Eicosanoids, mesangial contraction, and intracellular signal transduction.

The glomerular mesangial cell is a specialized pericyte with multiple functional capabilities including contraction. Mesangial contraction may reduce the glomerular filtration surface area and hence the ultrafiltration coefficient, Kf. Cultured mesangial cells convert arachidonic acid into biologically active eicosanoids which are either contractile (thromboxane A2 [TxA2], prostaglandin F2 alpha [PGE2 alpha]) or relaxant (PGE2, PGI2). The addition of TxA2 analogues, PGE2 or sulfidopeptide leukotrienes (LTC4 and LTD4) stimulated contraction of cultured mesangial cells with threshold responses at approximately 1 nM and maximum responses at 1 microM. PGE2 and PGI2 antagonized mesangial contraction induced by TxA2 analogues. Contraction was enhanced by inhibiting mesangial cyclooxygenase with nonsteroidal antiinflammatory drugs (NSAID). Contractile eicosanoids stimulated phospholipase C thereby elevating intracellular inositol trisphosphate and cytosolic free Ca2+ concentration ([Ca2+]i). Vasorelaxant prostanoids stimulated adenylate cyclase, increasing intracellular cyclic AMP. We conclude that eicosanoids control mesangial contractility by regulating [Ca2+]i and cAMP. NSAID increase mesangial reactivity by blocking the inhibitory effects of endogenous vasodilator eicosanoids, with potential consequences on glomerular hemodynamics.     

Calcium dependency of prostaglandin E2 production in rat glomerular mesangial cells. Evidence that protein kinase C modulates the Ca2+-dependent activation of phospholipase A2

Calcium has been implicated as an important factor in prostaglandin production. Phospholipase A2, the enzyme believed to be rate limiting for prostaglandin synthesis, is stimulated by Ca2+; however, the levels of Ca2+ necessary to stimulate phospholipase A2 in cell-free systems are higher than levels achieved in intact cells in response to agonists that stimulate prostaglandin synthesis. We examined the calcium dependency of prostaglandin E2 (PGE2) synthesis in the glomerular mesangial cell. Vasopressin enhanced PGE2 synthesis by mechanisms independent of extracellular Ca2+ concentration. The Ca2+ concentration dependency of PGE2 production was established by rendering cells permeable with digitonin and clamping Ca2+ concentration at various levels. When cytosolic free Ca2+ concentration ([Ca2+]f) was set at levels equal to those measured after stimulation with vasopressin in the intact cell, the PGE2 production by the Ca2+-clamped permeabilized cells was approximately one-half of that obtained in nonpermeabilized cells stimulated with vasopressin. Since stimulation of mesangial cells with vasopressin increases protein kinase C activation as well as [Ca2+]f the effects on PGE2 production of protein kinase C activation with phorbol myristate acetate (PMA) were examined. When permeabilized cells were exposed to Ca2+ concentrations in the range of [Ca2+]f measured in cells treated with vasopressin the addition of PMA approximately doubled PGE2 production. No increase in PGE2 production was observed with PMA when Ca2+ concentration was fixed at basal levels of less than 100 nM. Ca2+-dependent acylhydrolase activity and PGE2 production were inhibited by calmodulin inhibitors, W-7 and compound 48/80. Thus, vasopressin-induced PGE2 production could be explained by a synergistic effect of protein kinase C activation together with an increase in [Ca2+]f. A synergistic action of Ca2+ and PMA on acylhydrolase activity could also be observed in nonpermeabilized cells where A23187 was used to increase [Ca2+]f. The effect of PMA was mimicked by another stimulant of protein kinase C, 1-oleoyl 2-acetylglycerol, albeit with lower potency. Neither PMA nor 1-oleoyl 2-acetylglycerol alone had any effect on acylhydrolase activity. Vasopressin, in the presence of GTP gamma S, stimulated phospholipase C in permeabilized cells when [Ca2+]f was fixed at less than 100 nM, without an associated increase in acylhydrolase activity. This evidence, together with inhibition of acylhydrolase activity with phospholipase A2 inhibitors, dibucaine and mepacrine, indicates that the primary acylhydrolase activity was due to phospholipase A2. The enhanced phospholipase A2 activity observed with protein kinase C activation when [Ca2+]f is increased may be related to phosphorylation of phospholipase A2 itself or phospholipase A2 modulatory proteins. These experiments demonstrate that both Ca2+ and protein kinase C play important roles in the regulation of phospholipase A2 and PGE2 synthesis.     

Prostaglandin synthesis by rat glomerular mesangial cells in culture. Effects of angiotensin II and arginine vasopressin.

Arginine vasopressin (AVP) and angiotensin II (ANG II) reduce the glomerular filtration rate and ultrafiltration coefficient. Vasodilatory prostaglandins (PG) antagonize these effects. AVP and ANG II also cause mesangial cell contraction. Therefore, possible PG stimulation by these peptides and two vasopressin analogues was studied in cultured rat glomerular mesangial cells. The effect of altered calcium availability on PG production was also studied. Glomeruli from 75-100-g Sprague-Dawley rats were cultured in supplemented nutrient media for 28 d and experiments were performed on the first passage. Mesangial cell morphology was confirmed by electron microscopy. Cells produced PGE2 much greater than PGF2 alpha greater than 6-keto-PGF1 alpha greater than thromboxane B2 when incubated with the divalent cation ionophore, A23187, or arachidonic acid (C20:4). ANG II and AVP selectively stimulated PGE2 at threshold concentrations of 10 nM ANG II and 100 pM of AVP. The effects of the antidiuretic analogue 1-desamino-8-D-arginine vasopressin (dDAVP) and the antipressor analogue [1-(beta-mercapto-beta beta-cyclopentamethylene propionic acid)-4-valine, 8-D-arginine]-vasopressin (d[CH2]5VDAVP), were studied. Neither compound stimulated PGE2 and preincubation with d(CH2)5VDAVP abolished, and dDAVP blunted, AVP-enhanced PGE2 production. Incubation in verapamil, nifedipine, or zero calcium media blocked peptide-stimulated PGE2 production. Increasing extracellular calcium or adding A23187 increased PGE2 synthesis. Selective stimulation of PGE2 by ANG II or AVP in mesangial cells suggests a hormone-sensitive phospholipase and a coupled cyclooxygenase capable of synthesizing only PGE2. Since neither vasopressin analogue stimulated PGE2, but both blocked AVP-enhanced PGE2 production, we conclude that these cells respond to the pressor activity of AVP. This is a calcium-dependent process. Selective stimulation of PGE2 by ANG II and AVP may modulate their contractile effects on the glomerulus.     

Relationship between phospholipase C activation and prostaglandin E2 and cyclic adenosine monophosphate production in rabbit tubular epithelial cells. Effects of angiotensin, bradykinin, and arginine vasopressin.

By employing early-passaged rabbit kidney epithelial cells in tissue culture, we demonstrated that angiotensin II (AII) has unique mechanisms of signal transduction. First, unlike its action in other target tissues, micromolar concentrations of AII are required to induce small rises in cytosolic calcium, [Ca2+]i, an action which is not accompanied by the release of inositol phosphates (IP). In contrast, nanomolar bradykinin (BK) mobilizes [Ca2+]i through activation of phospholipase C and release of IP. Neither of these stimulated calcium responses exhibits pertussis toxin (PTx) sensitivity. Secondly, AII and BK at 10(-9) to 10(-7) M stimulate cAMP indirectly through PGE2 production in distal cells. AII- and BK-stimulated PGE2 release is PTx inhibitible, suggestive of the presence of a GTP binding protein mediating the response. By contrast, arginine vasopressin fails to elicit rises in [Ca2+]i but exerts its primary effect on cAMP production in distal cells via direct coupling to a stimulatory GTP binding protein, as evidenced by uncoupling with cholera toxin. Regulation of PGE2 synthesis appears to occur via phospholipase A2, not C, by all three peptides.     

Effect of platelet activating factor antagonists on cultured rat mesangial cells

Platelet activating factor (PAF; 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a lipid mediator of inflammation produced by inflammatory cells and also by renal glomerular mesangial cells. PAF may play a role in glomerular function and disease. Previously the authors reported that glomerular mesangial cells respond to PAF by increasing prostaglandin E2 (PGE2) synthesis and by cell contraction. This paper examines the specificity of BN52021, SRI 63-072 and kadsurenone on the effects of PAF on cultured rat glomerular mesangial cells. Both BN52021 and SRI 63-072 specifically decreased PAF (1 X 10(-6) M)-stimulated PGE2 production in a dose-dependent fashion (10(-7)-10(-5) M) without affecting the stimulation by angiotensin II (AII) or the calcium ionophore A23187. Kadsurenone also decreased PAF-stimulated PGE2 production, but in a less specific manner, as it also decreased AII- and A23187-stimulated PGE2 production. In order to examine the site of antagonism of PAF-induced PGE2 synthesis by BN52021 and SRI 63-072, the authors prelabeled cells with [14C]arachidonic acid. Both agents diminished the release of [14C]arachidonate from these prelabeled cells in response to PAF in a dose-dependent fashion, though not decreasing release in response to AII or A23187, indicating that they blocked the effect of PAF on phospholipase activation, consistent with receptor blocking activity. BN52021 and SRI 63-072 also inhibited PAF-induced contraction of cultured mesangial cells without an effect on basal tone of the cells or the contractile response to AII.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glomerular prostaglandins: synthesis, mechanism of action and interaction with ACE inhibitors]

Prostaglandins (PG) play an important role in the regulation of the renal blood flow and glomerular filtration rate. This study was designed to examine PG synthesis in the presence and absence of the ACE inhibitor captopril, PG binding to specific receptors and the ability of PG to stimulate cAMP accumulation in isolated glomeruli. Glomeruli were isolated from rat kidneys by a passive mechanical sieving technique. PG synthesis was determined by RTLC and RIA. The main eicosanoids synthesized by glomeruli were PGF2 alpha, thromboxane (TX) A2 (measured as TXB2), PGI2 (measured as 6-keto-PGF1 alpha) and PGE2. Binding experiments were performed with PGE1, PGE2 and the PGI2 analogue iloprost. Scatchard analysis revealed that the specific binding was highest for PGE1, followed by iloprost and PGE2. Adenylate cyclase was preferentially stimulated by PGE1 and PGE2, and to a lesser extent by PGI2, whereas PGF2 alpha had almost no effect. Captopril reduced mainly TXB2 concentrations. Glomerular TXB2 reduction, therefore, seems to be an additional hypotensive effect of captopril medication.     

Simultaneous measurements of cytosolic free calcium level and prostaglandin synthesis reveal a correlation between them in perfused monolayer of cultured rat vascular smooth muscle cells: effects of bradykinin and angiotensin II.

The level of cytosolic free calcium ([Ca2+]i) and the production rate of prostacyclin were simultaneously measured in perfused monolayers of cultured vascular smooth muscle (VSM) cells. After loading of fura-2 (a fluorescent calcium indicator), the monolayer of VSM cells (cultured on a cover glass) was fixed in the perfusion cuvette and the cuvette was placed in a fluorometer to monitor the change in [Ca2+]i. The monolayer was perfused and the fractionated perfusion solution was collected to determine 6-keto-PGF1 alpha (a metabolite of prostacyclin) production found in the solution. Afterwards, the time-dependent changes in [Ca2+]i and 6-keto-PGF1 alpha synthesis were compared. Bradykinin (BK, 10(-6) M), angiotensin (Ang) II (10(-7) M) as well as ionomycin (10(-6) M) induced simultaneous increases in [Ca2+]i and 6-keto-PGF1 alpha production. An inhibitor against prostaglandin synthesis, acetylsalicylic acid (ASA, 10(-6) M) abolished BK-induced 6-keto-PGF1 alpha synthesis, whereas ASA did not affect the increase in [Ca2+]i. BK-induced increases in [Ca2+]i and 6-keto-PGF1 alpha production occurred in a dose-dependent manner and the half-maximal response was observed at the same concentration of BK (10(-7) M). These results indicate that an increase in [Ca2+]i is closely associated with BK as well as AngII-induced prostacyclin synthesis. It is suggested that an increase in [Ca2+]i plays a prior role in prostacyclin synthesis. Thus, an interaction between phospholipase A2 (prostaglandin synthesis) and phospholipase C (inositol trisphosphate-Ca2+ mobilization) is suggested.     

Regulation of mesangial cell ion channels by insulin and angiotensin II. Possible role in diabetic glomerular hyperfiltration.

We used patch clamp methodology to investigate how glomerular mesangial cells (GMC) depolarize, thus stimulating voltage-dependent Ca2+ channels and GMC contraction. In rat GMC cultures grown in 100 mU/ml insulin, 12% of cell-attached patches contained a Ca(2+)-dependent, 4-picosiemens Cl- channel. Basal NPo (number of channels times open probability) was < 0.1 at resting membrane potential. Acute application of 1-100 nM angiotensin II (AII) or 0.25 microM thapsigargin (to release [Ca2+]i stores) increased NPo. In GMC grown without insulin, Cl- channels were rare (4%) and unresponsive to AII or thapsigargin in cell-attached patches, and less sensitive to [Ca2+]i in excised patches. GMC also contained 27-pS nonselective cation channels (NSCC) stimulated by AII, thapsigargin, or [Ca2+]i, but again only when insulin was present. In GMC grown without insulin, 15 min of insulin exposure increased NPo (insulin > or = 100 microU/ml) and restored AII and [Ca2+]i responsiveness (insulin > or = 1 microU/ml) to both Cl- and NSCC. GMC AII receptor binding studies showed a Bmax (binding sites) of 2.44 +/- 0.58 fmol/mg protein and a Kd (binding dissociation constant) of 3.02 +/- 2.01 nM in the absence of insulin. Bmax increased by 86% and Kd was unchanged after chronic (days) insulin exposure. In contrast, neither Kd nor Bmax was significantly affected by acute (15-min) exposure. Therefore, we concluded that: (a) rat GMC cultures contain Ca(2+)-dependent Cl- and NSCC, both stimulated by AII. (b) Cl- efflux and cation influx, respectively, would promote GMC depolarization, leading to voltage-dependent Ca2+ channel activation and GMC contraction. (c) Responsiveness of Cl- and NSCC to AII is dependent on insulin exposure; AII receptor density increases with chronic, but not acute insulin, and channel sensitivity to [Ca2+]i increases with both acute and chronic insulin. (d) Decreased GMC contractility may contribute to the glomerular hyperfiltration seen in insulinopenic or insulin-resistant diabetic patients.     

Effect of thrombin on proliferation, contraction and prostaglandin production of rat glomerular mesangial cells in culture.

The effect of thrombin on mesangial cell function was investigated. Thrombin caused a dose-dependent increase in [3H] thymidine incorporation (EC50 = 0.36 +/- 0.09 U/ml), intracellular calcium [(Ca++)i] mobilization (EC50 = 1.9 +/- 0.5 U/ml) and prostaglandin E2 (PGE2) production (EC50 = 0.25 +/- 0.02 U/ml) in rat glomerular mesangial cells. These effects were blocked by the thrombin inhibitor, hirudin (KB = 10.4 +/- 0.2 nM). The role of (Ca++)i mobilization and arachidonate metabolism in thrombin-stimulated proliferation was tested by the addition of the calcium channel blocker, nifedipine, and the cyclooxygenase inhibitor, indomethacin, to mesangial cell cultures. Indomethacin, at doses that completely inhibited the thrombin-mediated production of PGE2, had no significant effect on proliferation. The Ca++ channel blocker, nifedipine, inhibited both PGE2 production and [3H] thymidine incorporation in a dose-dependent fashion, but only at concentrations considered nonspecific. In addition to its effects on PGE2, thymidine incorporation and Ca++ mobilization, thrombin caused mesangial cell contraction as determined by a substrate distortion technique. This effect was not inhibited by indomethacin. These results indicate that thrombin can alter mesangial cell function in vitro.     

Endothelin and angiotensin II stimulation of Na+-H+ exchange is impaired in cardiac hypertrophy.

We compared the effects of endothelin-1 (ET-1) on intracellular pH, intracellular [Ca2+]i, and cell contraction in hypertrophied adult ventricular myocytes from ascending aortic banded rats and age-matched controls. Intracellular pH (pH(i)) was measured in individual myocytes with SNARF-1, and [Ca2+]i was measured with indo-1, simultaneous with cell motion. Experiments were performed at 36 degrees C in myocytes paced at 0.5 Hz in Hepes-buffered solution (pH(o) 7.40) containing 1.2 mM CaCl2. At baseline, calibrated pH(i), diastolic and systolic [Ca2+]i values, and the amplitude of cell contraction were similar in hypertrophied and control myocytes. Exposure of the control myocytes to 10 nM ET-1 caused an increase in the amplitude of cell contraction to 163+/-22% of baseline (P < 0.05), associated with intracellular alkalinization (pH(i) + 0.08+/-0.02 U, P < 0.05) and a slight increase in peak systolic [Ca2+]i (104+/-11% of baseline, P < 0.05). In contrast, in the hypertrophied myocytes, exposure to ET-1 did not increase the amplitude of cell contraction or cause intracellular alkalinization (-0.01+/-0.02 U, NS). Similar effects were observed in the hypertrophied and control myocytes in response to exposure to 10 nM angiotensin II. ET-1 also increased the rate of recovery from intracellular acidosis induced by the washout of NH4Cl in the control cells, but did not do so in the hypertrophied cells. In the presence of 10 microM 5-(N-ethyl-N-isopropyl)-amiloride, which inhibits Na+-H+ exchange, ET-1 did not cause a positive inotropic effect or intracellular alkalinization in control cells. The activation of protein kinase C by exposure to phorbol ester caused intracellular alkalinization and it increased the rate of recovery from intracellular acidification induced by an NH4Cl pulse in control cells but not in hypertrophied cells. ET-1, as well as angiotensin II, and phorbol ester, fail to stimulate forward Na+-H+ exchange in adult hypertrophied myocytes. These data suggest a defect in the coupling of protein kinase C signaling with Na+-H+ exchange in adult hypertrophied myocytes.     

Mechanism of chloride secretion induced by carbachol in a colonic epithelial cell line.

Serosal application of carbachol to T84 cell monolayers mounted in an Ussing chamber caused an immediate increase in short circuit current (Isc) that peaked within 5 min and declined rapidly thereafter, although a small increase in Isc persisted for approximately 30 min. The increase in Isc was detectable with 1 microM carbachol; half-maximal with 10 microM carbachol; and maximal with 100 microM carbachol. Unidirectional Na+ and Cl- flux measurements indicated that the increase in Isc was due to net Cl- secretion. Carbachol did not alter cellular cAMP, but caused a transient increase in free cytosolic Ca2+ ([Ca2+]i) from 117 +/- 7 nM to 160 +/- 15 nM. The carbachol-induced increase in Isc was potentiated by either prostaglandin E1 (PGE1) or vasoactive intestinal polypeptide (VIP), agents that act by increasing cAMP. Measurements of cAMP and [Ca2+]i indicated that the potentiated response was not due to changes in these second messengers. Studies of the effects of these agents on ion transport pathways indicated that carbachol, PGE1, or VIP each increased basolateral K+ efflux by activating two different K+ transport pathways on the basolateral membrane. The pathway activated by carbachol was not sensitive to barium, while that activated by PGE1 or VIP was; furthermore, their action on K+ efflux are additive. Our study indicates that carbachol causes Cl- secretion, and that this action may result from its ability to increase [Ca2+]i and basolateral K+ efflux. Carbachol's effect on Cl- secretion is greatly augmented in the presence of VIP or PGE1, which open a cAMP-sensitive Cl- channel on the apical membrane, accounting for a potentiated response.     

Effect of platelet-activating factor and serum-treated zymosan on prostaglandin E2 synthesis, arachidonic acid release, and contraction of cultured rat mesangial cells

The interaction of inflammatory cells and glomerular prostaglandins (PG) may be important during glomerulonephritis. We therefore examined the influence of platelet-activating factor (PAF), (a mediator of inflammation released from leukocytes) and of phagocytosis of zymosan on arachidonic acid metabolism and on cell contractility in rat glomerular mesangial cells in culture. PAF increased PGE2 synthesis (determined by radioimmunoassay) within minutes (threshold: 10(-10)M; maximal effect: 10(-7)M). Serum-treated zymosan also stimulated PGE2, but with a slower onset. In cells prelabeled with [14C]arachidonic acid both PAF and serum-treated zymosan released 14C from phospholipids and increased free [14C]arachidonate. The ratio of 14C-release to PGE2 was, however, different with PAF and serum-treated zymosan, indicating different phospholipid pools. Under phase-contrast microscopy, PAF caused contraction of mesangial cells with a dose-response and time-course parallel to that for PGE2 synthesis. Serum-treated zymosan caused no contraction. The PAF-induced contraction was enhanced by PG synthesis inhibition and was attenuated by addition of PGE2, indicating a feedback mechanism. The mesangial contraction by PAF may be important in favoring deposition of immune complexes, while the PGE2 synthesis stimulated by PAF and by phagocytosis of zymosan may counteract the deleterious effects of PAF during induction of glomerulonephritis.     

Histamine modulates contraction and cyclic nucleotides in cultured rat mesangial cells. Differential effects mediated by histamine H1 and H2 receptors.

Histamine influences the glomerular microcirculation and modulates immune-inflammatory responses. In the rat kidney, histamine is synthesized by glomeruli and stimulates cyclic nucleotide production specifically in glomeruli. We investigated the in vitro effect of histamine on cyclic nucleotide accumulation in rat cultured glomerular mesangial and epithelial cells. Histamine stimulated cyclic AMP (cAMP) accumulation in cultured mesangial cells (64.0 +/- 22.1 to 511.4 +/- 86.6 pmol/mg protein, n = 9) but had no effect on cAMP accumulation in epithelial cells. This effect was dose-dependent and time-dependent. Stimulation of cAMP accumulation occurred in the range of 5 X 10(-6) M-10(-4) M histamine with a half maximal stimulatory effect of 2 X 10(-5) M. Initial stimulation was noted by 30 s, and maximum stimulation was observed at 5 min. The H2 antagonist cimetidine (10(-4) M) abolished the stimulatory effect of histamine (10(-4) M), while equimolar concentrations of the H1 antagonist diphenhydramine had no significant effect on cAMP accumulation. Moreover, the specific H2 agonist dimaprit, but not the H1 agonist 2-pyridylethylamine, stimulated cAMP accumulation. Histamine had no effect on cAMP accumulation in epithelial cells or on cyclic guanosine monophosphate accumulation in epithelial or mesangial cells. Since the in vivo infusion of histamine reduces ultrafiltration coefficient and since mesangial cell contraction is thought to be responsible for the reduction in the ultrafiltration coefficient, we examined the effect of histamine on the contractile property of mesangial cells. Histamine (5 X 10(-6)-10(-4) M) contracted mesangial cells, and the H1 antagonist diphenhydramine (10(-4) M) but not the H2 antagonist cimetidine (10(-4) M) prevented histamine (10(-4) M) induced contraction. In addition, the H1 agonist 2-pyridylethylamine, but not the H2 agonist dimaprit, contracted mesangial cells. Histamine and its specific agonists and antagonists induced contraction of isolated glomeruli as assessed by glomerular planar surface area in a manner parallel to their effect on mesangial cells. Cinnarizine (10(-5) M), a Ca++ channel blocker, or Ca++, Mg++-free medium prevented histamine (10(-4) M) induced mesangial cell and glomerular contraction. Thus, histamine enhances cAMP accumulation specifically in mesangial cells via an H2 receptor. In contrast, histamine contracts mesangial cells and glomeruli via an H1 receptor, an effect that is dependent on extracellular Ca++ entry. These findings show that histamine potentially influences intraglomerular hemodynamics via effects on mesangial cell contraction. Moreover, our findings considered with the in vivo observation that histamine reduces kf via and H1 receptor provide further support of the hypothesis that mesangial cell contraction regulates the glomerular capillary surface area available for filtration. Our studies also show that this contractile effect of histamine is dependent on extracellular calcium. The presence of a cAMP system sensitive to histamine may have major implications in the pathogenesis of inflammatory glomerulopathies. Mesangial cells possess characteristics similar to circulating and tissue immune effector cells, including lysosomal enzyme release, oxygen radical production, and release of a number of immunomodulatory factors. Histamine and cAMP have been shown to modulate such characteristics of inflammatory cells. It is therefore conceivable that histamine, via its interaction with H2 receptors and subsequent generation cAMP, may have profound effects on such properties of mesangial cells, suggesting that this autacoid may modulate not only glomerular hemodynamics but also immune, inflammatory responses within the glomerulus.     

Effects of parathyroid hormone on cytosolic free calcium concentration in individual rabbit connecting tubules.

PTH stimulates active Ca reabsorption in isolated perfused rabbit kidney connecting tubules (CNTs). The existence of PTH-sensitive adenylate cyclase and the reproduction of increased epithelial Ca transport by dibutyryl-cAMP suggest that cAMP is the mediator. Accordingly, we studied the effects of PTH and 8-bromoadenosine 3',5'-cAMP (8-Br-cAMP) on cytosolic free calcium concentration [( Ca2+]i) in individual rabbit CNTs. [Ca2+]i was estimated by continuous epifluorescence microscopy of single fura-2-loaded tubules during dual wave-length excitation. In nonperfused controls at 37 degrees C, [Ca2+]i decreased with time. In contrast to vehicle controls, synthetic bovine (1-34) PTH (0.1 nM) increased [Ca2+]i within 4 min, produced a maximal effect in 7.2 min, and sustained its effect for at least 2 min after washout. 8-Br-cAMP (1 mM) mimicked the effect of PTH, but with an earlier onset of action. To test the hypothesis that lumen Ca is the predominant source of the rise in [Ca2+]i, we studied singly perfused CNTs. In the absence of bath and lumen Ca, PTH elicited no rise in [Ca2+]i, implying that intracellular Ca stores are not the major source. In contrast, there was a rise when Ca was replenished in both media. In the continuous presence of bath Ca, lumen Ca was estimated to contribute 65% of the total rise in [Ca2+]i in response to PTH when it was first deleted and then replenished. However, when the sequence of lumen Ca manipulation was reversed, the contributions by lumen and bath Ca were found to be essentially equal. We conclude (a) at a physiologic concentration, PTH increases [Ca2+]i in rabbit CNTs, (b) 8-Br-cAMP mimics this action, implicating cAMP as a second messenger, and (c) the PTH-stimulated rise in [Ca2+]i depends importantly on both bath and tubular luminal fluid Ca.     

Effects of sodium depletion on renal prostanoid synthesis in rats: influence of the converting enzyme inhibitor captopril

1. The synthesis of prostaglandin (PG) E2, PGF2 alpha, 6-keto-PGF1 alpha and thromboxane (TX) B2 by isolated glomeruli, cortical tubules, inner medullary slices and outer medullary slices was measured in salt-depleted (LNa) rats and in salt-depleted rats receiving captopril (LNa-CEI). Animals were studied before and after 4, 9 and 15 days of Na+ depletion. 2. Na+ balance was reached in LNa rats after 4 days. Blood pressure and creatinine clearance remained stable. Serum Na+ decreased from 140 +/- 1 to 126 +/- 1 mmol/l (mean +/- SEM, P less than 0.01). In contrast, LNa-CEI rats were unable to conserve Na+ adequately: fractional excretion of Na+ and natriuresis were constantly greater than in LNa animals. As a consequence, LNa-CEI rats developed severe hyponatraemia, lost weight and their creatinine clearance decreased. 3. The glomerular synthesis of PGE2, PGF2 alpha and 6-keto-PGF1 alpha, but not of TXB2, was significantly increased in LNa rats. In LNa-CEI rats, the synthesis of PGE2 and 6-keto-PGF1 alpha was similar to control values, but PGF2 alpha and TXB2 synthesis was elevated at day 9. In cortical tubules, PGE2 and PGF2 alpha were unaffected by Na+ depletion, but 6-keto-PGF1 alpha and TXB2 were increased and a similar trend was observed in LNa-CEI rats. In outer medulla of LNa rats, a decrease in all the eicosanoids measured was observed at day 4. In LNa-CEI animals, the synthesis of PGE2 and PGF2 alpha, but not of 6-keto-PGF1 alpha and TXB2, was significantly depressed. In inner medulla, Na+ depletion only tended to decrease PGF2 alpha and 6-keto-PGF1 alpha, but in the presence of captopril, the synthesis of all prostanoids was significantly decreased.     

Low density lipoprotein enhances the cellular action of arginine vasopressin in rat glomerular mesangial cells in culture.

The present study was undertaken to determine whether low density lipoprotein (LDL) modulates the cellular action of arginine vasopressin (AVP) in rat glomerular mesangial cells in culture. AVP increased cellular free calcium ([Ca2+]i) in a dose-dependent manner. When cells were preincubated for 24 h with 10 microgram/ml LDL, the 1 x 10(-7) M AVP-mobilized [Ca2+]i was 874 nM, a value significantly greater than that of 375 nM in the intact cells. AVP caused a biphasic change in cellular pH (pHi), namely, an early acidification followed by a sustained alkalinization, and the change in pHi produced by AVP was also enhanced by LDL. AVP stimulated a 2.2-fold increase in [3H]thymidine incorporation, an effect significantly greater in the presence of 10 micrograms/ml LDL. Furthermore, 1 x 10(-7) M AVP significantly activated mitogen-activated protein kinase from 14.0 to 24.5 pmol/mg protein. Such an activation was significantly enhanced by the LDL pretreatment. Both [3H]thymide incorporation and mitogen-activated protein kinase were not altered by 10 micrograms/ml LDL. [3H]AVP receptor binding was not affected by the LDL pretreatment. 1 x 10(-7) M AVP increased inositol trisphosphate production by 1.9-fold, an effect significantly greater in the presence of LDL. These results indicate that LDL enhances the cellular action of AVP and the AVP-stimulated cellular proliferation in glomerular mesangial cells. A site of action of LDL is the hydrolysis of phosphatidylinositol.     

Feedback inhibition of cyclic adenosine monophosphate-stimulated Na+ transport in the rabbit cortical collecting duct via Na(+)-dependent basolateral Ca++ entry.

Arginine vasopressin (AVP) transiently stimulates Na+ transport in the rabbit cortical collecting duct (CCD). However, the sustained effect of both AVP and its putative second messenger, cyclic adenosine monophosphate (cAMP), on Na+ transport in the rabbit CCD is inhibitory. Because maneuvers that increase [Ca++]i inhibit Na+ transport, the effects of AVP and cell-permeable cAMP analogues, on [Ca++]i were investigated in fura-2-loaded in vitro microperfused rabbit CCDs. Low-dose AVP (23-230 pM) selectively stimulated Ca++ influx, whereas 23 nM AVP additionally released calcium from intracellular stores. 8-chlorophenylthio-cAMP (8CPTcAMP) and 8-bromo-cAMP (8-Br-cAMP) also increased CCD [Ca++]i. The 8CPTcAMP-stimulated [Ca++]i increase was totally dependent on basolateral [Ca++]. In the absence of cAMP, peritubular Na+ removal produced a marked increase in [Ca++]i, which was also dependent on bath [Ca++], suggesting the existence of basolateral Na+/Ca++ exchange. Luminal Na+ removal in the absence of cAMP did not alter CCD [Ca++]i, but it completely blocked the cAMP-stimulated [Ca++]i increase. Thus the cAMP-dependent Ca++ increase is totally dependent on both luminal Na+ and basolateral Ca++, suggesting the [Ca++]i increase is secondary to cAMP effects on luminal Na+ entry and its coupling to basolateral Na+/Ca++ exchange. 8CPTcAMP inhibits lumen-to-bath 22Na flux [JNa(l-b)] in CCDs bathed in a normal Ca++ bath (2.4 mM). However, when bath Ca++ was lowered to 100 nM, a maneuver that also blocks the 8CPTcAMP [Ca++]i increase, 8CPTcAMP stimulated, rather than inhibited JNa(l-b). These results suggest that cAMP formation initially stimulates CCD Na+ transport, and that increased apical Na+ entry secondarily activates basolateral Ca++ entry. The cAMP-dependent [Ca++]i increase leads to inhibition Na+ transport in the rabbit CCD.     

Increased prostaglandin production by glomeruli isolated from rats with streptozotocin-induced diabetes mellitus.

Abnormalities in glomerular function have been observed frequently in the early stages of both clinical and experimental diabetes mellitus. Because prostaglandins (PGs) are present in the glomerulus and have profound effects on glomerular hemodynamics, and because abnormalities of PG metabolism have been noted in other tissues from diabetics, we studied PG biosynthesis in glomeruli obtained from rats in the early stages of experimental diabetes mellitus. Streptozotocin, 60 mg/kg, was administered intravenously to male Sprague-Dawley rats. Control rats received an equal volume of the vehicle. Glomeruli were isolated 9-23 d later. Production of eicosanoids was determined by two methods: by direct radioimmunoassay after incubation of glomeruli under basal conditions and in the presence of arachidonic acid (C20:4), 30 microM, and by radiometric high-performance liquid chromatography (HPLC) after incubation of glomeruli with [14C]C20:4. When assessed by radioimmunoassay, mean basal production of both prostaglandin E2 (PGE2) and prostaglandin F2 alpha (PGF2 alpha) was twofold greater in the diabetic animals whereas production of thromboxane B2 (TXB2) was not significantly greater than control. In response to C20:4, both PGE2 and PGF2 alpha were also greater in the diabetic animals, but these differences were not statistically significant. The increased rate of basal PG production did not appear to be related directly to the severity of the diabetic state as reflected by the degree of hyperglycemia at the time of sacrifice. In fact, the rates of glomerular PG production in the individual diabetic animals correlated inversely with the plasma glucose concentration. The increased rate of PG synthesis did not appear to be due to a nonspecific effect of streptozotocin inasmuch as glomerular PG production was not increased significantly in streptozotocin-treated rats which were made euglycemic by insulin therapy. Furthermore, addition of streptozotocin, 1-10 mM, to the incubation media had no effect on PGE2 production by normal glomeruli. PGE2 production by normal glomeruli was also not influenced by varying the glucose concentration in the incubation media over a range of 1-40 mM. When metabolism of [14C]C20:4 was evaluated by high-performance liquid chromatography conversion to labeled PGE2, PGF2 alpha, TXB2, and hydroxyheptadecatrienoic acid by diabetic glomeruli was two- to threefold greater compared with that in control glomeruli, whereas no significant difference in conversion to 12- and 15-hydroxyeicosatetraenoic acid occurred. These findings indicate that glomerular cyclooxygenase but not lipoxygenase activity was increased in the diabetic animals. A concomitant increase in glomerular phospholipase activity may also have been present to account for the more pronounced differences in PG production noted in the absence of exogenous unlabeled C20:4. These abnormalities in PG biosynthesis by diabetic glomeruli may contribute to the altered glomerular hemodynamics in this pathophysiologic setting.     

Role of Ca2+ in the action of adrenocorticotropin in cultured human adrenal glomerulosa cells.

The present report details the role of Ca2+ in the early events of ACTH action in human adrenal glomerulosa cells. Threshold stimulations of both aldosterone and cAMP production were obtained with a concentration of 10 pM ACTH, an ED50 of 0.1 nM, and maximal aldosterone stimulation (5.5-fold increase over control) at 10 nM ACTH. ACTH also induced a sustained increase of intracellular calcium ([Ca2+]i) with maximal stimulation of 1.6 +/- 0.1-fold over control values. This increase does not involve mobilization of calcium from intracellular pools since no response was observed in Ca2+-free medium or in the presence of nifedipine, suggesting the involvement of Ca2+ influx by L-type Ca2+ channels. This was confirmed by patch clamp studies that demonstrated that ACTH stimulates L-type Ca2+ channels. Moreover, the Ca2+ ion is not required for ACTH binding to its receptor, but is essential for sustained cAMP production and aldosterone secretion after ACTH stimulation. These results indicate that, in human adrenal glomerulosa cells, a positive feedback loop between adenylyl cyclase-protein kinase A-Ca2+ channels ensures a slow but sustained [Ca2+]i increase that is responsible for sustained cAMP production and aldosterone secretion.     

Inhibition of apical Na+ channels in rabbit cortical collecting tubules by basolateral prostaglandin E2 is modulated by protein kinase C.

We used the cell-attached patch clamp technique to investigate the interaction of exogenous prostaglandins (PG), intracellular [Ca2+]i, and protein kinase C (PKC) on the high selectivity, 4 pS Na+ channel found in the principal cell apical membrane of rabbit cortical collecting tubule (CCT) cultures grown on collagen supports with 1.5 microM aldosterone. Application of 0.5 microM PGE2 to the basolateral membrane decreased mean NP0 (number of channels times the open probability) for apical Na+ channels by 46.5% (n = 9). There was no consistent change in NP0 after apical 0.5 microM PGE2 (n = 12) or after apical or basolateral 0.5 microM PGF2 alpha (n = 8). Release of [Ca2+]i stores with 0.25 microM thapsigargin (n = 7), or activation of apical membrane PKC with apical 0.1 microM 4 beta-phorbol-12-myristate-13-acetate (n = 5) or 10 microM 1-oleyl-2-acetylglycerol (n = 4) also decreased NP0. Depletion of [Ca2+]i stores (0.25 microM thapsigargin pretreatment) (n = 7) or inhibition of apical PKC (100 microM D-sphingosine pretreatment) (n = 8) abolished the inhibitory effects of basolateral PGE2. Conclusions: (a) apical Na+ transport in rabbit CCT principal cells is modulated by basolateral PGE2; (b) the mechanism involves release of IP3-sensitive, [Ca2+]i stores; and (c) Ca(2+)-dependent activation of apical membrane PKC, which then inhibits apical Na+ channels.