Hydroosmotic Effect Research Articles

Apelin-13 Decreases Epithelial Sodium Channel (ENaC) Expression and Activity in Kidney Collecting Duct Cells.

Apelin and its G protein-coupled receptor APLNR (also known as APJ) are widely expressed within the central nervous system and peripheral organs including heart, lung and kidney. Several studies have shown that the apelin/APJ system is involved in various important physiological processes such as energy metabolism, cardiovascular functions and fluid homeostasis. In the kidney, the apelin/APJ system performs a wide range of activities. We recently demonstrated that apelin antagonises the hydro-osmotic effect of vasopressin on aquaporin-2 water channel (AQP-2) expression by reducing its mRNA and protein levels in collecting duct principal cells. The central role of these cells in water and sodium transport is governed by AQP-2 and the epithelial sodium channel (ENaC). The coordination of these channels is essential for the control of extracellular fluid volume, sodium homeostasis and blood pressure. This study aimed at investigating the role of apelin in the regulation of sodium balance in the distal nephron, and more specifically its involvement in modulating the expression and activity of ENaC in collecting duct principal cells. mpkCCD cells were incubated in the presence of aldosterone and treated with or without apelin-13. Transepithelial Na+ current was measured and the changes in ENaC expression determined by RT-PCR and immunoblotting. Our data show that apelin-13 reduces the transepithelial sodium amiloride-sensitive current in collecting duct principal cells after 8h and 24h treatment. This effect was associated with a decrease in αENaC subunit expression and mediated through the ERK pathway as well as SGK1 and Nedd4-2. Our findings indicate that apelin is involved in the fine regulation of sodium balance in the renal collecting duct by opposing the effects of aldosterone, likely by activation of ENaC ubiquitination.

Efficiency of Osmotic Concentration after Combined Treatment with Vasopressin and Blockage of Prostaglandin Synthesis.

We performed a complex functional study of the effects of prostaglandin synthesis blockage with diclofenac on manifestation of the hydroosmotic effect of vasopressin V2-receptor agonist desmopressin in the kidneys of Wistar rats with normal synthesis of endogenous vasopressin and homozygous Brattleboro rats with hereditary impaired synthesis of neurohypophyseal hormone vasopressin. Blockage of prostaglandin synthesis led to more pronounced increase in urine osmolality in Brattleboro rats than in Wistar rats due to elevation of not only urine but also sodium gradient at the expense of elimination of the inhibitory effect of prostaglandins on sodium reabsorption and membrane permeability for urine. During combined treatment, the effects of the hormone predominated: the increase in urine osmolality in Wistar and Brattleboro rats did not differ from that after desmopressin administration.

Analysis of the efficiency of osmotic concentration in Wistar and Brattleboro rats under the effect of desmopressin.

We studied the effect of vasopressin V2 receptor agonist (desmopressin, 5 μg/100 g body weight for 2 days) on the efficiency of osmotic concentration in Wistar rats and homozygous Brattleboro rats defective by the synthesis of antidiuretic hormone. Specific differences in the reaction of the osmotic concentration system components (urine osmolality and urea, sodium, and potassium content in the renal tissue) involved in the realization of the hydroosmotic effect of the hormone depend on genetically determined capacity to vasopressin synthesis in these animals.

Vasopressin: Molecular Mechanisms of Its Antidiuretic Effect

The body’s water balance is regulated by the neurohypophyseal hormone vasopressin. Numerous investigators have identified a significant role for aquaporins – water channels in cell membranes – in regulating permeability. This review presents recent published data on the molecular mechanisms underlying the short-term control by vasopressin of the water permeability of renal tubules via displacement of aquaporin 2 from vesicles to the apical membranes of principal cells and the long-term action of the hormone on gene expression and the levels of aquaporins types 2, 3, and 4 in epithelial cells. Data on the actions of modulators of the hydroosmotic effect of vasopressin (prostaglandin E2, bradykinin, dopamine, and endothelin 1) are summarized. Changes in the expression or displacement of aquaporin 2 are of particular importance in pathology in conditions of impairments to the concentrating function of the kidneys.

Glycogen synthase kinase-3 regulation of urinary concentrating ability

Glycogen synthase kinase-3 (GSK3) is an enzyme that is gaining prominence as a critical signaling molecule in the epithelial cells of renal tubules. This review will focus on recent findings exploring the role of GSK3 in renal collecting ducts, especially its role in urine concentration involving vasopressin signaling. Recent studies using inhibition or tissue-specific gene deletion of GSK3 revealed the mechanism by which GSK3 regulates aquaporin 2 water channels via adenylate cyclase or the prostaglandin-E2 pathway. In other studies, postnatal treatment with lithium, an inhibitor of GSK3, increased cell proliferation and led to microcyst formation in rat kidneys. These studies suggest that loss of GSK3 activity could interfere with renal water transport at two levels. In the short term, it could disrupt vasopressin signaling in collecting duct cells and in the long term it could alter the structure of the collecting ducts, making them less responsive to the hydro-osmotic effects of vasopressin. Ongoing studies reveal the crucial role played by GSK3 in the regulation of vasopressin action in the renal collecting ducts and suggest a possible use of GSK3 inhibitors in disease conditions associated with disrupted vasopressin signaling.

Expression of type II hyaluronan-synthase gene in kidneys Wistar and Brattleboro rats with diabetes Insipidus: Effect of vasopressin and its analogues

Vasopressin, a neurohypophyseal hormone, plays a key role in water balance regulation in mammals. One of the major effects of vasopressin is an increase in the water permeability of collecting duct epithelium, which is based on the cascade of reactions from the membrane receptor of the basolateral membrane to the incorporation of water channels, aquaporins-2, into the apical membrane of the principal cells [1]. The mechanism underlying the involvement of the extracellular matrix, the major component of which is hyaluronan, in the hydroosmotic effect of vasopressin, has been studied the least. Hyaluronan (hyaluronic acid, HA) is a negatively charged nonsulfated linear glycosaminoglycan that is synthesized on the plasma membrane and then translocated through the latter in the form of a growing chain to the extracellular space [2‐4]. In mammalian kidney, HA is distributed heterogeneously: the highest concentration is detected in the medulla, where vasopressin-regulated reabsorption of water takes place [5, 6]. The hypothesis on the role of HA contained in the extracellular matrix in the effect of vasopressin on water transport was first proposed by Ginetzinsky [7]. Later, a negative correlation between the HA content in the renal papilla and the osmolality of excreted urine and a positive correlation between the HA concentration and water content in tissue were found [6, 8]. The possible role of HA as a factor involved in the regulation of hydroosmotic effect of vasopressin stimulates further studies of the mechanisms regulating its metab

Vasopressin receptor antagonists

The first non-peptide vasopressin receptor antagonist (VRA) was recently approved by the United States Food and Drug Administration, and several others are now in late stages of clinical development. Phase 3 trials indicate that these agents predictably reduce urine osmolality, increase electrolyte-free water excretion, and raise serum sodium concentration. They are likely to become a mainstay of treatment of euvolemic and hypervolemic hyponatremia. Although tachyphylaxis to the hydro-osmotic effect of these agents does not appear to occur, their use is accompanied by an increase in thirst, and they do not always eliminate altogether the need for water restriction during treatment of hyponatremia. Experience with use of these agents for treatment of acute, severe, life-threatening hyponatremia as well as chronic hyponatremia is limited. Further studies are needed to determine how they are best used in these situations, but the risk of overly rapid correction of hyponatremia seems low. Results of long-term trials to determine the ability of VRAs to reduce morbidity or mortality in congestive heart failure or to slow the progression of polycystic kidney disease are awaited with great interest.

Urokinase (u‐PA) is produced by collecting duct principal cells and is post‐transcriptionally regulated by SV40 large‐T, arginine vasopressin, and epidermal growth factor

We have analyzed the expression and regulation of plasminogen activators (PA) in principal cells of the renal collecting duct. We used a rabbit principal cell line (RC.SVtsA58) infected with the temperature-sensitive SV40 strain tsA58. Transformed cells cultured at permissive temperature (33 degrees C) produced only tissue-type plasminogen activator (t-PA). Shifting the cells to nonpermissive temperature (39.5 degrees C) induced their differentiation and a marked increase in total fibrinolytic activity due to the induction of urokinase-type plasminogen activator (u-PA) synthesis and secretion. The effect on u-PA was post-transcriptional and it could be attributed to large-T inactivation at 39.5 degrees C since it was abolished by re-infecting the cells with wild-type SV40. Run-on assay and real-time RT-PCR of u-PA transcripts indicated that large-T altered post-transcriptional regulation. u-PA was also produced by primary cultures of collecting duct cells and was present in the rabbit urine. In the kidney, u-PA and its receptor (u-PAR) were almost exclusively expressed at the apex of collecting duct cells. We then analyzed the regulation of u-PA by arginine vasopressin (AVP) and epidermal growth factor (EGF), two key regulators of principal cell functions. We found that AVP and EGF, which have opposite hydro-osmotic effects in the collecting duct, also exhibited contrasted effects on u-PA synthesis in differentiated RC.SVtsA58 cells. EGF increased but AVP suppressed u-PA activity and protein, and these regulations occurred at post-transcriptional level. These results point to a physiological role of u-PA in principal cells of the renal collecting duct.

Nitric oxide inhibits arginine-vasotocin-induced increase of water osmotic permeability in frog urinary bladder.

The present study addressed the question of whether nitric oxide (NO) participates in regulation of osmotic water permeability in the urinary bladder of the frog Rana temporaria L. Experiments were carried out on isolated, paired hemi-bladders filled with amphibian Ringer solution diluted 1:10 with distilled water. Sodium nitroprusside (SNP, 125-250 micro M), an NO donor, markedly attenuated the increase of osmotic water flow elicited by arginine-vasotocin (AVT) (AVT 10(-10) M: 2.20+/-0.26; AVT plus 200 micro M SNP: 1.21+/-0.15 micro l/min cm(2), n=20, P<0.001). This effect of SNP was apparent only in the presence of 50 micro M zaprinast, an inhibitor of the cGMP-specific phosphodiesterase-5 (PDE5). In the presence of zaprinast, SNP elevated cGMP production significantly both in control and AVT-stimulated urinary bladders, but had no effect on the level of cAMP (AVT 5 x 10(-10) M: 7.6+/-0.6; AVT plus SNP 200 micro M: 7.5+/-0.4 pmol/mg protein, n=8, N.S.). 1 H-[1,2,4]-oxadiazole-[4,3-a]-quinoxalin-1-one (ODQ, 25-100 micro M), an inhibitor of soluble guanylate cyclase, enhanced the AVT-induced water flow, decreased the SNP-stimulated increase of cGMP in the bladder tissue and almost abolished the inhibitory effect of SNP on the AVT-induced hydroosmotic response. 8-( p-Chlorophenylthio)-cGMP (8-pCPT-cGMP, 25 or 50 micro M), a membrane-permeable cGMP analogue specific for cGMP-dependent protein kinase (PKG), inhibited, whereas 2 micro M KT-5823, an inhibitor of PKG, significantly stimulated the increase of water flow induced by AVT. The inhibitory effect of SNP on AVT-induced water flow was almost completely reversed by KT-5823, but not by 50-100 micro M erythro-9-[2-hydroxy-3-nonyl]adenine (EHNA), an inhibitor of cGMP-activated PDE2. Immunohistochemistry of urinary bladder slices with antibodies against different types of NO synthase (NOS) revealed a positive immunostaining for neuronal NOS (nNOS) in the mucosal epithelium. These results suggest that in the frog urinary bladder endogenous NO is involved in regulation of water osmotic permeability. NO inhibits the AVT-induced increase of water flow at least partly by activation of PKG, which interferes with the hydroosmotic effect of AVT probably at (a) post-cAMP step(s).

The origin of the hydroosmotic effect of arginine vasopressin: a hypothesis.

The origin of the hydroosmotic effect of arginine vasopressin: a hypothesis.

Effects of vasopressin on hyaluronate hydrolase activities and water permeability in the frog urinary bladder.

The involvement of enzymes catabolizing hyaluronic acid (hyaluronidase, beta-glucuronidase, N-acetyl-beta-D-hexosaminidase) in the hydroosmotic effect of vasopressin in the frog (Rana ridibunda) urinary bladder was studied. It was observed that vasopressin (50 nM), an agonist of V2 receptors, L-desamino-8-D-arginine-vasopressin (dDAVP, 1.5 microM) and forskolin (30 microM) activated the enzymes and caused their release into Ringer solution at the mucosal side, together with an increase in osmotic water flow. The effect of AVP on enzyme activity developed 10 min after the hydroosmotic response. Cytochalasin B (a specific inhibitor of actin filament elongation, 50 nM) blocked the hydroosmotic response to AVP; hyaluronate hydrolase activity increased in the bladder tissue but not in Ringer solution. It is suggested that the involvement of hyaluronate hydrolases in AVP's effect is mediated by a cAMP-dependent mechanism and provides favorable conditions for an increase in the permeability of glycosaminoglycan structures adjacent to the apical cell surface.

An enhanced effect of arginine vasopressin in bradykinin B2 receptor null mutant mice.

Under water restriction, arginine vasopressin (AVP) is released and promotes water reabsorption in the distal nephron, mainly through AVP V2-receptors. It has been proposed that renal kinins counteract the hydro-osmotic effect of AVP. We hypothesized that kinins acting through B2 receptors antagonize the urinary concentrating effect of AVP. To test this, bradykinin B2 receptor knockout mice (B2-KO) and 129/SvEv mice (controls) were placed in metabolic cages and urine collected for 24 hours (water ad libitum). After that, urine was again collected from the same mice during 24 hours of water restriction. Urinary volume (UV), urinary osmolarity (UOsm), and urinary Na+ (UNaV) and K+ (UKV) excretion were determined. On water restriction, UV in controls decreased by approximately 25%, whereas in B2-KO mice there was almost a 60% drop in urinary output (P=0.001 versus controls). In the controls, water restriction increased UOsm by 347 mOsm/kg H2O, approximately 14% above baseline (NS), whereas in knockout mice the increase was 3 times that seen in the controls: >1000 mOsm/kg H2O (P=0.001 versus controls). Compared with normohydration, UNaV and UKV in the water-restricted state increased more in controls than in B2-KO mice. This difference in electrolyte excretion could be explained by greater dehydration in the controls (dehydration natriuresis). In a second protocol, we tried to mimic the effect of endogenous AVP by exogenous administration of an AVP V2-receptor agonist, desmopressin (DDAVP). To suppress endogenous AVP levels before DDAVP administration, mice were volume-overloaded with dextrose and alcohol. UOsm was 685+/-125 and 561+/-58 mOsm/kg H2O in water-loaded controls and B2-KO mice, respectively. After DDAVP was injected subcutaneously at a dose of 1 microgram/kg, UOsm increased to 1175+/-86 mOsm/kg H2O (Delta+490 mOsm) in the controls and 2347+/-518 mOsm/kg H2O (Delta+1786 mOsm) in B2-KO mice (P<0.05 versus controls). We concluded that water restriction or exogenous administration of an AVP V2-receptor agonist has a greater urinary concentrating effect in B2-KO mice than in controls, suggesting that endogenous kinins acting through B2 receptors oppose the antidiuretic effect of AVP in vivo.

Matrix Metalloproteinase 2 (MMP2) and MMP9 Are Produced by Kidney Collecting Duct Principal Cells but Are Differentially Regulated by SV40 Large-T, Arginine Vasopressin, and Epidermal Growth Factor

We analyzed the expression and regulation of matrix metalloproteinase 2 (MMP2) and MMP9 gelatinases in a rabbit kidney collecting duct principal cell line (RC.SVtsA58) (Prié, D., Ronco, P. M., Baudouin, B., Géniteau-Legendre, M., Antoine, M., Piedagnel, R., Estrade, S., Lelongt, B., Verroust, P. J., Cassingéna, R., and Vandewalle, A. (1991) J. Cell Biol. 113, 951-962) infected with the temperature-sensitive (ts) SV40 strain tsA58. At the permissive temperature (33 degreesC), cells produced only MMP2. Shifting cells to a nonpermissive temperature (39.5 degreesC) induced a marked increase in total gelatinolytic activity due to an increase of MMP2 and an induction of MMP9 synthesis. This effect was attributed to large-T inactivation at 39.5 degreesC because it was abolished by re-infecting the cells with wild-type SV40 strain LP. Run-on experiments showed that negative regulation of MMP2 and MMP9 by large-T was transcriptional and posttranscriptional, respectively. MMP2 and MMP9 were also produced by primary cultures of collecting duct cells. In rabbit kidney, both MMP2 and MMP9 were almost exclusively expressed in collecting duct cells, where an unexpected apical localization was observed. Arginine vasopressin and epidermal growth factor, which exert opposite hydroosmotic effects in the collecting duct, also exhibited contrasted effects on MMP9 synthesis. Epidermal growth factor increased but arginine vasopressin suppressed MMP9 at a posttranscriptional level, whereas MMP2 was not affected. These results suggest a specific physiological role of MMP2 and MMP9 in principal cells of renal collecting duct.

Prostaglandin-dependent osmotic water permeability of the frog and trout urinary bladder

Washout of autacoids from serosal Ringer solution, using a repeated change of the solution of the frog and trout urinary bladder, was accompanied by a pronounced rise in the osmotic water permeability: the water transport in the frog rose from 0.05±0.02 to 1.21±0.26 μl min −1·cm −2, in the trout, from 0.041±0.011 to 0.26±0.034 μl min −1·cm −2. Such an increase in the osmotic water permeability in the trout and frog urinary bladder occurred in the background of a decrease in the prostaglandin E 2 concentration in the serosal Ringer solution. This permeability increase was accompanied by the formation of aggregates of intramembranous particles in the apical plasma membrane of the trout and frog urinary bladder. A decrease in the osmotic water permeability was achieved by the addition to the serosal Ringer solution of 10 −8 M prostaglandin. Experiments on the frog urinary bladder have shown that prostaglandins E 1, I 2 and F 2 α also decrease the osmotic water permeability. Vasotocin increased the osmotic water permeability in the frog urinary bladder but did not affect the osmotic water permeability of the trout urinary bladder. The data obtained indicates a role of the endogenous prostaglandin production in maintaining the low osmotic water permeability in the frog and trout urinary bladder. A suggestion is made that in the vertebrate evolution, colonisation of the fresh-water was connected with the maintenance of the low osmotic water permeability via participation of prostaglandins, whereas the vasotocin hydroosmotic effect developed in the vertebrate evolution later and provided for the possibility of the water absorption, osmotic homeostasis and animal migration from fresh-water to the land.

Modulation of the effect of arginine-vasopressin on water and ion transport in the newt early distal tubule and frog urinary bladder by V1-antagonists.

In the early distal tubule of the newt Triturus vulgaris L., 1 nM arginine-vasopressin (AVP) increased water reabsorption; the fractional reabsorption of Na+ was elevated from 46.2 +/- 6.9% to 67.8 +/- 3.9% (P < 0.001), of Cl- from 52.7 +/- 6.7% to 73.1 +/- 3.5% (P < 0.001), of Mg2+ from 48.0 +/- 7.7% to 71.7 +/- 6.3% (P < 0.001). When V1-receptors were blocked by 1 nM peptide V1-antagonist [1-(beta-mercapto-beta,beta-cyclopentamethylene propionic acid), 2-(O-methyl) Tyr]-[Arg8]vasopressin, 1 nM AVP increased the fractional reabsorption of fluid by 8.9% of Na+ by 10.7% and of Cl- by 11.2%, as compared with the effect of AVP alone. The fractional reabsorption of Ca2+ after addition of AVP did not differ from control; when V1-receptors were blocked in the presence of AVP, the fractional reabsorption of Ca2+ was increased by AVP. The V1-receptor block in the presence of AVP did not change the fractional reabsorption of Mg2+. Experiments on the urinary bladder of the frog Rana temporaria L. showed that 1 nM SR 49059, a non-peptide antagonist of V1a-receptors, like the peptide V1-antagonist, enhanced the AVP effect by 29%. Inhibition of protein kinase C activity by calphostin C (1 nM) mimicked the effect of V1-antagonists; the AVP hydroosmotic effect was increased by 60%. The results obtained indicate that V1-receptors modulate the effects of V2-receptor activation: their block is accompanied by an enhancement of the AVP hydroosmotic effect in the frog urinary bladder and by an increase of Na+ and Cl- reabsorption in the newt early distal tubule. The enhancement of the AVP effect owing to the V1-receptor activation seems to be mediated by a decrease in protein kinase C activity.

Epidermal growth factor antagonizes vasopressin in vivo and in vitro

Since EGF causes diuresis through a renal action and may antagonize the hydroosmotic effect of AVP in vitro we investigated the antagonistic action of EGF with AVP in vivo and the mechanism of the antagonism in vitro. Conscious ewes received i.m. injections of a selective AVP V2-receptor agonist (1-desamino, D-Arg8 vasopressin acetate, DDAVP) every 12 hours for days 5 to 16. All ewes received an i.v. isotonic saline infusion (100 ml/day) for days 1 to 8 and days 13 to 16, and i.v. EGF in 100 ml saline/day at doses of 0 (N = 8) or 10 (N = 8) micrograms/hr for days 9 to 12. DDAVP reduced both urine volume and water intake, and increased urine osmolality. In contrast, simultaneous infusion of EGF reversed the DDAVP-induced responses, resulting in a transient negative fluid balance, kaliuresis and a transient natriuresis (all P < 0.05). When EGF treatment ceased, the effects of DDAVP treatment alone gradually became apparent. From the in vitro studies, the AVP-related peptides displaced specific AVP V1- and V2-receptor antagonist radioligands from rat renal inner medullary membranes, whereas EGF had no effect. However, EGF antagonized AVP V2-stimulated cAMP production in a dose-dependent way (IC50 = 2 x 10(-7) M). Therefore, the diuretic effect of EGF is not via direct antagonism of the antidiuretic AVP V2-receptor but seems mediated by inhibition of the antidiuretic AVP V2-receptor second messenger system.

The effect of arachidonic acid on the hydroosmotic action of vasopressin in frog urinary bladder

In experiments on frog urinary bladder it was found that 23 nM arginine-vasopressin (antidiuretic hormone, AVP), 10 min after addition, increased the content of free arachidonic acid (AA) in bladder tissue from 18.6 ± 1.2 to 33.3 ± 3.9 ng/mg protein. The exogenous AA (10 −8– −5 M) added to the serosal Ringer solution did not change the basal level of osmotic water flow, but significantly inhibited the hydroosmotic effect of 23 nM AVP in a dose-dependent manner. The inhibitory effect of AA was not eliminated in the presence of 1 μM indomethacin, 1 μM nordihydroguaiaretic acid, 100 μM ketoconazole, nor if the nonmetabolized analog of AA, eicosatetraynoic acid (1 μM), was used instead of AA. AA inhibited only the action of vasopressin, but had no effect on osmotic water flow induced by 2 mM cAMP + 50 μM 3-isobutyl-1-methylxanthine or 20 μM forskolin. These data suggest that AA is another phospholipid-derived messenger, which modulates the hydroosmotic action of vasopressin in frog urinary bladder at a point prior to cAMP formation.

Hydrocortisone-induced enhancement of the hydroosmotic effects of vasopressin and cAMP on the wall of frog urinary bladder

Hydrocortisone-induced enhancement of the hydroosmotic effects of vasopressin and cAMP on the wall of frog urinary bladder

Vasopressin V1-antagonist increases the hydroosmotic response to arginine vasopressin in frog urinary bladder.

V1-antagonist, [Mca1, D-Phe2, Sar7] arginine vasopressin, at a concentration 0.5 nM to 5 nM increased hydroosmotic effect of 5 nM arginine vasopressin in frog urinary bladder. 1 nM V1-antagonist [Mca1, O-Me-Tyr2] arginine vasopressin does not changed hydroosmotic effect of 0.1 nM dibutyryl cAMP. We suggest that-both V1- and V2-receptors are present on epithelial cells of frog urinary bladder and that V1-receptors are involved in the modulation of the hydroosmotic effect of arginine vasopressin-mediated by V2-receptors. by V2-receptors.

Cytochrome P450 metabolites of arachidonic acid are potent inhibitors of vasopressin action on rabbit cortical collecting duct.

AA is metabolized by a cytochrome P450, NADPH-dependent epoxygenase to four regioisomeric epoxyeicosatrienoic acids (EETs). The EETs are further hydrated enzymatically to their respective diols, vic-dihydroxyeicosatrienoic acids (DHETs). We studied the effect of pretreatment with DHETs on 10 microU/cm2 arginine vasopressin (AVP)-stimulated hydraulic conductivity (Lp) (Lp x 10(-7) cm/atm/s, mean +/- SE) in rabbit cortical collecting ducts (CCDs) perfused in vitro at 37 degrees C. At 10(-6) M all four DHETs were potent inhibitors of the hydroosmotic effect of AVP. 14,15-DHET was the most potent isomer; it reduced AVP-induced Lp from a control value of 234.75 +/- 11.7, n = 17, to a value of 95.2 +/- 8.39, n = 5, P less than 0.0001, a reduction of AVP-mediated water flow of 60%. The inhibitory effect of 14,15-DHET was dose dependent and significant to nanomolar concentrations. 14,15-DHET at 10(-7) M was as potent an inhibitor of AVP's activity as was 10(-7) M PGE2. AVP's hydroosmotic effect is mediated through its intracellular second messenger, cAMP. 8-p-Chlorophenylthio-cAMP (CcAMP) at 10(-4) M induced a peak Lp of 189.6 +/- 11.0, n = 8; pretreatment with 10(-6) M 14,15-DHET reduced CcAMP-peak Lp to 132.0 +/- 13.4, n = 5, P less than 0.01, demonstrating a post-cAMP effect. Gas chromatography/mass spectroscopy suggests that EETs are present in extracts purified from CCDs. We conclude that cytochrome P450 epoxygenase eicosanoids are potent inhibitors of the hydroosmotic effect of vasopressin and are endogenous constituents of normal CCDs, the major target tissue for AVP.