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.     

Interaction of prostaglandins with adenosine diphosphate induced increase in cytosolic free calcium in human platelets.

The interaction of prostaglandins with changes in cytosolic Ca2+ concentration ([Ca2+]) and aggregation of human platelets induced by adenosine diphosphate (ADP) were investigated. Cytosolic [Ca2+] was measured with the fluorescent dye Quin2. Addition of ADP (0.25-2.5 mumol l-1) to platelet suspensions produced a dose dependent increase in cytosolic [Ca2+] from a basal level of 51 +/- 1 nmol l-1 to maximum levels exceeding 1 mumol l-1 and induced platelet aggregation. Chelation of extracellular calcium with 100 mumol l-1 EGTA markedly reduced the increase in cytosolic [Ca2+] induced by 0.25 mumol l-1 ADP, while pretreatment with the calcium entry blocker verapamil was without effect. Stimulation of cyclic AMP with prostaglandins (PGD2, PGE1, PGE2, PGI2, but not PGF2 alpha) and forskolin, or incubation with dibutyryl-cAMP, inhibited the rise in cytosolic [Ca2+] and platelet aggregation following ADP. We conclude that prostaglandins inhibit the increase in cytosolic [Ca2+] and aggregation of human platelets induced by ADP, probably by stimulation of cyclic AMP generation, thereby opposing the mechanism by which ADP increases cytosolic [Ca2+] and subsequently induces platelet aggregation.     

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.     

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.     

Endothelin-1 stimulates contraction of rat glomerular mesangial cells and potentiates beta-adrenergic-mediated cyclic adenosine monophosphate accumulation.

The newly isolated peptide, endothelin-1 (ET-1), is a potent pressor agent that reduces GFR and the glomerular ultrafiltration coefficient. Recent evidence demonstrates that ET-1 mobilizes intracellular Ca2+ [( Ca2+]i) in glomerular mesangial cells by activating the phosphoinositide cascade. The present experiments were designed to examine whether ET-1 stimulates mesangial cell contraction and regulates the synthesis of PGE2 and cAMP, which dampen vasoconstrictor-induced mesangial contraction. ET-1 (greater than or equal to 1 nM) reduced the cross-sectional area of rat mesangial cells cultured on three-dimensional gels of collagen type I. ET-1 also caused complex rearrangements of F-actin microfilaments consistent with a motile response. Contraction in response to ET-1 occurred only at concentrations that activate phospholipase C, and contraction was unaffected by blockade of dihydropyridine-sensitive Ca2+ channels. Elevation of [Ca2+]i with ionomycin, to equivalent concentrations of [Ca2+]i achieved with ET-1, also reduced mesangial cell cross-sectional area. ET-1 (0.1 microM) also evoked [3H]arachidonate release and a fivefold increase in PGE2 synthesis as well as increased synthesis of PGF2 alpha and small changes of TXB2. ET-1 caused a minor increase in intracellular cAMP accumulation only in the presence of 3-isobutyl-1-methylxanthine. ET-1 also amplified cAMP production in response to isoproterenol. TPA and ionomycin, alone and in combination, failed to mimic the potentiating effect of ET-1; however, indomethacin blocked ET-1-induced potentiation of isoproterenol-stimulated cAMP, which was restored by addition of exogenous 10 nM PGE2. Thus the present data demonstrate that ET-1 stimulates mesangial cell contraction via pharmacomechanical coupling and activates phospholipase A2 to produce PGE2, PGF2 alpha, and TXB2. ET-1 also amplified beta adrenergic-stimulated cAMP accumulation by a PGE2-dependent mechanism.     

Interleukin-6 attenuates agonist-mediated calcium mobilization in murine osteoblastic cells.

Interleukin-6 (IL-6) is a multifunctional cytokine which is made by osteoblasts and has diverse effects on bone metabolism. We studied the interaction of IL-6 with the Ca2+ and cAMP signaling systems in the osteoblastic cell line UMR-106 and in primary osteoblastic cultures derived from neonatal rat calvariae. IL-6 did not alter basal intracellular calcium concentration ([Ca2+]i) but inhibited Ca2+ transients induced by parathyroid hormone (PTH), prostaglandin E2 (PGE2), and endothelin-1 in both dose- (100-400 U/ml) and time- (4-48 h) dependent manners. The effect of the cytokine was abolished by the tyrosine kinase inhibitor, herbimycin A (50 ng/ml). The IL-6 effect on the Ca2+ message system was related to suppressed production of hormonally induced inositol 1,4,5-triphosphate and inhibition of Ca2+ release from intracellular stores. Hormonally induced calcium entry pathways (estimated by using Mn2+ as a surrogate for Ca2+) were not, however, altered by the cytokine. IL-6 did not modulate cAMP generation in osteoblasts. With respect to osteoblast function, IL-6, although having no effect on cell proliferation by itself, greatly enhanced the antiproliferative effect of PGE2 and PTH. Because the production of IL-6 in osteoblasts is stimulated by calciotropic hormones (e.g., PTH and PGE2), the suppressive effect of the cytokine on hormonally induced Ca2+ transients may serve as an autocrine/paracrine mechanism for modulating the effect of hormones on bone metabolism.     

The regulatory mechanism of free Ca2+ concentration in activated platelets]

The change in intracellular Ca2+ concentration ([Ca2+]i) following platelet stimulation results from mobilization, influx and restoration of Ca2+. To determine whether inositol 1,4,5 trisphosphate (IP3) is involved in Ca2+ influx, the relationship between IP3 formation (IP3) and Ca2+ influx ( delta [Ca2+]i) was investigated in platelets stimulated wtih various agonists (thrombin, ADP, PAF, STA2, etc). The ratio of IP3 to delta [Ca2+]i varied among the agonists, although delta [Ca2+]i was increased, depending on the amount of agonist. Furthermore, in spite of the similar delta [Ca2+]i, IP3 was smaller at 20 degrees C compared with that at 37 degrees C in thrombin-stimulated platelets. These results indicate that Ca2+ influx in platelets might be regulated by receptor-operated Ca2+ channel rather than by an IP3 mediated mechanism. As for Ca2+ restoration, calpain was demonstrated to play a role through Ca(2+)-ATPase activation by limited proteolysis.     

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.     

Vasopressin V1 receptors on the principal cells of the rabbit cortical collecting tubule. Stimulation of cytosolic free calcium and inositol phosphate production via coupling to a pertussis toxin substrate.

The effects of arginine vasopressin (AVP) on the cytosolic free calcium concentration ([Ca2+]f) were examined in freshly immunodissected rabbit cortical collecting tubule cells using fluorescent Ca2+ indicators fura-2 and indo-1. The addition of AVP to a cell suspension resulted in a rapid and transient increase in the [Ca2+]f. The 1-deamino-8-D-AVP (dDVP), a V2 receptor agonist of AVP that stimulated adenosine 3',5' cAMP production in these cells, had no effect on [Ca2+]f and did not affect AVP-induced increase in [Ca2+]f. The AVP-induced increase in [Ca2+]f but not cAMP production was blocked by the V1 receptor antagonist, [1-(beta-mercapto-beta-beta-cyclopentamethylene propionic acid), 2-(O-methyl)tyrosine] Arg8-vasopressin. The AVP-stimulated increase in [Ca2+]f appeared to be largely due to Ca2+ release from intracellular stores as reduction of extracellular Ca2+ with EGTA had little if any effect on the AVP-induced increase in [Ca2+]f. This AVP-induced increase in [Ca2+]f was associated with an increase in inositol-1,4,5-trisphosphate production and appeared to involve a guanine nucleotide-binding protein (G), since the pretreatment of cells with pertussis toxin for 4-6 h inhibited this effect. Finally, measurements of [Ca2+]f in single cells suggest that only the principal cells of the collecting tubules respond to AVP with an increase in [Ca2+]f. In summary, these results demonstrate that the principal cells of the cortical collecting tubule possess two distinct receptor systems for vasopressin, the well-known V2 receptor coupled to adenylate cyclase, and a V1 receptor system that leads to the mobilization of cytosolic calcium, coupled through a pertussis toxin substrate (G protein) to a production of inositol phosphates.     

Calcium-dependent action of osmolality on adenosine 3'',5''-monophosphate accumulation in rat renal inner medulla: evidence for a relationship to calcium-responsive arachidonate release and prostaglandin synthesis.

When urea and NaCl are employed as the major solutes of high osmolality buffers, the cyclic AMP (cAMP) content of oxygenated slices of rat renal inner medulla increases three- to fivefold as osmolality is decreased from 1,650 to 305 mosM. Incubation of slices in Ca2+-free media containing 2 mM EGTA largely abolished this action of osmolality on cAMP, whereas exclusion of Mg2+ or 5+ from the incubation media was without effect. Addition of Ca2+ to Ca2+-deprived inner medulla incubated at 750 mosM (175 mM Na+, 380 mM urea) significantly increased tissue cAMP and media prostaglandin (PG)E accumulation. Ca2+ also stimulated the release of 14C-fatty acid from Ca2+-deprived slices prelabeled with [14C]arachidonate, but not from those labeled with [14C]palmitate. The divalent cation ionophore A23187 enhanced the actions of Ca2+ to increase tissue cAMP, media PGE accumulation, and the release of [14C]-arachidonate from prelabeled inner medulla. By contrast, when slices were incubated at 1,650 mosM (365 mM Na+, 900 mM urea) in the presence or absence of A23187, all of these actions of Ca2+ were markedly suppressed or abolished. Addition of exogenous arachidonate increased tissue cAMP and media PGE at both 750 and 1,650 mosM, whereas palmitate and stearate had no effect on cAMP at either osmolality. The actions of Ca2+ and arachidonate to increase cAMP and PGE accumulation were abolished by the cyclo-oxygenase inhibitors, indomethacin and meclofenamate. They were also abolished by exclusion of molecular O2, which serves as cosubstrate with arachidonate in prostaglandin synthesis. At maximally effective concentrations, exogenous PGE2 and arachidonate produced similar increases in inner medullary cAMP. The maximal effects of the two agents on cAMP were not additive, but were expressed in the absence of Ca2+ at both 750 and 1,650 mosM. However, in marked contrast to the O2-dependent action of arachidonate, PGE2 increased cAMP in the presence or absence of O2. Comparison of the separate actions of urea and NaCl indicated that suppression of Ca2+-responsive [14C]arachidonate release, PGE, and cAMP accumulation at 1,650 mosM reflected primarily an effect of urea, whereas hypertonic NaCl, mannitol, and sucrose alone stimulated inner medullary cAMP and PGE accumulation by a pathway which did not require extracellular Ca2+. Analogous to the actions of hypertonic urea, tetracaine and mepacrine inhibited the actions of Ca2+ plus A23187 to stimulate [14C]-arachidonate release, PGE, and cAMP accumulation. Inhibition of PGE and cAMP accumulation by tetracaine and mepacrine was also overcome by arachidonate. The results suggest that high osmolaity media with urea as a major solute reduces inner medullary cAMP content, at least in part, through effects on Ca2+-dependent prostaglandin synthesis. Inhibition of PGE synthesis, in turn, may be the result of osmotic suppression of Ca2+-dependent release of arachidonate, the availability of which is often rate limiting to prostaglandin generation.     

Prostaglandin E2 inhibits sodium transport in rabbit cortical collecting duct by increasing intracellular calcium.

The mechanism by which prostaglandin E2 (PGE2) inhibits sodium absorption (JNa) in the rabbit cortical collecting duct (CCD) was explored. PGE2 activates at least three signaling mechanisms in the CCD: (a) by itself PGE2 increases cAMP generation (b) PGE2 also inhibits vasopressin-stimulated cAMP accumulation, and (c) PGE2 raises intracellular calcium([Ca++]i). We tested the contribution of these signaling pathways to PGE2's effect on Na+ absorption, measuring 22Na flux (JNa) and [Ca++]i (using fura-2) in microperfused rabbit CCDs. In control studies PGE2 reduced JNa from 28.2 +/- 3.4 to 15.6 +/- 2.6 pmol.mm-1.min-1. Lowering bath calcium from 2.4 to 45 nM did not by itself alter JNa but in this setting PGE2 failed to inhibit JNa (28.6 +/- 5.4 to 38.5 +/- 4.0). In separate tubules, PGE2 raised [Ca++]i in a spike-like fashion followed by a sustained elevation. However, in 45 nM bath Ca++, PGE2 failed to produce a sustained [Ca++]i elevation. While pretreatment of CCDs with pertussis toxin blocked PGE2 inhibition of vasopressin-stimulated water permeability, it did not block the effect of PGE2 on JNa. To see if cAMP generation contributes to the effect of PGE2 on JNa, we tested the effect of exogenous cAMP, (8-chlorophenylthio(CPT)cAMP) on JNa. 0.1 mM 8-CPTcAMP reduced JNa from 35.75 +/- 2.3 to 21.6 +/- 2.2. However, the addition of PGE2 further blunted JNa to 15.9 +/- 1.3. In CCDs pretreated with indomethacin, 8-CPTcAMP did not significantly decrease JNa 33.6 +/- 2.8 vs. 28.4 +/- 2. However, superimposed PGE2 reduced JNa to 19.0 +/- 3.0. We conclude that PGE2 inhibits sodium transport predominantly by increasing intracellular calcium. This action is not mediated by a pertussis toxin-sensitive G protein. Finally, cAMP, through a cyclooxygenase-dependent mechanism, also inhibits CCD JNa and may contribute to the effects of PGE2 on JNa in the rabbit CCD.     

Luminal NaCl delivery regulates basolateral PGE2 release from macula densa cells

Macula densa (MD) cells express COX-2 and COX-2-derived PGs appear to signal the release of renin from the renal juxtaglomerular apparatus, especially during volume depletion. However, the synthetic machinery and identity of the specific prostanoid released from intact MD cells remains uncertain. In the present studies, a novel biosensor tool was engineered to directly determine whether MD cells release PGE2 in response to low luminal NaCl concentration ([NaCl]L). HEK293 cells were transfected with the Ca2+-coupled E-prostanoid receptor EP1 (HEK/EP1) and loaded with fura-2. HEK/EP1 cells produced a significant elevation in intracellular [Ca2+] ([Ca2+]i) by 29.6 +/- 12.8 nM (n = 6) when positioned at the basolateral surface of isolated perfused MD cells and [NaCl]L was reduced from 150 mM to zero. HEK/EP1 [Ca2+]i responses were observed mainly in preparations from rabbits on a low-salt diet and were completely inhibited by either a selective COX-2 inhibitor or an EP1 antagonist, and also by 100 microM luminal furosemide. Also, 20-mM graduated reductions in [NaCl]L between 80 and 0 mM caused step-by-step increases in HEK/EP1 [Ca2+]i. Low-salt diet greatly increased the expression of both COX-2 and microsome-associated PGE synthase (mPGES) in the MD. These studies provide the first direct evidence that intact MD cells synthesize and release PGE2 during reduced luminal salt content and suggest that this response is important in the control of renin release and renal vascular resistance during salt deprivation.     

Calcium- and CaMKII-dependent chloride secretion induced by the microsomal Ca(2+)-ATPase inhibitor 2,5-di-(tert-butyl)-1,4-hydroquinone in cystic fibrosis pancreatic epithelial cells.

Microsomal Ca(2+)-ATPase inhibitors such as thapsigargin (THG), cyclopiazonic acid (CPA) and 2,5-di-(tert-butyl)-1,4-hydroquinone (DBHQ) have been shown to inhibit Ca2+ reuptake by the intracellular stores and increase cytosolic free Ca2+ ([Ca2+]i). DBHQ is a commercially available non-toxic synthetic compound chemically unrelated to THG and CPA. In this study, we tested the feasibility of utilizing DBHQ to improve Cl- secretion via the Ca(2+)-dependent pathway, in the cystic fibrosis (CF)-derived pancreatic epithelial cell line CFPAC-1. DBHQ stimulated 125I efflux and mobilized intracellular free Ca2+ in a dose-dependent manner. The maximal effects were seen at concentrations of 25-50 microM. DBHQ (25 microM) caused a short-term rise in [Ca2+]i in the absence of ambient Ca2+, and a sustained elevation of [Ca2+]i in cell monolayers bathed in the efflux solution (1.2 mM Ca2+), which was largely attenuated by Ni2+ (5 mM). Bath-application of DBHQ induced an outwardly-rectifying whole-cell Cl- current, which was abolished by pipette addition of BAPTA (5 mM) or CaMK [273-302] (20 microM), an inhibitory peptide of multifunctional Ca2+/calmodulin-dependent protein kinase (CaMKII). Pretreatment of monolayers of CFPAC-1 cells with DBHQ for 4-5 min significantly increased the Ca(2+)-independent or autonomous activity of CaMKII assayed in the cell homogenates. Thus, DBHQ appears to enhance Cl- channel activity via a Ca(2+)-dependent mechanism involving CaMKII. Pretreatment of CFPAC-1 cells with up to 50 microM DBHQ for 6 h did not cause any detectable change in cell viability and did not significantly affect the cell proliferation rate. These results suggest that appropriate selective microsomal Ca(2+)-ATPase inhibitors may be therapeutically useful in improving Cl- secretion in CF epithelial cells.     

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.     

Apoptosis in insulin-secreting cells. Evidence for the role of intracellular Ca2+ stores and arachidonic acid metabolism.

This study investigated the role of intracellular free Ca2+ concentration ([Ca2+]i) in apoptosis in MIN6 cells, an insulin secreting cell line, and in mouse islets. Thapsigargin, an inhibitor of sarcoendoplasmic reticulum Ca2+-ATPases (SERCA), caused a time- and concentration-dependent decrease in the viability of MIN6 cells and an increase in DNA fragmentation and nuclear chromatin staining changes characteristic of apoptosis. Two structurally distinct SERCA inhibitors, cyclopiazonic acid and 2,5-di-[t-butyl]-1,4-hydroquinone also caused apoptosis, but agents that increased [Ca2+]i by other mechanisms did not induce apoptosis in MIN6 cells. Carbachol- or ionomycin-releasible intracellular Ca2+ stores were completely depleted in cells treated by SERCA inhibitors, but not by other agents that increase [Ca2+]i. The ability of thapsigargin to induce cell death was not affected by blocking Ca2+ influx or by clamping [Ca2+]i with a cytosolic Ca2+ buffer suggesting that the process did not depend on changes in [Ca2+]i per se. However, application of the lipoxygenase inhibitors 5,8,11-eicosatrienoic acid and nordihydroguaiaretic acid partially prevented MIN6 cell apoptosis, while exposure of cells to the product of lipoxygenase, 12-hydroxy-[5,8,10,14]-eicosatetraenoic acid, caused apoptosis. In contrast, inhibition of cyclooxygenase with indomethacin did not abolish thapsigargin-induced apoptosis in MIN6 cells. Our findings indicate that thapsigargin causes apoptosis in MIN6 cells by depleting intracellular Ca2+ stores and leading to release of intermediate metabolites of arachidonic acid metabolism.     

Pulsatile insulin release from pancreatic islets with nonoscillatory elevation of cytoplasmic Ca2+.

The relationship between insulin release and cytoplasmic Ca2+ concentration ([Ca2+]i) was studied in isolated pancreatic islets from ob/ob mice. Although [Ca2+]i was low and stable in the presence of 3 mM glucose, basal insulin release exhibited low amplitude pulsatility, with a frequency of 0.32 +/- 0.04 min-1. Depolarization by raising K+ from 5.9 to 30.9 mM or by the addition of 1 mM tolbutamide caused a pronounced initial insulin pulse followed by declining pulses, but there was no change in frequency. This decline in amplitude of the insulin pulses was prevented in similar experiments performed in the presence of 11 mM glucose. Corresponding measurements of [Ca2+]i in islets exposed to tolbutamide or the high K+ concentration revealed stable elevations without oscillations. Although the [Ca2+]i level is an important determinant for the rate of secretion, the results indicate that pulsatile insulin release does not always depend on [Ca2+]i oscillations. It is suggested that cyclic generation of ATP may fuel pulsatile release under conditions when [Ca2+]i remains stable.     

Alpha-2 adrenergic inhibition of intestinal secretion induced by prostaglandin E1, vasoactive intestinal peptide and dibutyryl cyclic AMP in rat jejunum

Effects of alpha adrenergic agents on intestinal secretion induced by prostaglandin E1 (PGE1), vasoactive intestinal peptide (VIP) and dibutyryl cyclic AMP (Bt2cAMP) were investigated in rat jejunum in vivo. Oxymetazoline and clonidine were more potent than epinephrine in inhibiting the PGE1-induced secretion. Methoxamine failed to inhibit the PGE1-induced secretion even with a 100-fold higher dose than that of clonidine. A high dose (1 mumol/kg) of oxymetazoline not only inhibited the PGE1- induced secretion but also enhanced net fluid absorption. Yohimbine reversed the inhibitory effect of clonidine, whereas phenoxybenzamine did not. These antagonists per se did not produce any effects on PGE1-induced secretion. Clonidine inhibited the intestinal secretion induced by VIP or Bt2cAMP, whereas methoxamine did not. The inhibitory effect of clonidine was reversed by yohimbine. Phenoxybenzamine per se inhibited intestinal secretion induced by either VIP or Bt2cAMP. Clonidine did not produce any significant effects on PGE1- augmented cAMP levels in jejunal mucosa in vivo. These results suggest that stimulation of alpha-2 adrenoceptors in rat jejunal mucosa inhibits some mechanisms distal to cAMP generation and in turn results in the inhibition of net water intestinal secretion. These findings also raise the question of general validity of a hypothesis that alpha-2 adrenoceptors regulate cellular functions through the inhibition of adenylate cyclase activity.     

Therapies to slow polycystic kidney disease

Advances in the understanding of cystogenesis and availability of animal models orthologous to human autosomal dominant polycystic kidney disease (ADPKD) and recessive polycystic kidney disease (ARPKD) will likely facilitate the development of treatments for these diseases. Proteins mutated in ADPKD and ARPKD, as well as in several animal models, are localized to renal primary cilia. These are thought to have a sensory function and contribute to the regulation of the intracellular calcium ([Ca2+]i). It seems likely that the maintenance of a differentiated renal epithelial phenotype, characterized by controlled fluid secretion and cell proliferation, requires precise functional coordination of cAMP and Ras/Raf/MEK/ERK signaling by [Ca2+]i. [Ca2+]i alterations, linked to genetic defects causing polycystic kidney disease, may hinder negative feedback mechanisms that control cAMP and Ras/Raf/MEK/ERK signaling, and result in increased fluid secretion and cell proliferation. cAMP levels, Raf kinase activities and ERK phosphorylation are increased in polycystic kidneys. There is also evidence of abnormal cross-talk between cAMP and MAPK pathways, that can be reproduced in wild-type cells by altering [Ca2+]i. While cAMP inhibits Ras-Raf-1-stimulated phosphorylation of ERK in normal kidney cells, it markedly increases B-Raf kinase activity and ERK phosphorylation in polycystic kidney cells. Treatment strategies should probably be aimed at increasing [Ca2+]i, inhibiting Ras/Raf/MEK/ERK signaling or lowering cAMP in the distal nephron and collecting duct. Vasopressin is the major adenylyl cyclase agonist in the collecting duct principal cells via a V2 receptor. OPC31260, a V2 receptor antagonist, lowers renal cAMP and markedly inhibits cystogenesis in four animal models of polycystic kidney disease, three of which are orthologous to human diseases (PCK rat, ARPKD; pcy mouse, adolescent nephronophthisis; Pkd2WS25/- mouse, ADPKD). The renal selectivity and safety profile of this class of drugs make it an excellent candidate for clinical trials.     

Glucose-stimulated insulin secretion correlates with changes in mitochondrial and cytosolic Ca2+ in aequorin-expressing INS-1 cells.

Nutrient-stimulated insulin secretion is dependent upon the generation of metabolic coupling factors in the mitochondria of the pancreatic B cell. To investigate the role of Ca2+ in mitochondrial function, insulin secretion from INS-1 cells stably expressing the Ca2+-sensitive photoprotein aequorin in the appropriate compartments was correlated with changes in cytosolic calcium ([Ca2+]c) and mitochondrial calcium ([Ca2+]m). Glucose and KCl, which depolarize the cell membrane, as well as the Ca2+-mobilizing agonist, carbachol (CCh), cause substantial increases in [Ca2+]m which are associated with smaller rises in [Ca2+]c. The L-type Ca2+-channel blocker, SR7037, abolished the effects of glucose and KCl while attenuating the CCh response. Glucose-induced increases in [Ca2+]m, [Ca2+]c, and insulin secretion all demonstrate a pronounced initial peak followed by a sustained plateau. All three parameters are increased synergistically when glucose and CCh are combined. Finally, [Ca2+]m, [Ca2+]c, and insulin secretion also display desensitization phenomena following repeated additions of the three stimuli. The high sensitivity of [Ca2+]m to Ca2+ influx and the desensitization-resensitization effects can be explained by a model in which the mitochondria of INS-1 cells are strategically located to sense Ca2+ influx through plasma membrane Ca2+ channels. In conclusion, the correlation of [Ca2+]m and [Ca2+]c with insulin secretion may indicate a fundamental role for Ca2+ in the adaptation of oxidative metabolism to the generation of metabolic coupling factors and the energy requirements of exocytosis.     

Insulin attenuates agonist-mediated calcium mobilization in cultured rat vascular smooth muscle cells.

Insulin has been shown to attenuate pressor-induced vascular contraction, but the mechanism for this vasodilatory action is unknown. This study examines the effect of insulin on angiotensin II (ANG II)-induced increments in cytosolic calcium in cultured rat vascular smooth muscle cells (VSMC). 20-min incubations with insulin (10 microU/ml to 100 mU/ml) did not alter basal intracellular calcium concentration ([Ca2+]i), but inhibited the response to 100 nM ANG II in a dose-dependent manner (ANG II alone, 721 +/- 54 vs. ANG II + 100 mU/ml insulin, 315 +/- 35 nM, P < 0.01). A similar effect of insulin on ANG II action was observed in calcium poor buffer. Moreover, insulin did not effect calcium influx. ANG II receptor density and affinity were not affected by 24-h incubation with insulin. To further clarify the mechanisms of these observations, we measured ANG II-induced production of inositol 1,4,5-triphosphate (IP3), and IP3-releasable 45Ca. Insulin treatment did not alter ANG II-stimulated IP3 production. However, IP3-stimulated release of 45Ca in digitonin permeabilized cells was significantly reduced after 5-min incubations with 100 mU/ml insulin. Thapsigargin induced release of calcium stores was also blocked by insulin. Thus, insulin attenuates ANG II-stimulated [Ca2+]i primarily by altering IP3-releasable calcium stores. Insulin effects on ANG II-induced [Ca2+]i were mimicked by preincubation of VSMC with either sodium nitroprusside or 8-bromo-cGMP. As elevations in cGMP in vascular tissue lower [Ca2+]i, it is possible that insulin affects IP3 release of calcium by a cGMP-dependent mechanism that would contribute to its vasodilatory effects.