Effect of phosphate deprivation on enzymes of the cyclic adenosine monophosphate metabolism in rat proximal convoluted and proximal straight tubules.

Phosphate deprivation causes a resistance to the phosphaturic effect of parathyroid hormone (PTH). The present study determined whether acute phosphate deprivation alters basal or stimulated activities of key enzymes of the cyclic adenosine monophosphate (cAMP) metabolism in microdissected proximal convoluted and proximal straight tubules, since blunted cAMP levels in these proximal subsegments might account for refractoriness to the effect of PTH on phosphate reabsorption in the proximal convoluted and proximal straight tubule segments. In the proximal convoluted tubules of rats fed a normal-phosphate diet (NPD), PTH increased the adenylate cyclase activity by tenfold. In the proximal convoluted tubule of rats fed a low-phosphate diet (LPD), PTH also increased the adenylate cyclase activity by tenfold. In addition, forskolin increased the adenylate cyclase activity to levels similar to PTH in the proximal convoluted tubule of rats fed NPD or LPD. In the proximal straight tubule of rats fed NPD, PTH resulted in an approximately fivefold increase in adenylate cyclase activity. In the proximal straight tubule of rats fed LPD, PTH resulted in a fourfold increase in adenylate cyclase activity. The forskolin-stimulated adenylate cyclase activity was markedly decreased (46%) in the proximal straight tubule of phosphate-deprived rats. The cAMP-phosphodiesterase activity in the proximal convoluted tubule was significantly increased by 26% in phosphate-deprived rats. The cAMP-phosphodiesterase activities in the proximal straight tubules from rats fed NPD or LPD were similar. We conclude that distinct differences in key enzymes of cAMP metabolism exist in the proximal convoluted and proximal straight tubule subsegments. Further, phosphate deprivation affects the cAMP-phosphodiesterase and adenylate cyclase activities differently in these nephron subsegments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ring-deleted analogs of atrial natriuretic factor inhibit adenylate cyclase/cAMP system. Possible coupling of clearance atrial natriuretic factor receptors to adenylate cyclase/cAMP signal transduction system

We have recently shown that atrial natriuretic factor (ANF) inhibits adenylate cyclase activity in rat platelets where only one population of ANF receptors (ANF-R2) is present, indicating that ANF-R2 receptors may be coupled to the adenylate cyclase/cAMP system. In the present studies, we have used ring-deleted peptides which have been reported to interact with ANF-R2 receptors also called clearance receptors (C-ANF) without affecting the guanylate cyclase/cGMP system, to examine if these peptides can also inhibit the adenylate cyclase/cAMP system. Ring-deleted analog C-ANF4-23 like ANF99-126 inhibited the adenylate cyclase activity in a concentration-dependent manner in rat aorta, brain striatum, anterior pituitary, and adrenal cortical membranes. The maximal inhibition was about 50-60% with an apparent Ki between 0.1 and 1 nM. In addition, C-ANF4-23 also decreased the cAMP levels in vascular smooth muscle cells in a concentration-dependent manner without affecting the cGMP levels. The maximal decrease observed was about 60% with an apparent Ki of about 1 nM. Furthermore, C-ANF4-23 was also able to inhibit cAMP levels and progesterone secretion stimulated by luteinizing hormone in MA-10 cell line. Other smaller fragments of ANF with ring deletions were also able to inhibit the adenylate cyclase activity as well as cAMP levels. Furthermore, the stimulatory effects of various agonists such as 5'-(N-ethyl)carboxamidoadenosine, dopamine, and forskolin on adenylate cyclase activity and cAMP levels were also significantly inhibited by C-ANF4-23. The inhibitory effect of C-ANF4-23 on adenylate cyclase was dependent on the presence of GTP and was attenuated by pertussis toxin treatment. These results indicate that ANF-R2 receptors or so-called C-ANF receptors are coupled to the adenylate cyclase/cAMP signal transduction system through inhibitory guanine nucleotide regulatory protein.     

Atrial natriuretic factor inhibits adenylate cyclase activity

The synthetic atrial natriuretic factor (ANF) (8- 33AA ) inhibited adenylate cyclase activity in aorta washed particles, mesenteric artery, and renal artery homogenates in a concentration dependent manner with an apparent Ki between 0.1 to 1nM . The extent of inhibition of adenylate cyclase by ANF varied from tissue to tissue. The adenylate cyclase from mesenteric artery and renal artery was inhibited to a greater extent as compared to that from aorta. ANF was also able to inhibit the stimulatory effects of hormones on adenylate cyclase activity and of agents such as F- and forskolin which activate adenylate cyclase by receptor- independent mechanism. In addition, ANF showed an additive effect with the inhibitory response of angiotensin II on adenylate cyclase from rat aorta. These studies for the first time demonstrate that ANF is an inhibitor of adenylate cyclase of several systems.     

Evidence for particulate guanylate cyclase in rat kidney after stimulation by atrial natriuretic factor. An ultracytochemical study

Summary Cytochemical localization of particulate guanylate cyclase (GC) in rat kidney, after stimulation with atrial natriuretic factor (ANF), was studied by electron microscopy. In the renal corpuscle GC reaction product was localized on podocytes. Other segments of the nephron that showed ultracytochemical evidence of GC activity were the proximal convoluted tubule, the thick ascending limb of the loop of Henle and the collecting tubule. All GC positivity was associated with plasma membranes. Samples incubated in basal conditions (without ANF) did not reveal any GC reaction product. These results indicate that ANF is a strong activator of particulate GC. Our data also suggests that, through the enzyme, ANF acts directly on epithelial cells of tubules where Na⁺ reabsorption occurs. This is in agreement with the hypothesis that ANF has a direct tubular effect on natriuresis.     

The increase of cGMP by atrial natriuretic factor correlates with the distribution of particulate guanylate cyclase

We have demonstrated previously that atrial natriuretic factor (ANF) augments urinary, plasma and kidney cGMP levels but has no significant effect upon cAMP. Using cGMP as a marker, we searched for specific target sites involved in the action of ANF in the dog kidney, and observed no change of cGMP in the proximal tubules, a 2-fold increase over basal levels in the thick loop of Henle and a 3-fold elevation in the collecting duct. The most striking action on cGMP occurred in the glomeruli with a rise of up to 50-fold being evident at 1-2 min. after the addition of ANF. The results obtained in the absence or presence of a phosphodiesterase inhibitor support the notion that the effects of ANF were exerted at the level of guanylate cyclase stimulation rather than cGMP phosphodiesterase inhibition. The action of sodium nitroprusside (SNP), a direct stimulator of soluble guanylate cyclase, differed from that of ANF. The ability of the factor to enhance cGMP levels was correlated with the distribution of particulate guanylate cyclase. This study identifies the glomeruli and the distal part of the nephron as specific targets of ANF and implicates particulate guanylate cyclase as the enzyme targetted for the expression of its action.     

Atrial natriuretic factor receptors are negatively coupled to adenylate cyclase in cultured atrial and ventricular cardiocytes

We have studied the effect of synthetic rat atrial natriuretic factor (ANF) on adenylate cyclase activity in cultured cardiocytes from atria (left and right) and ventricles from neonatal rats. ANF (Arg 101-Tyr 126) inhibited adenylate cyclase activity in a concentration dependent manner in cultured atrial (right and left atria) and ventricular cells. However the inhibition was greater in atrial cells as compared to ventricular cells. The maximal inhibition observed in ventricular cells was about 35% with an apparent Ki of about 10(-10) M, whereas about 55% inhibition with an apparent Ki between 5 X 10(-10) M and 65% inhibition with an apparent Ki of 10(-9) M were observed in right and left atrial cardiocytes respectively. The inhibitory effect of ANF was dependent on the presence of guanine nucleotides. Various hormones and agents such as isoproterenol, prostaglandins, adenosine, forskolin and sodium fluoride stimulated adenylate cyclase activities to various degrees in these atrial and ventricular cardiocytes. ANF inhibited the stimulatory responses of all these agonists, however the degree of inhibition varied for each agent. In addition ANF also inhibited cAMP levels in these cells. These data indicate that ANF receptors are present in cardiocytes and are negatively coupled to adenylate cyclase.     

Distribution of prostaglandin E2-sensitive adenylate cyclase along the rat nephron

To define sites of prostaglandin action of renal tubules, the distribution of adenylate cyclase sensitive to prostaglandin E₂ (PGE₂) was examined in single nephron segments dissected from rat kidney. Further, the interaction between PGE₂ and vasopressin on adenylate cyclase activity in nephron segments sensitive to vasopressin was evaluated. Procedures involved in isolating nephron segments were without effects on adenylate cyclase stimulation by PGE₂. PGE₂ stimulated adenylate cyclase activity of the thin descending limb of Henle (tDL), cortical collecting tubules (CCT), and medullary collecting tubules (MCT) at concentrations of 1.4 × 10^?5 to 2.8 × 10^?5 M. PGE₂ was without effects in other nephron segments tested including proximal convoluated tubules, proximal pars recta, the thin and thick ascending limb of Henle's loop, and distal and connecting tubules. PGE₂, at both high (2.8 × 10^?5 M) and low (2.8 × 10^?8 M) concentrations, did not inhibit adenylate cyclase activity stimulated by submaximal doses of vasopressin in medullary thick ascending limb of Henle (MTAL), CCT, and MCT. These data define the distribution of PGE₂-sensitive adenylate cyclase in the rat nephron, i.e., tDL, CCT, and MCT, and show the lack of direct inhibitory actions of PGE₂ on vasopressin sensitive adenylate cyclase in MTAL, CCT, and MCT.     

Inhibition of pituitary adenylate cyclase by atrial natriuretic factor

The effect of synthetic rat atrial natriuretic factor (ANF) on adenylate cyclase activity was studied in rat anterior and posterior pituitary homogenates. ANF (Arg 101-Tyr 126) inhibited adenylate cyclase activity in anterior and posterior pituitary homogenates in a concentration dependent manner. The maximum inhibitions observed were 42% in anterior pituitary with an apparent Ki of 10(-10) M, and 25% with an apparent Ki of 10(-11) M in posterior pituitary. Corticotropin-releasing factor (CRF), vasoactive intestinal peptide (VIP) and prostaglandins (PGE1) stimulated adenylate cyclase to various degrees in anterior pituitary homogenates and ANF inhibited the stimulatory effect of all these hormones. In addition ANF was also able to inhibit the stimulation exerted by NaF and forskolin which activate adenylate cyclase by receptor independent mechanism. Similarly, the stimulatory effects of N-Ethylcarboxamide adenosine (NECA), NaF and forskolin on adenylate cyclase in posterior pituitary homogenates were also inhibited by ANF. This is the first study demonstrating the inhibitory effect of ANF on pituitary adenylate cyclase.     

Islet activating protein inhibits kinin-stimulated inositol phosphate production, calcium mobilization, and prostaglandin E2 synthesis in renal papillary collecting tubule cells independent of cyclic AMP

The effects of islet-activating protein (pertussis toxin) on bradykinin-mediated inositol trisphosphate labeling, prostaglandin E2 production, and calcium mobilization in rabbit renal papillary collecting tubule cells were assessed. Islet-activating protein induced time and concentration-dependent decreases in bradykinin-stimulated prostaglandin E2 production. Islet-activating protein induced increases in basal cyclic AMP levels but not in arginine vasopressin-stimulated cAMP. This effect could be inhibited by prior incubation with 2',5'-dideoxyadenosine, an inhibitor of adenylate cyclase. Although cAMP and cAMP analogues were able to inhibit both basal and bradykinin-stimulated prostaglandin E2 formation, the inhibitory effects of islet-activating protein on prostaglandin E2 formation and inositol trisphosphate labeling were observed in the presence of dideoxyadenosine. Moreover, islet-activating protein lowered both the basal and kinin-stimulated cytosolic calcium concentration as assessed by Quin 2 fluorescence. Finally, incubation of a membrane fraction of papillary cells with islet-activating protein resulted in the ADP-ribosylation of a 39/41-kDa doublet. These data support the role of a guanine nucleotide regulatory protein in bradykinin-mediated signal transduction in rabbit papillary collecting tubule cells.     

Amiloride interacts with guanine nucleotide regulatory proteins and attenuates the hormonal inhibition of adenylate cyclase

The effect of amiloride on the hormonal regulation of adenylate cyclase was studied in the rat anterior pituitary. The diuretic did not alter basal adenylate cyclase but augmented the enzyme activity in an irreversible manner in the presence of guanosine 5'-O-(thiotriphosphate) (GTP gamma S) stimulated adenylate cyclase at lower concentrations and inhibited at higher concentrations. Amiloride treatment enhanced the stimulatory and abolished the inhibitory phase of GTP gamma S action. In addition, amiloride also attenuated the inhibitory effects of atrial natriuretic factor (ANF 99-126) and angiotensin II on cAMP levels and adenylate cyclase activity. On the other hand, amiloride showed an additive effect on the stimulation exerted by corticotropin-releasing factor and vasoactive intestinal peptide on adenylate cyclase in anterior pituitary and on isoproterenol-stimulated cAMP levels in cultured vascular smooth muscle cells. Pertussis toxin, in the presence of [alpha-32 P]NAD, catalyzed the ADP-ribosylation of two protein bands of Mr 41,000 and 39,000, referred to as Gi and Go, respectively, in the anterior pituitary, and 40,000-Da protein in the aorta, referred to as Gi. Amiloride treatment inhibited the labeling of all these bands in a concentration- and time-dependent manner. Similarly, the pertussis toxin-catalyzed ADP-ribosylation of purified Gi from bovine brain was also inhibited by amiloride treatment. However, amiloride had no significant effect on the cholera toxin-catalyzed ADP-ribosylation of Gs. These data suggest that amiloride interacts with the guanine nucleotide regulatory proteins Gi and Go. Modification of Gi results in the attenuation of hormone-induced adenylate cyclase and cAMP inhibition. However, the interaction between amiloride and Go and the consequent Ca2+ mobilization and phosphatidylinositol turnover have to be investigated.     

Activation of adenylate cyclase by molybdate.

The effect of molybdate on adenylate cyclase (EC 4.6.1.1) in rat liver plasma membranes has been examined. The apparent K alpha for molybdate activation of the enzyme is 4.5 mM, and maximal, 7-fold stimulation is achieved at 50 mM. The observed increase in cAMP formation in the adenylate cyclase assay is not due to: (a) an inhibition of ATP hydrolysis; (b) a molybdate-catalyzed conversion of ATP to cAMP; (c) an inhibition of cAMP hydrolysis; or (d) an artifact in the isolation of cAMP formed in the reaction. Molybdate activation of adenylate cyclase is a general phenomenon exhibited by the enzyme in brain, cardiac, and renal tissue homogenates and in erythrocyte ghosts. However, like fluoride and guanyl-5'-yl imidodiphosphate (Gpp(NH)p), molybdate does not activate the soluble rat testicular adenylate cyclase. Molybdate is a reversible activator of adenylate cyclase. Activation is not due to an increase in ionic strength and is independent of the salt used to introduce molybdate. Molybdate does not activate adenylate cyclase previously stimulated with Gpp(NH)p or fluoride. At concentration greater than 20 mM, molybdate inhibits fluoride-stimulated adenylate cyclase, and at concentrations greater than 100 mM, molybdate stimulation of basal adenylate cyclase activity is diminished.     

Mechanisms of release and renal tubular action of atrial natriuretic factor

Inasmuch as atrial natriuretic factor (ANF) is apparently involved causally in the renal response to acute hypervolemia, it became of interest to study cellular mechanisms of release and renal tubular action. To study release mechanisms, freshly excised rat heart atria were incubated in vitro. Activation of the cellular adenylate cyclase system by either beta-adrenergic stimulation or the vasopressin analog deamino-8-D-arginine vasopressin did not result in ANF release. By contrast, activation of the polyphosphoinositide system by alpha-adrenergic stimulation or stimulation of the V1-type vasopressin receptors, and by a calcium ionophore or active phorbol ester, significantly increased natriuretic activity in the medium and reduced it in tissue. It is concluded, therefore, that activation of this latter system is the mechanism for ANF secretion from atrial myocytes. To test the effect of ANF on tubular transport in the medullary collecting duct, microcatheterization was used in rats before and during i.v. infusion of synthetic atrial peptide (23 amino acids). It was found that tubular delivery of salt to this part of the nephron was increased, and that reabsorption in the duct itself was reduced. In control experiments, increased delivery was associated with proportionately increased reabsorption, which demonstrated glomerulotubular balance in the nephron segment under normal conditions. The natriuretic effect of ANF, therefore, was not caused solely by enhanced tubular load, but included specific inhibition of duct sodium reabsorption as an essential feature of the renal response.     

Neuropeptide Y inhibits cAMP accumulation in renal proximal convoluted tubules

The effect of neuropeptide Y (NPY) on cAMP accumulation in various segments of the rabbit nephron was examined. NPY inhibited parathyroid hormone-stimulated cAMP accumulation in the proximal convoluted tubule in a concentration-dependent manner. NPY also inhibited forskolin-stimulated cAMP production in this segment of the nephron. In contrast, NPY had no effect on parathyroid hormone or forskolin-stimulated cAMP accumulation in the proximal straight tubule. Similarly, NPY had no effect on forskolin-stimulated cAMP levels along the rest of the nephron. These results are consistent with previous studies which have localized NPY receptors to the proximal convoluted tubule, and suggest that NPY via its effects on cAMP metabolism may play a role in proximal tubule transport.     

Histochemical localization of hormone sensitive adenylate cyclase in defined nephron epithelia in culture

Summary Distal nephron epithelia of defined anatomical origin were microdissected from rabbit kidneys and individually explanted into an in vitro culture system. The 7 day monolayers grown from four different nephron epithelia were studied for the presence and amount of adenylate cyclase reaction product. In each case basal adenylate cyclase was compared with the enzyme reaction product after stimulation by arginine vasopressin, calcitonin, parathyroid hormone (PTH) and isoproterenol. In cortical collecting tubule cultures, the reaction was stimulated by vasopressin >isoproterenol>calcitonin. PTH had no effect. In cortical thick ascending loop of Henle cells, the stimulation was by calcitonin>vasopressin=PTH. Isoproterenol had no effect. In medullary ascending loop epithelia, stimulation was by vasopressin=calcitonin. Neither isoproterenol nor PTH had an effect.These observations indicate that adenylate cyclase is histochemically demonstrable in cultivated cells from rabbit distal nephron segments and that the enzyme activation by hormones is differential according to the epithelium of origin.     

Histochemical localization of hormone sensitive adenylate cyclase in defined nephron epithelia in culture

Distal nephron epithelia of defined anatomical origin were microdissected from rabbit kidneys and individually explanted into an in vitro culture system. The 7 day monolayers grown from four different nephron epithelia were studied for the presence and amount of adenylate cyclase reaction product. In each case basal adenylate cyclase was compared with the enzyme reaction product after stimulation by arginine vasopressin, calcitonin, parathyroid hormone (PTH) and isoproterenol. In cortical collecting tubule cultures, the reaction was stimulated by vasopressin greater than isoproterenol greater than calcitonin. PTH had no effect. In cortical thick ascending loop of Henle cells, the stimulation was by calcitonin greater than vasopressin = PTH. Isoproterenol had no effect. In medullary ascending loop epithelia, stimulation was by vasopressin = calcitonin. Neither isoproterenol nor PTH had an effect. These observations indicate that adenylate cyclase is histochemically demonstrable in cultivated cells from rabbit distal nephron segments and that the enzyme activation by hormones is differential according to the epithelium of origin.     

Pertussis toxin attenuates atrial natriuretic factor-mediated inhibition of adenylate cyclase. Involvement of inhibitory guanine nucleotide regulatory protein

The effect of pertussis toxin treatment was studied on the inhibitory effect of atrial natriuretic factor (ANF) on adenylate cyclase activity in rat aorta. The incubation of rat aorta washed particles with pertussis toxin and [alpha-32P]NAD resulted in the ADP-ribosylation of a single 40-kDa protein. In addition, pertussis toxin treatment enhanced guanosine 5'-O-(thiotriphosphate) and isoproterenol-stimulated adenylate cyclase activities and attenuated the ANF-mediated inhibition of basal, isoproterenol-, and forskolin-stimulated adenylate cyclase activities. These data suggest that ANF receptors are coupled to adenylate cyclase through inhibitory guanine nucleotide regulatory protein.     

Renal tubular actions of ANF.

Many of the earliest investigations of the renal effects of atrial natriuretic factor (ANF) pointed to the glomerulus as a major site of the peptide's action. More recently, there have been many reports showing various effects of ANF on renal tubular epithelia, including collecting ducts, thick ascending limbs of Henle's loop, thin limbs of Henle's loops, and proximal tubules. The purpose of this review is to summarize the evidence for renal tubular actions of ANF and analyze it from the perspective of the specialized functions of the individual nephron segments, addressing the question: can renal tubule effects of ANF play a significant role in the precise day-to-day regulation of renal NaCl and water excretion? Based on these considerations, we propose that long-term renal tubular action of ANF may be distinct from its short-term natriuretic effect. The short-term action of ANF to accelerate salt and water excretion may play a role in the overall response to acute volume overload. This action of ANF appears to be largely due to an ANF-mediated increase in glomerular filtration rate accompanied by a blunting of the tubuloglomerular feedback mechanism, perhaps with some contribution from ANF-mediated inhibition of fluid absorption in the proximal tubule. In contrast, contributions of ANF to the precise day-to-day regulation of salt and water excretion are likely to be chiefly due to ANF-mediated inhibition of NaCl and water absorption in collecting ducts, but may also involve actions of ANF on the loop of Henle.     

A microfluorometric method for alkaline phosphatase: Application to the various segments of the nephron

A microtechnique has been developed for the measurement of alkaline phosphatase in minute amounts of renal tissue. This microtechnique utilizes the known fluorescent property of 4-methylumbelliferyl phosphate following enzymatic hydrolysis. The reaction is sensitive and reproducible and is inhibited by l-bromotetramisole, a specific alkaline phosphatase inhibitor. The microdetermination of alkaline phosphatase activity in the various segments of the mouse nephron allowed the localization of the enzyme in the glomeruli, and in the proximal convoluted tubule where the activity progressively decreases from the capsule of Bowman to the more distal segments. The enzyme was absent from the pars recta or S3 and from the rest of the nephron. This technique is applicable to very small amounts (0.1 μg of protein) of any tissue containing alkaline phosphatase.     

Regulation of cyclic AMP metabolism in rabbit cortical collecting tubule cells by prostaglandins

Prostaglandin E1 (PGE1) at 1 nM inhibits arginine-vasopressin (AVP)-induced water reabsorption in the rabbit cortical collecting tubule (RCCT), while 100 nM PGE1, by itself, stimulates water reabsorption (Grantham, J. J., and Orloff, J. (1968) J. Clin. Invest. 47, 1154-1161). To investigate the basis for these two responses, we measured the effects of prostaglandins on cAMP metabolism in purified RCCT cells. In freshly isolated cells, PGE2, PGE1, and 16,16-dimethyl-PGE2 acting at high concentrations (0.1-10 microM) stimulated cAMP accumulation; however, one PGE2 analog, sulprostone (16-phenoxy-17,18,19,20-tetranor-PGE2 methylsulfonilamide), failed to stimulate cAMP accumulation or to antagonize PGE2-induced cAMP formation; PGD2, PGF2 alpha, and a PGI2 analog, carbacyclin (6-carbaprostaglandin I2), also failed to stimulate cAMP synthesis. These results suggest that there is a PGE-specific stimulatory receptor in RCCT cells which mediates activation of adenylate cyclase. Occupancy of this receptor would be anticipated to cause water reabsorption by the collecting tubule. At lower concentrations (0.1-100 nM) PGE2, PGE1, 16,16-dimethyl-PGE2, and, in addition, sulprostone inhibited AVP-induced cAMP accumulation by fresh RCCT cells in the presence of cAMP phosphodiesterase inhibitors. Pertussis toxin pretreatment of RCCT cells blocked the ability of both PGE2 and sulprostone to inhibit AVP-induced cAMP accumulation. In membranes prepared from RCCT cells, sulprostone prevented stimulation of adenylate cyclase by AVP. These results suggest that E-series prostaglandins (including sulprostone) can act through an inhibitory PGE receptor(s) coupled to the inhibitory guanine nucleotide regulatory protein, Gi, to block AVP-induced cAMP synthesis by RCCT cells. Occupancy of this receptor would be expected to cause inhibition of AVP-induced water reabsorption in the intact tubule. Curiously, after RCCT cells were cultured for 5-7 days, PGE2 no longer inhibited AVP-induced cAMP accumulation, but PGE2 by itself could still stimulate cAMP accumulation. In contrast to PGE2, epinephrine acting via an alpha 2-adrenergic, Gi-linked mechanism did block AVP-induced cAMP formation by cultured RCCT cells. This implies that some component of the inhibitory PGE response other than Gi is lost when RCCT cells are cultured.     

Augmentation of forskolin-induced cAMP production by ouabain in rat renal papillary collecting tubule cells in culture

The effect of extracellular calcium (Ca2+) on the cellular action of forskolin was studied using a Na+, K(+)-ATPase inhibitor ouabain in rat renal papillary collecting tubule cells in culture. Forskolin-induced cAMP production was enhanced by the pretreatment of cells with ouabain, providing that a dose-dependent curve with forskolin shifted to the left. The enhancement by ouabain of cellular cAMP production in response to forskolin was totally blunted by cotreatment with cobalt, verapamil, or Ca2(+)-free medium containing 1 mM EGTA. In addition, two dissimilar antagonists of calmodulin, namely trifluoperazine and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W - 7), attenuated the ouabain's effect on cAMP production in response to forskolin. These results therefore indicate that ouabain enhances the activation of adenylate cyclase by forskolin, mediated through cellular free Ca2+, in renal papillary collecting tubule cells, and that extracellular Ca2+ is an important source for cellular Ca2+ mobilization by ouabain.