Ca2+-mediated generation of inositol 1,4,5-triphosphate and inositol 1,3,4,5-tetrakisphosphate in pancreatic islets. Studies with K+, glucose, and carbamylcholine

The role of Ca2+ in the generation of inositol phosphates was investigated using rat pancreatic islets after steady state labeling with myo-[2-3H]inositol. Depolarizing K+ concentrations (24 mM) evoked early (2 s) increases in inositol 1,4,5-trisphosphate (Ins-1,4,5-P3) and inositol 1,3,4,5-tetrakisphosphate (Ins-1,3,4,5-P4) as measured by high performance anion-exchange chromatography. The increase in Ins-1,4,5-P3 was transient and was followed by a more pronounced rise in Ins-1,3,4-P3. These effects were dependent on the presence of extracellular Ca2+ but were not secondary to release of either neurotransmitters or metabolites of arachidonic acid. K+ also promoted the breakdown of phosphatidylinositol 4,5-bisphosphate (PtdIns-4,5-P2) and of the other phosphoinositides. Glucose (16.7 mM) was less marked in its effects but still promoted rapid increases in Ins-1,3,4,5-P4 (2 s) and Ins-1,4,5-P3 (10 s) and a slower rise in Ins-1,3,4-P3 (30 s). The levels of all three metabolites rose steadily over 10 min stimulation. These responses to glucose could be largely, although not entirely, inhibited by depletion of extracellular Ca2+ or by Ca2+ channel blockade with verapamil (20 microM). Carbamylcholine (0.5 mM) was the most potent stimulus used evoking early rises in Ins-1,4,5-P3 and Ins-1,3,4,5-P4 (2 s) followed by Ins-1,3,4-P3 (10 s), effects which were only partially dependent on extracellular Ca2+. The results suggest that a Ca2+-mediated PtdIns-4,5-P2 hydrolysis accounts for most of the Ins-1,4,5-P3 generated in response to glucose but not carbamylcholine. In addition, glucose may exert effects on inositol phosphate metabolism which are Ca2+ independent.     

Ca2+ regulates the inositol tris/tetrakisphosphate pathway in intact and broken preparations of insulin-secreting RINm5F cells

The two-step isomerization of inositol 1,4,5-trisphosphate (Ins-1,4,5-P3) to Ins-1,3,4-P3 via the intermediate inositol 1,3,4,5-tetrakisphosphate (Ins-P4) was studied in intact RINm5F cells and in subcellular fractions. Muscarinic stimulation with carbamylcholine leads to a rapid (2 s) rise in both Ins-1,4,5-P3 and Ins-P4, whereas Ins-1,3,4-P3 was produced only after a lag of at least 5 s. In cells with depleted Ca2+ stores, the rise in Ins-1,4,5-P3 was nearly tripled, and that of Ins-1,3,4-P3 markedly diminished as compared to control cells. Raising the free Ca2+ concentration from 10(-7) to 10(-5) M increased inositol 1,4,5-triphosphate-3-kinase activity in cytosolic fractions by 2 1/2-fold (EC50 for Ca2+ approximately 0.8 microM) but had no effect on the activity of inositol 1,4,5-triphosphate-5-phosphomonoesterase. At 10(-7) M Ca2+ these two enzymes displayed comparable activity when assayed at concentrations of Ins-1,4,5-P3 occurring in stimulated cells; however, at 10(-5) M Ca2+, kinase activity predominates. These results suggest that Ins-1,4,5-P3 counter-regulates its own levels through the activity of inositol 1,4,5-trisphosphate 3-kinase and that the increase in [Ca2+]i may account for the transience of the rise in Ins-1,4,5-P3 seen during muscarinic stimulation of RINm5F cells.     

Catalytic properties of inositol trisphosphate kinase: activation by Ca2+ and calmodulin

Inositol 1,4,5-trisphosphate (Ins-1,4,5-P3) is an important second-messenger molecule that mobilizes Ca2+ from intracellular stores in response to the occupancy of receptor by various Ca2+-mobilizing agonists. The fate of Ins-1,4,5-P3 is determined by two enzymes, a 3-kinase and a 5-phosphomonoesterase. The first enzyme converts Ins-1,4,5-P3 to Ins-1,3,4,5-P4, whereas the latter forms Ins-1,4-P2. Recent studies suggest that Ins-1,3,4,5-P4 might modulate the entry of Ca2+ from an extracellular source. In the current report, we describe the partial purification of the 3-kinase [approximately 400-fold purified, specific activity = 0.12 mumol/(min.mg)] from the cytosolic fraction of bovine brain and studies of its catalytic properties. We found that the 3-kinase activity is significantly activated by the Ca2+/calmodulin complex. Therefore, we propose that Ca2+ mobilized from endoplasmic reticulum by the action of Ins-1,4,5-P3 forms a complex with calmodulin, and that the Ca2+/calmodulin complex stimulates the conversion of Ins-1,4,5-P3, an intracellular Ca2+ mobilizer, to Ins-1,3,4,5-P4, an extracellular Ca2+ mobilizer. A rapid assay method for the 3-kinase was developed that is based on the separation of [3-32P]Ins-1,3,4,5-P4 and [gamma-32P]ATP by thin-layer chromatography. Using this new assay method, we evaluated kinetic parameters (Km for ATP = 40 microM, Km for Ins-1,4,5-P3 = 0.7 microM, Ki for ADP = 12 microM) and divalent cation specificity (Mg2+ much greater than Mn2+ greater than Ca2+) for the 3-kinase. Studies with various inositol polyphosphates indicate that the substrate-binding site is quite specific to Ins-1,4,5-P3. Nevertheless, Ins-2,4,5-P3 could be phosphorylated at a velocity approximately 1/20-1/30 that of Ins-1,4,5-P3.     

Generation of inositol phosphates, cytosolic Ca2+, and ionic fluxes in PC12 cells treated with bradykinin

Accumulation of inositol phosphates (Ins-Ps, revealed by high performance liquid chromatography), changes of the cytosolic free Ca2+ [( Ca2+]i, revealed by fura-2), membrane potential and ionic currents (revealed by bis-oxonol and patch clamping) were investigated in PC12 cells treated with bradykinin (BK). The phenomena observed were (a) due to the activation of a B2 receptor (inhibitor studies) and (b) unaffected by pertussis toxin, cAMP analogs, and inhibitors of either cyclooxygenase or voltage-gated Ca2+ channels. During the initial tens of s, three interconnected events predominated: accumulation of Ins-1,4,5-P3, Ca2+ release from intracellular stores and hyperpolarization due to the opening of Ca2+-activated K+ channels. Phorbol myristate acetate partially inhibited Ins-1,4,5-P3 accumulation at all [BK] investigated, and the [Ca2+]i increase at [BK] less than 50 nM. In PC12 cells treated with maximal [BK] in the Ca2+-containing incubation medium, Ins-1,4,5-P3 peaked at 10 s, dropped to 20% of the peak at 30 s, and returned to basal within 5 min; the peak increase of Ins-1,3,4-P3 was slower and was variable from experiment to experiment, while Ins-P4 rose for 2 min, and remained elevated for many min thereafter. Meanwhile, influx of Ca2+ from the extracellular medium, plasma membrane depolarization (visible without delay when hyperpolarization was blocked), and increased plasma membrane conductance were noticed. Evidence is presented that these last three events (which were partially inhibited by phorbol myristate acetate at all [BK]) were due to the activation of a cation influx, which was much more persistent than the elevation of the two Ins-P3 isomers. Our results appear inconsistent with the possibility that in intact PC12 cells the BK-induced activation of cation influx is accounted for entirely by the increases of either Ins-1,3,4-P3 or Ins-1,4,5-P3 (alone or in combination with Ins-1,3,4,5-P4), as previously suggested by microinjection studies in different cell types.     

Inositol polyphosphate production and regulation of cytosolic calcium during the biphasic activation of adrenal glomerulosa cells by angiotensin II

Stimulation of aldosterone production by angiotensin II in the adrenal glomerulosa cell is mediated by increased phosphoinositide turnover and elevation of intracellular Ca2+ concentration. In cultured bovine glomerulosa cells, angiotensin II caused rapid increases in inositol-1,4,5-trisphosphate (Ins-1,4,5-P3) levels and cytosolic Ca2+ during the first minute of stimulation, when both responses peaked between 5 and 10 s and subsequently declined to above-baseline levels. In addition to this temporal correlation, the dose-response relationships of the angiotensin-induced peak increases in cytosolic Ca2+ concentrations and Ins-1,4,5-P3 levels measured at 10 s were closely similar. However, at later times (greater than 1 min) there was a secondary elevation of Ins-1,4,5-P3, paralleled by increased formation of inositol 1,3,4,5-tetrakisphosphate that was associated with cytosolic Ca2+ concentrations only slightly above the resting value. These results are consistent with the primary role of Ins-1,4,5-P3 in calcium mobilization during activation of the glomerulosa cell by angiotensin II. They also suggest that Ins-1,4,5-P3 participates in the later phase of the target-cell response, possibly by acting alone or in conjunction with its phosphorylated metabolites to promote calcium entry and elevation of cytosolic Ca2+ during the sustained phase of aldosterone secretion.     

Second messenger function of inositol 1,4,5-trisphosphate. Early changes in inositol phosphates, cytosolic Ca2+, and insulin release in carbamylcholine-stimulated RINm5F cells

The second messenger function of inositol 1,4,5-trisphosphate (Ins-1,4,5-P3) was investigated in carbamylcholine-stimulated RINm5F cells by analysis of the early changes in inositol phosphates, cytosolic free Ca2+ concentration ([Ca2+]i), and insulin secretion. After a lag of 2 s, [Ca2+]i rose to a peak at 13 +/- 2 s, a response which was due mainly to mobilization from intracellular stores since it persisted even in the absence of extracellular Ca2+. The Ca2+ response had already declined toward prestimulatory levels by the time insulin secretion reached its maximal rate (2-3 min). Although the rises in inositol trisphosphate preceded those of both inositol bisphosphate and monophosphate, all three attained maximal concentrations after 1 min and remained elevated for at least 10 min. The accumulation of inositol trisphosphate was truly Ca2+-independent since it persisted under conditions in which the rise in [Ca2+]i was abolished by prior depletion of intracellular Ca2+ pools. Further analysis by high performance liquid chromatography revealed the presence of the two isomers, Ins-1,4,5-P3 and Ins-1,3,4-P3 in stimulated cells. The latter was virtually absent under nonstimulatory conditions but started to accumulate after a 5-s lag and reached maximal levels after 30 s of stimulation. Ins-1,4,5-P3 doubled within 1 s of carbamylcholine addition, reached a peak after 5 s, and, although declining thereafter, remained slightly elevated for at least 3 min. Hence, both the onset and peak of the rise of Ins-1,4,5-P3 preceded that of [Ca2+]i, which in turn preceded the peak in insulin release. These results strongly suggest that Ins-1,4,5-P3 acts as the second messenger by which carbamylcholine mobilizes intracellular Ca2+ during the initiation of insulin release.     

Hormone-evoked calcium release from intracellular stores is a quantal process

Ca2+ mobilization by hormones, ionomycin, and inositol 1,4,5-trisphosphate (Ins-1,4,5-P3) were studied to determine whether Ca2+ release is a continuous or a quantal process. Hormone-mediated Ca2+ release occurs only during the first 2-4 s of stimulation. Stimulation of acini with a maximal hormone concentration following stimulation with a submaximal concentration resulted in free cytosolic Ca2+ concentration ([Ca2+]i) increase and 45Ca efflux. The peak [Ca2+]i increase induced by a maximal concentration of agonist was nearly constant when cells were prestimulated with a submaximal dose for 1-15 min. Submaximal hormone concentrations release only a fraction of intracellular 45Ca2+, after which intracellular Ca2+ content remains constant. The partially released stores remain depleted until cell stimulation is terminated, at which time the stores reload with Ca2+. For comparison, increasing concentrations of ionomycin resulted in increasing rates of Ca2+ release. Each ionomycin concentration released all the Ca2+ from intracellular stores. We therefore conclude that hormone-evoked Ca2+ release is a quantal rather than a continuous process. In permeabilized cells, increasing concentrations of Ins-1,4,5-P3 resulted in an increased fraction of Ca2+ release. No submaximal Ins-1,4,5-P3 concentration was capable of releasing all the Ins-1,4,5-P3-mobilizable Ca2+. Therefore, it appears that the quantal properties of hormone-evoked Ca2+ release reflect the quantal properties of Ins-1,4,5-P3-mediated Ca2+ release from intracellular stores.     

Calcium-calmodulin stimulates inositol 1,4,5-trisphosphate kinase activity from insulin-secreting RINm5F cells

In a cytosolic fraction derived from insulin-secreting RINm5F cells, the rate of conversion of inositol 1,4,5-trisphosphate (Ins-1,4,5-P3) to inositol 1,3,4,5-tetrakisphosphate (Ins-1,3,4,5-P4) was half-maximally stimulated by 0.8 microM Ca2+ (Biden, T. J., and Wollheim, C. B. (1986) J. Biol. Chem. 261, 11931-11934). In the present study we show that after initial purification by anion exchange chromatography, the Ins-1,4,5-P3 kinase activity responsible for that conversion is stimulated by Ca2+-calmodulin, but not by Ca2+ alone. This is almost certainly due to a specific interaction of the enzyme and its activator since kinase activity was retained on a calmodulin-linked Sepharose 6B column in the presence of Ca2+ but eluted upon chelation of the cation. After this two-step purification, Ins-1,4,5-P3 kinase activity was maximally stimulated 5-fold by 10 microM calmodulin in the presence of 10(-5) M Ca2+, and 2 1/2-fold at 10(-6) M Ca2+. Under these conditions the minimum concentrations of calmodulin needed to stimulate activity were in the 10-50 nM range. At 10(-7) M Ca2+, calmodulin (up to 30 microM) was without effect. Stimulated Ins-1,4,5-P3 kinase activity was inhibited in a dose-dependent fashion by N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W7) although the calmodulin antagonist had no effect on the residual activity seen at 10(-7) M Ca2+. These results strongly support our previous suggestion that alterations in cytosolic free Ca2+ concentrations play an important role in regulating the levels of Ins-1,4,5-P3 and Ins-1,3,4,5-P4 during cellular stimulation.     

Metabolism of inositol 1,3,4-trisphosphate to a new tetrakisphosphate isomer in angiotensin-stimulated adrenal glomerulosa cells

Angiotensin stimulates rapid and prominent increases in inositol polyphosphates and their metabolites in bovine glomerulosa cells labeled with [3H]inositol. In addition to the early formation of inositol 1,4,5-trisphosphate (Ins-1,4,5-P3) and inositol 1,3,4-trisphosphate (Ins-1,3,4-P3), as well as their intermediate product, inositol 1,3,4,5-tetrakisphosphate (Ins-1,3,4,5-P4), delayed increases in two new InsP4 isomers were consistently observed by high resolution high performance liquid chromatography. Studies on the metabolism of purified Ins-1,3,4,5-P4 preparations, labeled with [3H]inositol and 32P to monitor sites of dephosphorylation, were performed in permeabilized glomerulosa cells. In addition to rapid degradation of Ins-1,3,4,5-P3 to Ins-1,3,4-P3 and then to Ins-3,4-P2, there was delayed formation of one of the putative InsP4 isomers observed during AII stimulation in intact cells. The kinetics of formation of the new InsP4 isomer, and the lack of phosphate in its 5 position based on isotope ratios, were consistent with its origin from Ins-1,3,4-P3. This was confirmed by the conversion of [3H]Ins-1,3,4-P3 to the new InsP4 isomer in permeabilized cells by a kinase distinct from that which phosphorylates Ins-1,4,5-P3. These results have demonstrated that the dephosphorylation sequence of Ins-1,4,5-P3 metabolism is accompanied by a complex cycle of higher phosphorylations with formation of new intermediates of potential significance in cellular regulation.     

Regulation of epidermal growth factor-stimulated formation of inositol phosphates in A-431 cells by calcium and protein kinase C

Epidermal growth factor (EGF) treatment of A-431 cells induces a biphasic increase in the levels of inositol phosphates. The growth factor produces an initial, rapid increase in the level of inositol 1,4,5-trisphosphate (Ins-1,4,5-P3) due to hydrolysis of phosphatidyl-inositol-4,5-bisphosphate (Wahl, M., Sweatt, J. D., and Carpenter, G. (1987) Biochem. Biophys. Res. Commun. 142, 688-695). The level of inositol 1,3,4,5-tetrakisphosphate (Ins-1,3,4,5-P4) also rises rapidly in response to treatment with EGF. The initial formation (less than 1 min) of Ins-1,4,5-P3 and Ins-1,3,4,5-P4 does not require Ca2+ present in the culture medium. However, the addition of Ca2+ to the medium at levels of 100 microM or greater potentiates the growth factor-stimulated increases in the levels of all inositol phosphates at later times after EGF addition (1-60 min). The data suggest that EGF-receptor complexes initially stimulate the enzyme phospholipase C in a manner that is independent of an influx of extracellular Ca2+. The presence of Ca2+ in the medium allows prolonged growth factor activation of phospholipase C. Treatment of A-431 cells with Ca2+ ionophores (A23187 and ionomycin) did not mimic the activity of EGF in producing a rapid increase in the formation of the Dowex column fraction containing Ins-1,4,5-P3, Ins-1,3,4,5-P4, and inositol 1,3,4-trisphosphate (InsP3). However, the initial EGF-stimulated formation of inositol phosphates was substantially diminished in cells loaded with the Ca2+ chelator Quin 2/AM. EGF receptor occupancy studies indicated that maximal stimulation of InsP3 accumulation by EGF requires nearly full (75%) occupancy of available EGF binding sites, while half-maximal stimulation requires 25% occupancy. 12-O-Tetradecanoylphorbol-13-acetate (TPA), an exogenous activator of Ca2+/phospholipid-dependent protein kinase (protein kinase C), causes a dramatic, but transient, inhibition of the EGF-stimulated formation of inositol phosphates. Tamoxifen and sphingosine, reported pharmacologic inhibitors of protein kinase C activity, potentiate the capacity of EGF to induce formation of inositol phosphates. Neither TPA nor tamoxifen significantly affects the 125I-EGF binding capacity of A-431 cells; however, TPA appeared to enhance internalization of the ligand. Ligand occupation of the EGF receptor on the A-431 cell appears to initiate a complex signaling mechanism involving production of intracellular messengers for Ca2+ mobilization and activation of protein kinase C.(ABSTRACT TRUNCATED AT 400 WORDS)     

Does the inositol tris/tetrakisphosphate pathway exist in rat heart?

Appearance of two isomers of inositol trisphosphate (InsP3) was observed when [3H]inositol prelabelled rat heart ventricles were stimulated for 10 and 30 s with noradrenaline. In contrast, inositol tetrakisphosphate (InsP4) could not be detected. However the existence of the inositol tris/tetrakisphosphate pathway was demonstrated by studying [3H]inositol 1,4,5-trisphosphate (Ins-1,4,5-P3) metabolism in a soluble fraction of rat heart. There, [3H]Ins-1,4,5-P3 was phosphorylated to form [3H]Ins-1,3,4,5-P4. Raising [Ca2+] from 1 nM to 1 microM increased InsP3 kinase activity by 2-fold (EC50 for Ca2+ approx. 56 nM). This effect appeared to be due to an increase of the apparent Vmax of the enzyme while the apparent Km was unchanged.     

Mechanisms of receptor-mediated Ca2+ signaling in rat hepatocytes.

The Ca2+ signal observed in individual fura-2-loaded hepatocytes stimulated with the alpha 1-adrenergic agonist phenylephrine consisted of a variable latency period, a rapid biphasic increase in the cytosolic free Ca2+, followed by a period of maintained elevated cytosolic Ca2+ (plateau phase) that depended on the continued presence of both agonist and external Ca2+. Microinjection of guanosine-5'-O-(3-thiophosphate) elicited a Ca2+ transient with the same basic features. The Ca2+ transient resulting from microinjecting inositol 1,4,5-trisphosphate (Ins-1,4,5-P3) occurred with essentially no latency period and consisted of a rapid spike that decayed back to preinjection levels within 15 s. Microinjection of inositol 1,4,5-trisphosphorothioate (thio-IP3), a nonmetabolizable analog of Ins-1,4,5-P3, elicited a Ca2+ transient that was initially identical to that observed with Ins-1,4,5-P3, except that the cytosolic Ca2+ remained elevated. The maintained thio-IP3-induced Ca2+ increase was dependent on the presence of external Ca2+, suggesting an activation of Ca2+ influx. Reintroduction of external Ca2+ in the presence of 5 microM phenylephrine to Ca(2+)-depleted cells resulted in a 2-fold greater rate of rise in the cytosolic Ca2+ compared to the rate observed upon Ca2+ addition to cells Ca(2+)-depleted by preatement with thapsigargin. The rate of Ca2+ rise upon Ca2+ addition to cells microinjected with thio-IP3 was similar to that observed with phenylephrine. Coinjection of the cells with thio-IP3 plus heparin reduced the rate of Ca2+ rise upon Ca2+ addition to that observed in thapsigargin-treated cells. These data indicate that the mechanism responsible for receptor-mediated stimulation of Ca2+ entry into hepatocytes involves not only capacitative Ca2+ entry but also an additional component mediated directly by Ins-1,4,5-P3.     

Structures and metabolism of inositol tetrakisphosphates and inositol pentakisphosphate in bovine adrenal glomerulosa cells

In adrenal glomerulosa cells, angiotensin II stimulates rapid increases in inositol 1,4,5-trisphosphate (Ins-1,4,5-P3) and inositol 1,3,4,5-tetrakisphosphate (Ins-1,3,4,5-P4), followed by slower increases in two additional inositol tetrakisphosphate (InsP4) isomers. One of these InsP4 isomers was previously identified as Ins-1,3,4,6-P4 and shown to be a precursor of inositol pentakisphosphate (InsP5). Analysis of the third InsP4 isomer, purified from cultured bovine adrenal cells labeled with [3H]inositol and stimulated by angiotensin II, revealed that the polyol produced by periodate oxidation, borohydrate reduction, and dephosphorylation was [3H]iditol. This finding is consistent with precursor structures of either Ins-1,4,5,6-P4 or Ins-3,4,5,6-P4 (= L-Ins-1,4,5,6-P4) for the third InsP4 isomer. The [3H]iditol was readily converted to [3H]sorbose by the stereospecific enzyme, L-iditol dehydrogenase, indicating that it originated from Ins-3,4,5,6-P4. Chicken erythrocytes labeled with [3H]inositol also contained high levels of Ins-1,3,4,6-P4 and Ins-3,4,5,6-P4, as well as InsP5, but only small amounts of Ins-1,3,4,5-P4. Both [3H]Ins-1,3,4,6-P4 and [3H]Ins-3,4,5,6-P4, but not [3H]Ins-1,3,4,5-P4, were phosphorylated to form InsP5 in permeabilized bovine glomerulosa cells. In addition, InsP5 itself was slowly dephosphorylated to Ins-1,4,5,6-P4, indicating that its structure is Ins-1,3,4,5,6-P5. These results demonstrate that the higher inositol phosphates are metabolically interrelated and are linked to the receptor-regulated InsP3 response by the conversion of Ins-1,3,4-P3 through Ins-1,3,4,6-P4 to Ins-1,3,4,5,6-P5. The source of Ins-3,4,5,6-P4, the other precursor of InsP5, is not yet known but its elevation in angiotensin II-stimulated glomerulosa cells suggests that its formation is also influenced by agonist-regulated processes.     

Agonist-induced regulation of inositol tetrakisphosphate isomers and inositol pentakisphosphate in adrenal glomerulosa cells

In adrenal glomerulosa cells, angiotensin II (AII) rapidly stimulates the formation of inositol 1,4,5-trisphosphate (Ins-1,4,5-P3) and causes marked long-term changes in the levels of highly phosphorylated inositols. Glomerulosa cells prelabeled with [3H]inositol for 48 h and exposed to AII for 10 min showed prominent increases in inositol 1,3,4,5-tetrakisphosphate (Ins-1,3,4,5-P4) and smaller increases in two additional tetrakisphosphates, Ins-1,3,4,6-P4 and another (Ins-3,4,5,6-P4) eluting in the position of Ins-3,4,5,6-P4 and its stereoisomer, Ins-1,4,5,6-P4, on anion exchange liquid chromatography. A concomitant decrease in InsP5 indicates that an increase in Ins-1,4,5,6-P4, the breakdown product of InsP5, is probably responsible for the initial rise in Ins-3,4,5,6-P4 during 10 min stimulation by AII. During prolonged stimulation by AII, Ins-1,3,4,5-P4 began to decline from its high, stimulated level after the first hour but the level of Ins-1,3,4,6-P4 remained elevated for several hours. There were also progressive increases in the levels of Ins-3,4,5,6-P4 and InsP5 during stimulation for up to 16 h with AII. Treatment of adrenal cells for 16 h with the cyclic AMP-mediated secretagogue, adrenocorticotropic hormone (ACTH), slightly increased basal levels of Ins-1,3,4,6-P4, Ins-3,4,5,6-P4, and InsP5, and enhanced the subsequent AII-stimulated increases in the two additional tetrakisphosphate isomers but not of inositol trisphosphates or Ins-1,3,4,5-P4. This change in the pattern of the higher inositol phosphate response to AII was manifested within 2 h after exposure to ACTH, and was mimicked by treatment with 8-bromo cyclic AMP or forskolin. Treatment with 50 microM cycloheximide abolished the ACTH-induced increases in inositol polyphosphate responses during AII stimulation, but had no effect on the responses of untreated cells to AII. The conversion of [3H]Ins-1,3,4-P3 to [3H]Ins-1,3,4,6-P4, a reaction linking the receptor-mediated InsP3 response to higher inositol phosphates, was enhanced in permeabilized cells that were pretreated for 16 h with either ACTH or AII. These results demonstrate that the reactions by which Ins-1,3,4,6-P4 and Ins-3,4,5,6-P4 are formed and converted to InsP5 are influenced by agonist-stimulated regulatory processes that include both calcium-dependent and cyclic AMP-dependent mechanisms of target cell activation. They also reveal changes consistent with agonist-induced conversion of InsP5 to its dephosphorylated metabolite, Ins-1,4,5,6-P4, during short-term stimulation by AII.     

Phosphoinositide and calcium signalling responses in smooth muscle cells: comparison between lipoproteins, Ang II, and PDGF.

The effects of low density lipoprotein (LDL) and high density lipoprotein (HDL3) on second messenger systems were investigated in cultured human vascular smooth muscle cells (VSMC) and compared with those of angiotensin II (Ang II) and platelet-derived growth factor (PDGF-BB). Phosphoinositide metabolism was studied in myo-[2-3H]-inositol prelabelled VSMC using high performance liquid anion-exchange chromatography. The spectra of inositol phosphate isomers increased after stimulation with either Ang II, LDL, HDL3 or PDGF-BB were qualitatively identical. Major increases occurred in 4-IP1, 1,4-IP2, 1,3,4-IP3 and 1,3,4,5-IP4. These are metabolic conversion products of 1,4,5-IP3 for which only a minor increase was found. Thus lipoproteins, like Ang II and PDGF-BB, activate polyphosphatidylinositol-specific phospholipase C. Intracellular Ca2+ concentrations ([Ca2+]i) were studied in fura-2 loaded VSMC. In monolayer cultures LDL and HDL3 increased [Ca2+]i with kinetics comparable to those for Ang II. Relative to the effects of these agonists, the PDGF-BB-induced increase in [Ca2+]i was slower in onset and the decay from peak [Ca2+]i levels more gradual. Fluorescence recordings from single cells exposed to LDL and HDL3 revealed a prolonged series of transient oscillations of [Ca2+]i, a phenomenon typical for calcium-mobilizing hormones. Additionally, as found for Ang II, preincubation of VSMC with either phorbol 12-myristate, 13-acetate, forskolin or 8-bromo-cyclic GMP inhibited LDL- and HDL-induced accumulation of [3H]-inositol monophosphate. We propose that LDL and HDL3 stimulate signal transduction in VSMC via mechanisms analogous to those of Ca(2+)-mobilizing hormones.     

Epidermal growth factor (EGF) stimulates inositol trisphosphate formation in cells which overexpress the EGF receptor

EGF is a low molecular weight polypeptide hormone which acts as a regulator of cell growth and differentiation. The A-431 cell line has been used frequently to examine receptor-mediated biochemical effects of EGF, since this cell line has an increased (20-50 fold) level of EGF receptors. We have utilized A-431 cells to examine the influence of EGF on formation of an intracellular second messenger, inositol, 1,4,5-trisphosphate (Ins-1,4,5-P3), and other inositol phosphates. The results show that EGF induces rapid formation of Ins-1,4,5-P3 as well as Ins-1,3,4-P3 and Ins-1,3,4,5-P4. There is a concurrent decrease in the level of the lipid precursor for Ins-1,4,5-P3, phosphatidylinositol 4,5-biphosphate (PIP2). Furthermore, we have examined five other cell lines that overexpress the EGF receptor and find that EGF treatment induces formation of inositol polyphosphates in those cell lines also.     

Metabolism of inositol-1,3,4,6-tetrakisphosphate to inositol pentakisphosphate in adrenal glomerulosa cells

Angiotensin II stimulates rapid formation of inositol-1,4,5-trisphosphate (Ins-1,4,5-P3) in bovine adrenal glomerulosa cells. In addition to being rapidly metabolized to lower inositol phosphates, Ins-1,4,5-P3 is converted to Ins-1,3,4,5-tetrakisphosphate (Ins-1,3,4,5-P4) and Ins-1,3,4-P3 which is in turn phosphorylated to a further Ins-P4 isomer, namely Ins-1,3,4,6-P4. In bovine adrenocortical cytosol [3H]Ins-1,3,4,5-P4 and [3H]Ins-1,3,4-P3 were converted to Ins-1,3,4,6-P4 and inositol pentakisphosphate (Ins-P5) in a metabolic sequence suggesting that unlike Ins-1,3,4,5-P4, Ins-1,3,4,6-P4 is a direct precursor of Ins-P5. Consistent with this assumption, [3H]Ins-1,3,4,6-P4 was converted to Ins-P5 in electropermeabilized adrenal glomerulosa cells. These findings demonstrate that Ins-1,3,4,6-P4 is an intermediate link between InsP3 metabolism and the higher inositol phosphates detected in several tissues.     

Angiotensin-induced formation and metabolism of inositol polyphosphates in bovine adrenal glomerulosa cells

The actions of angiotensin II (AII) on inositol polyphosphate production and metabolism were analyzed in cultured bovine adrenal glomerulosa cells. In cells labeled for 24 hr with [3H]inositol, AII caused a rapid and prominent rise in formation of Ins-P3 (mainly the Ins-1,3,4,-P3 isomer) and of Ins-P4, with marked increases in two isomers of Ins-P2 and Ins-P. These findings are consistent with rapid formation and turnover of Ins-1,4,5-P3, partly via conversion to Ins-1,3,4,5-P4 with subsequent metabolism to Ins-1,3,4-P3 and lower inositol phosphates. The demonstration of a cytosolic Ins-P3-kinase gave further evidence for the presence of the tris/tetrakisphosphate pathway and Ins-P4 synthesis during AII action in the bovine adrenal cortex.     

Crystal structures of the Dab homology domains of mouse disabled 1 and 2

Disabled (Dab) 1 and 2 are mammalian homologues of Drosophila DAB. Dab1 is a key cytoplasmic mediator in Reelin signaling that controls cell positioning in the developing central nervous system, whereas Dab2 is an adapter protein that plays a role in endocytosis. DAB family proteins possess an amino-terminal DAB homology (DH) domain that is similar to the phosphotyrosine binding/phosphotyrosine interaction (PTB/PI) domain. We have solved the structures of the DH domains of Dab2 (Dab2-DH) and Dab1 (Dab1-DH) in three different ligand forms, ligand-free Dab2-DH, the binary complex of Dab2-DH with the Asn-Pro-X-Tyr (NPXY) peptide of amyloid precursor protein (APP), and the ternary complex of Dab1-DH with the APP peptide and inositol 1,4,5-trisphosphate (Ins-1,4,5-P3, the head group of phosphatidylinositol-4,5-diphosphate (PtdIns-4,5-P2)). The similarity of these structures suggests that the rigid Dab DH domain maintains two independent pockets for binding of the APP/lipoprotein receptors and phosphoinositides. Mutagenesis confirmed the structural determinants specific for the NPXY sequence and PtdIns-4,5-P2 binding. NMR spectroscopy confirmed that the DH domain binds to Ins-1,4,5-P3 independent of the NPXY peptides. These findings suggest that simultaneous interaction of the rigid DH domain with the NPXY sequence and PtdIns-4,5-P2 plays a role in the attachment of Dab proteins to the APP/lipoprotein receptors and phosphoinositide-rich membranes.     

Critical role of NADPH oxidase-derived reactive oxygen species in generating Ca2+ oscillations in human aortic endothelial cells stimulated by histamine

There is increasing evidence that intracellular reactive oxygen species (ROS) play a role in cell signaling and that the NADPH oxidase is a major source of ROS in endothelial cells. At low concentrations, agonist stimulation of membrane receptors generates intracellular ROS and repetitive oscillations of intracellular Ca(2+) concentration ([Ca(2+)](i)) in human endothelial cells. The present study was performed to examine whether ROS are important in the generation or maintenance of [Ca(2+)](i) oscillations in human aortic endothelial cells (HAEC) stimulated by histamine. Histamine (1 microm) increased the fluorescence of 2',7'-dihydrodichlorofluorescin diacetate in HAEC, an indicator of ROS production. This was partially inhibited by the NADPH oxidase inhibitor diphenyleneiodonium (DPI, 10 microm), by the farnesyltransferase inhibitor H-Ampamb-Phe-Met-OH (2 microm), and in HAEC transiently expressing Rac1(N17), a dominant negative allele of the protein Rac1, which is essential for NADPH oxidase activity. In indo 1-loaded HAEC, 1 microm histamine triggered [Ca(2+)](i) oscillations that were blocked by DPI or H-Ampamb-Phe-Met-OH. Histamine-stimulated [Ca(2+)](i) oscillations were not observed in HAEC lacking functional Rac1 protein but were observed when transfected cells were simultaneously exposed to a low concentration of hydrogen peroxide (10 microm), which by itself did not alter either [Ca(2+)](i) or levels of inositol 1,4,5-trisphosphate (Ins-1,4,5-P(3)). Thus, histamine generates ROS in HAEC at least partially via NADPH oxidase activation. NADPH oxidase-derived ROS are critical to the generation of [Ca(2+)](i) oscillations in HAEC during histamine stimulation, perhaps by increasing the sensitivity of the endoplasmic reticulum to Ins-1,4,5-P(3).