin in Septic Shock

Septic shock continues to be one of the leading causes of death in the intensive care unit today. The confluence of many factors contributes to the deterioration of patients’ condition in septic shock. Increased levels of nitric oxide, in part, mediate the cardiovascular effects of septic shock. Nitric oxide is major mediator of vasodilation and hypotension as well as myocardial depression. It also contributes to decreased production and release of endogenous vasopressin. Vasopressin effects are actualized by stimulation of V1, V2, and V3 receptors located in various parts of the body. The response is dose dependent. Endogenous vasopressin and angiotensin II act synergistically to preserve and restore blood pressure levels. Decreased circulating vasopressin contributes to adrenal insufficiency via hypothalamic-pituitary-adrenal axis suppression and increased catecholamine resistance to vasopressors. Exogenous vasopressin supplementation in physiologic doses has been shown to improve blood pressure levels and decrease vasopressor needs in patients with septic shock.

Effects of Vasopressin in Septic Shock

Septic shock continues to be one of the leading causes of death in the intensive care unit today. The confluence of many factors contributes to the deterioration of patients' condition in septic shock. Increased levels of nitric oxide, in part, mediate the cardiovascular effects of septic shock. Nitric oxide is major mediator of vasodilation and hypotension as well as myocardial depression. It also contributes to decreased production and release of endogenous vasopressin. Vasopressin effects are actualized by stimulation of V1, V2, and V3 receptors located in various parts of the body. The response is dose dependent. Endogenous vasopressin and angiotensin II act synergistically to preserve and restore blood pressure levels. Decreased circulating vasopressin contributes to adrenal insufficiency via hypothalamic-pituitary-adrenal axis suppression and increased catecholamine resistance to vasopressors. Exogenous vasopressin supplementation in physiologic doses has been shown to improve blood pressure levels and decrease vasopressor needs in patients with septic shock.

The role of central vasopressin receptors in the modulation of autonomic cardiovascular controls: a spectral analysis study

Although it has been suggested that vasopressin (VP) acts within the central nervous system to modulate autonomic cardiovascular controls, the mechanisms involved are not understood. Using nonpeptide, selective V(1a), V(1b), and V(2) antagonists, in conscious rats, we assessed the roles of central VP receptors, under basal conditions, after the central application of exogenous VP, and after immobilization, on cardiovascular short-term variability. Equidistant sampling of blood pressure (BP) and heart rate (HR) at 20 Hz allowed direct spectral analysis in very-low frequency (VLF-BP), low-frequency (LF-BP), and high-frequency (HF-BP) blood pressure domains. The effect of VP antagonists and of exogenous VP on body temperature (T(b)) was also investigated. Under basal conditions, V(1a) antagonist increased HF-BP and T(b), and this was prevented by metamizol. V(1b) antagonist enhanced HF-BP without affecting T(b), and V(2) antagonist increased VLF-BP variability which could be prevented by quinapril. Immobilization increased BP, LF-BP, HF-BP, and HF-HR variability. V(1a) antagonist prevented BP and HR variability changes induced by immobilization and potentiated tachycardia. V(1b) antagonist prevented BP but not HR variability changes, whereas V(2) antagonist had no effect. Exogenous VP increased systolic arterial pressure (SAP) and HF-SAP variability, and this was prevented by V(1a) and V(1b) but not V(2) antagonist pretreatment. Our results suggest that, under basal conditions, VP, by stimulation of V(1a), V(1b), and cognate V(2) receptors, buffers BP variability, mostly due to thermoregulation. Immobilization and exogenous VP, by stimulation of V(1a) or V(1b), but not V(2) receptors, increases BP variability, revealing cardiorespiratory adjustment to stress and respiratory stimulation, respectively.

Stimulation of the Epithelial Sodium Channel (ENaC) by cAMP Involves Putative ERK Phosphorylation Sites in the C Termini of the Channel's β- and γ-Subunit

The mechanisms involved in the regulation of the epithelial sodium channel (ENaC) via the cAMP pathway are not yet completely understood. The aim of the present study was to investigate cAMP-mediated ENaC regulation in Xenopus laevis oocytes heterologously expressing the three subunits (alphabetagamma) of rat ENaC and to determine the ENaC regions important for mediating the stimulatory effect of cAMP. In oocytes treated for about 24 h with 1 mm 3-isobutyl-1-methylxanthine (IBMX) and 1 microm forskolin (FSK) so as to increase intracellular cAMP, the amiloride-sensitive whole cell current (DeltaI(Ami)) was on average 10-fold larger than DeltaI(Ami) in matched control oocytes. This effect on DeltaI(Ami) was paralleled by an increase in ENaC surface expression caused by a reduced rate of ENaC retrieval. In addition, IBMX/FSK also enhanced ENaC open probability from about 0.2 to 0.5. The stimulatory effect of IBMX/FSK was dependent on the presence of intact PY motifs in the C termini of the channel. Mutagenesis of putative protein kinase A and CK-2 consensus motifs in the cytosolic domains of the channel did not reveal critical sites involved in mediating the stimulatory effect of IBMX/FSK. In contrast, site-directed mutagenesis of two putative ERK-consensus motifs (T613A in betaENaC and T623A in gammaENaC) largely reduced the stimulatory effect of IBMX/FSK. Phosphorylation of these ERK sites has previously been reported to enhance the interaction of ENaC and Nedd4 (Shi, H., Asher, C., Chigaev, A., Yung, Y., Reuveny, E., Seger, R., and Garty, H. (2002) J. Biol. Chem. 277, 13539-13547). Using co-expression experiments we demonstrated that mutating the two ERK sites attenuates the inhibitory effect of Nedd4-2 on ENaC currents. We conclude that an increase in intracellular cAMP favors the dephosphorylation of the two ERK sites, which reduces channel retrieval and increases P(O) by modulating ENaC/Nedd4 interaction. This defines a novel regulatory pathway likely to be relevant for cAMP-induced stimulation of ENaC in vivo.