Bradykinin-stimulated changes in inositol phosphate mass in renal papillary collecting tubule cells

The effect of bradykinin on changes in the chemical levels of myo-inositol polyphosphates in renal papillary collecting tubules was investigated. Myo-inositol phosphate mass was determined by means of an enzymatic, fluorometric assay. Bradykinin induced increases in myo-inositol mono-, bis-, and trisphosphate which were both time and concentration dependent. Furthermore, the magnitude of the chemical levels of myo-inositol monophosphate formed were unlikely to be accounted for solely by the formation and degradation of myo-inositol trisphosphate. These observations are consistent with the concomitant hydrolysis of phosphatidylinositol and phosphatidylinositol bisphosphate. This study also confirms, in freshly isolated renal tubules, observations regarding bradykinin-induced phosphatidylinositol bisphosphate hydrolysis made previously in radiolabeled cultures.     

Vasopressin does not hydrolyze polyphosphoinositides in rabbit papillary collecting tubule cells

Changes in phosphatidylinositol metabolism are suggested to be involved in the mechanism of action of many membrane active hormones. We studied the effect of vasopressin on polyphosphoinositide metabolism in rabbit papillary collecting tubule cells to assess if the hydrolysis of these phospholipids is involved in transmembrane signaling. Rabbit papillary collecting tubule cells grown in monolayers for 5 days were labeled to constant specific activity with [3H]inositol. The temporal changes in [3H]inositol-labeled phospholipids were assessed in response to vasopressin. Similarly, water-soluble inositides were monitored after separation by ion exchange chromatography. Intracellular Ca2+ was monitored by use of the fluorescent indicator dye, quin2. Vasopressin (10(-7) M) did not increase the hydrolysis of phosphoinositides over a 5 min period when compared with controls. Similarly, there was no increase in water-soluble phosphoinositols during the same interval. Pretreating the cells with LiCl (10 mM) did not produce any increase in inositol 1-phosphate when stimulated with vasopressin but did in response to bradykinin. Finally, vasopressin did not increase cytosolic Ca2+ and did not increase the release of prostaglandin E2 into the media under our experimental conditions. We conclude that vasopressin does not stimulate prostaglandin E2 in rabbit papillary collecting tubule cells, does not initiate hydrolysis of polyphosphoinositides and does not increase cytosolic Ca2+. Thus these cells lack V1 receptor coupling mechanisms.     

Observation of myo-inositol 1,2-(cyclic) phosphate in a Morris hepatoma by 31P NMR

We have identified an unusual resonance at 16.5 ppm in the 31P NMR spectrum of a Morris (7777) hepatoma grown in the inguinal fossa of a Buffalo rat as myoinositol 1,2-(cyclic) phosphate. This compound has been observed in all of the 32 tumors examined as well as in cultured cells derived from the tumor, but it has not been observed in normal rat tissues. Its level in the aqueous phase of chloroform/methanol/water extracts of the tumor is 70 +/- 40 nmol/g, wet weight (n = 4). The presence of a breakdown product of phosphatidylinositol at such high levels in a fast growing tumor may provide an important clue for understanding the metabolic defect that results in the malignant growth of this tumor.     

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.     

Atrial natriuretic factor increases cyclic GMP and inhibits cyclic AMP in rat renal papillary collecting tubule cells in culture

The present study was undertaken to determine whether human atrial natriuretic factor (hANF) produces guanosine-3', 5'-monophosphate (cGMP) and alters arginine vasopressin (AVP)- and forskolin (F)- induced adenosine-3', 5'-monophosphate (cAMP) production in the cultured rat renal papillary collecting tubule cells. hANF increased cellular cGMP levels in a dose dependent manner. AVP and F, however, did not affect cGMP production. hANF significantly inhibited AVP- and F-stimulated cAMP levels, but hANF by itself did not affect cellular cAMP production. Since F activates adenylate cyclase at a step of catalytic unit and the cellular action of AVP to activate adenylate cyclase is mediated through receptor-catalytic units, the present results indicate that hANF may directly inhibit the AVP- and F-stimulated adenylate cyclase in renal papillary collecting tubules.     

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.     

Interrelationships among prostaglandins, vasopressin and cAMP in renal papillary collecting tubule cells in culture

To determine the influence of prostaglandins on cAMP metabolism in renal papillary collecting tubule (RPCT) cells, intracellular cAMP levels were measured after incubating cells with prostaglandins (PGs) alone or in combination with arginine vasopressin (AVP). PGE1, PGE2 and PGI2, but not PGD2 or PGF2 alpha, increased intracellular cAMP concentrations. At maximal concentrations (10(-5) M) the effects of PGE2 plus PGI2 (or PGE1), but not of PGI2 plus PGE1, were additive suggesting that at least two different PG receptors may be present in RPCT cell populations. Bradykinin treatment of RPCT cells caused an accumulation of intracellular cAMP which was blocked by aspirin and was quantitatively similar to that observed with 10(-5) M PGE2. PGs, when tested at concentrations (e.g. 10(-9) M) which had no independent effect on intracellular cAMP levels, did not inhibit the AVP-induced accumulation of intracellular cAMP in RPCT cells. These results indicate that PGs do not block AVP-induced accumulation of intracellular cAMP in RPCT cells at concentrations of PGs which have been shown to inhibit the hydroosmotic effect of AVP on perfused collecting tubule segments. However, at higher concentrations of PGs (e.g. 10(-5) M), the effects of AVP plus PGE1, PGE2, PGI2 or bradykinin on intracellular cAMP levels were not additive. Thus, under certain conditions, there is an interaction between PGs and AVP at the level of cAMP metabolism in RPCT cells.     

Basolateral Na-H exchange in the rabbit cortical collecting tubule

We used the intracellular absorbance spectrum of the dye 4',5'-dimethyl-5- (and -6-) carboxyfluorescein (Me2CF) to measure intracellular pH (pHi) in the isolated, perfused cortical collecting tubule (CCT) of the rabbit nephron. The incident spot of light was generally 10 micron in diameter, large enough to illuminate from two to six cells. No attempt was made to distinguish principal from intercalated cells. All experiments were carried out in HCO3- -free Ringer to minimize HCO3- transport. When cells were acid-loaded by briefly exposing them to Ringer containing NH+4 and then withdrawing the NH+4, pHi spontaneously recovered from the acid load. The pHi recovery was best fit by the sum of two exponentials. When the acid loading was performed in the absence of Na+, the more rapid of the two phases of pHi recovery was absent. The remaining slow phase never returned pHi to normal and was sometimes absent. Returning Na+ to the lumen had only a slight effect on the pHi recovery. However, when Na+ was returned to the basolateral (i.e., blood-side) solution, pHi recovered rapidly and completely. The apparent Km for basolateral Na+ was 27.3 +/- 4.5 mM. The basolateral Na-dependent pHi recovery was reversibly inhibited by amiloride. We conclude that the mechanism responsible for the rapid phase of pHi recovery is an Na-H exchanger confined primarily, if not exclusively, to the basolateral membrane of the CCT.     

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.     

Ouabain-induced cell swelling in rabbit cortical collecting tubule: NaCl transport by principal cells

Summary Ouabain had no effect on the volume of intercalated cells of DOCA-stimulated rabbit cortical collecting tubules, but caused principal cells to swell rapidly at an initial rate of 67% min., Principal cells swelled 133% then activated regulatory volume decrease mechanisms and shrank at an initial rate of –3%/min to a new volume 13% above control. The initial rate of ouabain swelling was completely inhibited by perfusate Na⁺ removal or reduced 95% by luminal addition of 10^–5 m amiloride. Luminal peritubular, or bilateral Cl^– removal each caused cell shrinkages of 10% and reduced the rate of ouabain swelling by 70, 85, and 99%, respectively. The presence of an apical Cl^– transport step in principal cells was confirmed by increasing luminal K⁺ from 5 to 53mm, which caused cell swelling of 22%. This volume increase was completely blocked by luminal Cl^– removal, but was unaffected by peritubular Cl^– substitution. Perfusion of CCT with 0.1mm acetazolomide, 0.1mm DPC or 0.5mm SITS caused principal cell shrinkages of 7–9% and reduced the rate of ouabain swelling by 60, 70, and 40%, respectively. The initial rate of ouabain swelling was inhibited 70% by bilateral CO₂/HCO₃ removal and 50% by whole animal acid loading. Taken together these results demounstrate that ouabain swelling is due to cellular NaCl accumulation and that Na⁺ enters the cell primarily through apical Na⁺ channels. Cellular Cl^– entry occurs at least partially through the apical membrane and may be mediated by a Cl^–/HCO ₃ ^– exchanger. Brief (45–90 sec) exposure of principal cells to ouabain is associated with a rapid inhibition of Na⁺ and/or Cl^– entry steps, whereas long-term (>5min) ouabvain exposure completely blocks one or both of these transport pathways.     

Vasopressin-induced increases in cellular free calcium concentration measured in single cells of rat renal papillary collecting tubule

We determined the cellular free calcium concentration [Ca2+]i in response to arginine vasopressin (AVP) using single cells of cultured rat renal papillary collecting tubule cells. AVP at a concentration of 1 x 10(-10) M or higher significantly increased [Ca2+]i in a dose-dependent manner. The prompt increase in [Ca2+]i induced by AVP was completely blocked by the V1V2 antagonist, but not by the V1 antagonist. Also, an antidiuretic agonist of 1-deamino-8-D-arginine vasopressin (dDAVP) increased [Ca2+]i, which was blocked by the pretreatment with the V1 V2 antagonist. An AVP-induced increase in [Ca2+]i was still demonstrable in cells pretreated with Ca2(+)-free medium containing 1 x 10(-3) M EGTA, or a blocker of cellular Ca2+ uptake, 5 x 10(-5) M verapamil. These results indicate that AVP increases [Ca2+]i through the V2 receptor in renal papillary collecting tubule cells where cAMP is a well-known second messenger for AVP, and that cellular free Ca2+ mobilization depends on both the intracellular and extracellular Ca2+.     

Augmentation of vasopressin-induced cAMP production by high potassium in rat renal papillary collecting tubule cells in culture.

The effect of high potassium, 60 mM KCl, on the cellular action of arginine vasopressin (AVP) was studied in rat renal papillary collecting tubule cells in culture. In the presence of 0.5 mM 3-isobutyl-1-methylxanthine AVP-induced cAMP production was enhanced by pretreatment of the cells with 60 mM KCl. Such an enhancement was not found in cells pretreated with Ca(2+)-free medium containing 1 mM EGTA or in Na(+)-free medium, which rather reduced AVP-induced cAMP production. Similar results were obtained with the blockers of cellular Ca2+ uptake, 1 x 10(-4) M verapamil and 1 x 10(-5) M nifedipine. The 60 mM KCl elevated the cellular sodium concentration ([Na+]i) from 15.1 to 18.8 mM, cellular pH (pHi) from 7.18 to 7.32, and basal cellular free calcium concentration ([Ca2+]i). These results indicate that high potassium promptly augments AVP-induced cAMP production in renal papillary collecting tubule cells. This effect is based on the alkalinized pHi and the increased [Ca2+]i.     

Inhibitio of Na transport by prostacyclin (PGI2) in rabbit cortical collecting tubule

The effects of prostacyclin (PGI₂) on transepithelial potential difference (PD) and sodium transport were examined in rabbit cortical collecting tubules (CCT) perfused in vitro. Addition of PGI₂ (10^?6M) to the bathing medium, which was bubbled with 95% O₂ – 5% CO₂, caused a reversible decrease in PD averaging 49±9.4 (SE)%. Maximal effect was evident between 5–10 min. After addition of PGI₂ and PD returned spontaneously towards control values within 30 min., corresponding to the rapid degradation of PGI₂. In a more alkaline bathing solution achieved by bubbling with 100% O₂, in which the degradation of PGI₂ is known to be delayed markedly, the decrease in PD by PGI₂ was continuous and dose-dependent, with half-maximal and maximal effects achieved at 10^?7 M and 10^?5 M, respectively. Neither 10^?8 M PGI₂ nor vehicle alone exerted significant effects on PD. 6-keto-PGF_1α (10^?5M), believed to be the major metabolite of PGI₂, had no effect on PD. Lumen-to-bath flux of Na decreased with PGI₂ from 9.0 to 5.6 pEq/cm/sec (n=4, p<0.005), although bath-to-lumen flux did not change significantly. In summary, PGI₂ caused a dose-dependent decrease in PD of rabbit CCT and inhibited Na absorption in this segment in vitro. These results suggest that PGI₂ may play an important role in regulating Na transport in CCT.     

Bradykinin-induced changes in phosphoinositides, inositol phosphate production and intracellular free calcium in cultured bovine aortic endothelial cells

Acute hydrolysis of phosphoinositides has been demonstrated in bovine aortic endothelial cells (BAEC) treated with bradykinin (BK) (10(-7)M). The first phosphoinositide to decrease was phosphatidylinositol-4,5-bisphosphate (PIP2) indicating this to be the initial substrate of phospholipase action. Other lipid changes associated with the stimulation of BAEC were an increase in diacylglycerol (DAG) and arachidonic acid (AA) with a sustained production of phosphatidic acid (PA). The changes in cell phospholipids were accompanied by the release of inositol phosphates. Inositol-1,4,5-trisphosphate (Ins-1,4,5-P3) was produced within 10 s of stimulation with BK. There was no evidence for the production of inositol-1,3,4-trisphosphate. The release of ionic calcium (Ca2+) intracellularly was demonstrated. The timecourse of the rise in intracellular Ca2+ was consistent with the timecourse of production of IP3. Intracellular Ca2+ rose from 127 +/- 21 nM to 462 +/- 27 nM. The Ca2+ peak was at 7.0 +/- 0.4 s and took 3 min to reach a steady state which remained above the basal level. When extracellular Ca2+ was depleted in the extracellular medium a spike of intracellular Ca2+ release was measured with an immediate return to basal. Entry of extracellular Ca2+ into the cell after ionophore A23187 treatment does not induce inositol phosphate release, indicating that phosphoinositide hydrolysis is likely to be the cause rather than consequence of the elevation in cytosolic Ca2+. These data indicate action of phospholipase C (PLC) on PIP2 after BK stimulation of BAEC with the subsequent production of InsP3 causing the resulting intracellular Ca2+ release.(ABSTRACT TRUNCATED AT 250 WORDS)