Calcium regulates inositol 1,3,4,5-tetrakisphosphate production in lysed thymocytes and in intact cells stimulated with concanavalin A.

Lysed mouse thymocytes release [3H]inositol 1,4,5 trisphosphate from [3H]inositol-labelled phosphatidyl inositol 4,5-bisphosphate in response to GTP gamma S, and rapidly phosphorylate [3H]inositol 1,4,5-trisphosphate to [3H]inositol 1,3,4,5-tetrakisphosphate. The rate of phosphorylation is increased approximately 7-fold when the free [Ca2+] in the lysate is increased from 0.1 to 1 microM, the range in which the cytosolic free [Ca2+] increases in intact thymocytes in response to the mitogen concanavalin A. Stimulation of the intact cells with concanavalin A also results in a rapid and sustained increase in the amount of inositol 1,3,4,5-tetrakisphosphate, and a much smaller transient increase in 1,4,5-trisphosphate. Lowering [Ca2+] in the medium from 0.4 mM to 0.1 microM before addition of concanavalin A reduces accumulation of inositol 1,3,4,5-tetrakisphosphate by at least 3-fold whereas the increase in inositol 1,4,5-trisphosphate is sustained rather than transient. The data imply that in normal medium the activity of the inositol 1,4,5-trisphosphate kinase increases substantially in response to the rise in cytosolic free [Ca2+] generated by concanavalin A, accounting for both the transient accumulation of inositol 1,4,5-trisphosphate and the sustained high levels of inositol 1,3,4,5-tetrakisphosphate. Inositol 1,3,4,5-tetrakisphosphate is a strong candidate for the second messenger for Ca2+ entry across the plasma membrane. This would imply that the inositol polyphosphates regulate both Ca2+ entry and intracellular Ca2+ release, with feedback control of the inositol polyphosphate levels by Ca2+.     

Formation of inositol 1,4,5-trisphosphate and inositol 1,3,4-trisphosphate from inositol 1,3,4,5-tetrakisphosphate and their pathways of degradation in RBL-2H3 cells

Previous studies with antigen-stimulated rat basophilic leukemia (RBL-2H3) cells indicated the formation of multiple isomers of each of the various categories of inositol phosphates. The identities of the different isomers have been elucidated by selective labeling of [3H]inositol 1,3,4,5-tetrakisphosphate with [32P]phosphate in the 3'-or 4',5'-positions and by following the metabolism of different radiolabeled inositol phosphates in extracts of RBL-2H3 cells. We report here that inositol 1,3,4,5-tetrakisphosphate, when incubated with the membrane fraction of extracts of RBL-2H3 cells, was converted to inositol 1,4,5-trisphosphate and inositol 1,3,4-trisphosphate. Further dephosphorylation of the inositol polyphosphates proceeded rapidly in whole extracts of cells, although the process was significantly retarded when ATP (2 mM) levels were maintained by an ATP-regenerating system. The degradation of inositol 1,4,5-trisphosphate proceeded with the sequential formation of inositol 1,4-bisphosphate, the inositol 4-monophosphate (with smaller amounts of the 1-monophosphate), and finally inositol. Inositol 1,3,4-trisphosphate, on the other hand, was converted to inositol 1,3-bisphosphate and inositol 3,4-bisphosphate and subsequently to inositol 4-monophosphate and inositol 1-monophosphate (stereoisomeric forms were undetermined). The possible implications of the apparent interconversion between inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate in regulating histamine secretion in the RBL-2H3 cells are discussed.     

Formation and metabolism of [3H]inositol phosphates in AR42J pancreatoma cells. Substance P-induced Ca2+ mobilization in the apparent absence of inositol 1,4,5-trisphosphate 3-kinase activity

After 2 days of incubation of AR42J pancreatoma cells with 400 microM [3H]inositol, the specific radioactivity of [3H]phosphatidylinositol 4,5-bisphosphate and the specific radioactivity of [3H]inositol were similar, indicating that isotopic equilibrium had been achieved. The inositol 1,4,5-trisphosphate (1,4,5-IP3) level in cells was estimated to be approximately 2 microM and was increased by substance P receptor activation to about 25 microM. HPLC analysis of [3H]inositol phosphates indicated that only 1,4,5-IP3, inositol 1,4-bisphosphate, and inositol 4-monophosphate were increased upon receptor activation. There was no increase in inositol 1,3,4,5-tetrakisphosphate (1,3,4,5-IP4), or in any of its metabolites. Incubation of [3H]1,4,5-IP3 with a cell homogenate did not result in the formation of [3H]1,3,4,5-IP4. Therefore, it appears that 1,4,5-IP3 3-kinase is either not present or not functional under these assay conditions. Substance P increased cytosolic calcium levels in fura-2-loaded cells from about 600 nM to 2.5 microM. This increase in Ca2+ was partially attenuated in the absence of extracellular calcium, indicating that in AR42J cells, substance P stimulation appears to activate calcium signaling through both Ca2+ entry and intracellular Ca2+ release. These modes of Ca2+ mobilization occur without an increase in 1,3,4,5-IP4 or any of its metabolites.     

Formation and metabolism of inositol 1,3,4,5-tetrakisphosphate in liver

The inositol lipid pools of isolated rat hepatocytes were labeled with [3H]myo-inositol, stimulated maximally with vasopressin and the relative contents of [3H]inositol phosphates were measured by high performance liquid chromatography. Inositol 1,4,5-trisphosphate accumulated rapidly (peak 20 s), while inositol 1,3,4-trisphosphate and a novel inositol phosphate (ascribed to inositol 1,3,4,5-tetrakisphosphate) accumulated at a slower rate over 2 min. Incubation of hepatocytes with 10 mM Li+ prior to vasopressin addition selectively augmented the levels of inositol monophosphate, inositol 1,4-bisphosphate, and inositol 1,3,4-trisphosphate. A kinase was partially purified from liver and brain cortex which catalyzed an ATP-dependent phosphorylation of [3H]inositol 1,4,5-trisphosphate to inositol 1,3,4,5-tetrakisphosphate. Incubation of purified [3H]inositol 1,3,4,5-tetrakisphosphate with diluted liver homogenate produced initially inositol 1,3,4-trisphosphate and subsequently inositol 1,3-bisphosphate, the formation of which could be inhibited by Li+. The data demonstrate that the most probable pathway for the formation of inositol 1,3,4,5-tetrakisphosphate is by 3-phosphorylation of inositol 1,4,5-trisphosphate by a soluble mammalian kinase. Degradation of both compounds occurs first by a Li+-insensitive 5-phosphatase and subsequently by a Li+-sensitive 4-phosphatase. The prolonged accumulation of both inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate in vasopressin-stimulated hepatocytes suggest that they have separate second messenger roles, perhaps both relating to Ca2+-signalling events.     

Quantitative changes in inositol 1,4,5-trisphosphate in chemoattractant-stimulated neutrophils

myo-Inositol 1,4,5-trisphosphate is an intracellular second messenger generated from the hydrolysis of phosphatidylinositol 4,5-bisphosphate by phospholipase C. In the present study, we have used the abilities of inositol 1,4,5-trisphosphate to inhibit inositol 1,4,5-tris[32P]phosphate binding and to stimulate release of sequestered stores of 45Ca2+ to assay the mass of inositol 1,4,5-trisphosphate in extracts derived from [3H]inositol-prelabeled chemoattractant-stimulated neutrophils. These assays are specific for inositol 1,4,5-trisphosphate since the relative capacity of the extracts to compete with inositol 1,4,5-tris[32P]phosphate binding and to release 45Ca2+ correlated well with the [3H]inositol 1,4,5-trisphosphate content of the extract as determined by high pressure liquid chromatography. No correlation of these activities was observed with the content in the extract of either [3H]inositol 1,3,4-trisphosphate or [3H]inositol 1,3,4,5-tetrakisphosphate, whose formation exhibited kinetics distinct from [3H]inositol 1,4,5-trisphosphate. Thus, within 10 s of stimulation with 10 nM formyl-methionyl-leucyl-phenylalanine, the inositol 1,4,5-trisphosphate content of the extract increased from 0.05 to 0.55 pmol/10(6) cells, equivalent to a change in intracellular concentration from 100 nM to 1.1 microM. These studies demonstrate that neutrophils produce sufficient quantities of inositol 1,4,5-trisphosphate to mobilize Ca2+ from intracellular stores.     

Source of 3H-labeled inositol bis- and monophosphates in agonist-activated rat parotid acinar cells

The kinetics of [3H]inositol phosphate metabolism in agonist-activated rat parotid acinar cells were characterized in order to determine the sources of [3H]inositol monophosphates and [3H]inositol bisphosphates. The turnover rates of D-myo-inositol 1,4,5-trisphosphate and its metabolites, D-myo-inositol 1,4-bisphosphate and D-myo-inositol 1,3,4-trisphosphate, were examined following the addition of the muscarinic receptor antagonist, atropine, to cholinergically stimulated parotid cells. D-myo-Inositol 1,4,5-trisphosphate declined with a t1/2 of 7.6 +/- 0.7 s, D-myo-inositol 1,3,4-trisphosphate declined with a t1/2 of 8.6 +/- 1.2 min, and D-myo-inositol 1,4-bisphosphate was metabolized with a t1/2 of 6.0 +/- 0.7 min. The sum of the rates of flux through D-myo-inositol 1,4-bisphosphate and D-myo-inositol 1,3,4-trisphosphate (2.54% phosphatidylinositol/min) did not exceed the calculated rate of breakdown of D-myo-inositol 1,4,5-trisphosphate (2.76% phosphatidylinositol/min). Thus, there is no evidence for the direct hydrolysis of phosphatidylinositol 4-phosphate in intact cells since D-myo-inositol 1,4-bisphosphate formation can be attributed to the dephosphorylation of D-myo-inositol 1,4,5-trisphosphate. The source of the [3H]inositol monophosphates also was examined in cholinergically stimulated parotid cells. When parotid cells were stimulated with methacholine, D-myo-inositol 1,4,5-trisphosphate, D-myo-inositol 1,3,4,5-tetrakisphosphate, D-myo-inositol 1,4-bisphosphate, and D-myo-inositol 4-monophosphate levels increased within 2 s, whereas D-myo-inositol 1-monophosphate accumulation was delayed by several seconds. Rates of [3H]inositol monophosphate accumulation also were examined by the addition of LiCl to cells stimulated to steady state levels of [3H]inositol phosphates. The sum of the rates of accumulation of D-myo-inositol 1-monophosphate and D-myo-inositol 4-monophosphate did not exceed the rate of breakdown of D-myo-inositol 1,4,5-trisphosphate or the sum of the rates of flux through D-myo-inositol 1,4-bisphosphate and D-myo-inositol 1,3,4-trisphosphate. These kinetic analyses suggest that agonist-stimulated [3H]inositol bis- and monophosphate formation in intact rat parotid acinar cells can be accounted for by the metabolism of D-myo-[3H]inositol 1,4,5-trisphosphate rather than by phospholipase C-catalyzed hydrolysis of phosphatidylinositol or phosphatidylinositol 4-phosphate.     

Actions of inositol phosphates on Ca2+ pools in guinea-pig hepatocytes.

In permeabilized hepatocytes, inositol 1,4,5-trisphosphate, inositol 2,4,5-trisphosphate and inositol 4,5-bisphosphate induced rapid release of Ca2+ from an ATP-dependent, non-mitochondrial vesicular pool, probably endoplasmic reticulum. The order of potency was inositol 1,4,5-trisphosphate greater than inositol 2,4,5-trisphosphate greater than inositol 4,5-bisphosphate. The Ca2+-releasing action of inositol 1,4,5-trisphosphate is not inhibited by high [Ca2+], nor is it dependent on [ATP] in the range of 50 microM-1.5 mM. These results suggest a role for inositol 1,4,5-trisphosphate as a second messenger in hormone-induced Ca2+ mobilisation, and that a specific receptor is involved in the Ca2+-release mechanism.     

Effects of Ca2+ on phosphoinositide breakdown in exocrine pancreas.

Recent studies have established that inositol 1,4,5-trisphosphate [I(1,4,5)P3] provides the link between receptor-regulated polyphosphoinositide hydrolysis and mobilization of intracellular Ca2+. Here, we report the effects of Ca2+ on inositol trisphosphate (IP3) formation from phosphatidylinositol bisphosphate (PIP2) catalysed by phospholipase C in intact and electrically permeabilized rat pancreatic acinar cells. In permeabilized cells, the Ca2+-mobilizing agonist caerulein stimulated [3H]IP3 formation when the free [Ca2+] was buffered at 140 nM, the cytosolic free [Ca2+] of unstimulated pancreatic acinar cells. When the free [Ca2+] was reduced to less than 10 nM, caerulein did not stimulate [3H]IP3 formation. Ca2+ in the physiological range stimulated [3H]IP3 formation and reduced the amount of [3H]PIP2 in permeabilized cells. The effects of Ca2+ and the receptor agonist caerulein were additive, but we have not established whether this reflects independent effects on the same or different enzymes. The effect of Ca2+ on [3H]IP3 formation by permeabilized cells was unaffected by inhibitors of the cyclo-oxygenase and lipoxygenase pathways of arachidonic acid metabolism; nor were the effects of Ca2+ mimicked by addition of arachidonic acid. These results suggest that the effects of Ca2+ on phospholipase C activity are not a secondary consequence of Ca2+ activation of phospholipase A2. Changes in free [Ca2+] (less than 10 nM-1.2 mM) did not affect the metabolism of exogenous [3H]I(1,4,5)P3 by permeabilized cells. In permeabilized cells, breakdown of exogenous [3H]IP3 to [3H]IP2 (inositol bisphosphate), and formation of [3H]IP3 in response to receptor agonists were equally inhibited by 2,3-bisphosphoglyceric acid. This suggests that the [3H]IP2 formed in response to receptor agonists is entirely derived from [3H]IP3. In intact cells, [3H]IP3 formation was stimulated when ionomycin was used to increase the cytosolic free [Ca2+]. However, a maximal concentration of caerulein elicited ten times as much IP3 formation as did the highest physiologically relevant [Ca2+]. We conclude that the major effect of receptor agonists on IP3 formation does not require an elevation of cytosolic free [Ca2+], although the increase in free [Ca2+] that normally follows IP3 formation may itself have a small stimulatory effect on phospholipase C.     

Intracellular receptors for inositol 1,4,5-trisphosphate in angiotensin II target tissues

Many cells (including angiotensin II target cells) respond to external stimuli with accelerated hydrolysis of phosphatidylinositol 4,5-bisphosphate, generating 1,2-diacylglycerol and inositol 1,4,5-trisphosphate, a rapidly diffusible and potent Ca2+-mobilizing factor. Following its production at the plasma membrane level, inositol 1,4,5-trisphosphate is believed to interact with specific sites in the endoplasmic reticulum and triggers the release of stored Ca2+. Specific receptor sites for inositol 1,4,5-trisphosphate were recently identified in the bovine adrenal cortex (Baukal, A. J., Guillemette, G., Rubin, R., Sp?t, A., and Catt, K. J. (1985) Biochem. Biophys. Res. Commun. 133, 532-538) and have been further characterized in the adrenal cortex and other target tissues. The inositol 1,4,5-trisphosphate-binding sites are saturable and present in low concentration (104 +/- 48 fmol/mg protein) and exhibit high affinity for inositol 1,4,5-trisphosphate (Kd 1.7 +/- 0.6 nM). Their ligand specificity is illustrated by their low affinity for inositol 1,4-bisphosphate (Kd approximately 10(-7) M), inositol 1-phosphate and phytic acid (Kd approximately 10(-4) M), fructose 1,6-bisphosphate and 2,3-bisphosphoglycerate (Kd approximately 10(-3) M), with no detectable affinity for inositol 1-phosphate and myo-inositol. These binding sites are distinct from the degradative enzyme, inositol trisphosphate phosphatase, which has a much lower affinity for inositol trisphosphate (Km = 17 microM). Furthermore, submicromolar concentrations of inositol 1,4,5-trisphosphate evoked a rapid release of Ca2+ from nonmitochondrial ATP-dependent storage sites in the adrenal cortex. Specific and saturable binding sites for inositol 1,4,5-trisphosphate were also observed in the anterior pituitary (Kd = 0.87 +/- 0.31 nM, Bmax = 14.8 +/- 9.0 fmol/mg protein) and in the liver (Kd = 1.66 +/- 0.7 nM, Bmax = 147 +/- 24 fmol/mg protein). These data suggest that the binding sites described in this study are specific receptors through which inositol 1,4,5-trisphosphate mobilizes Ca2+ in target tissues for angiotensin II and other calcium-dependent hormones.     

Interconversion of inositol (1,4,5)-trisphosphate to inositol (1,3,4,5)-tetrakisphosphate and (1,3,4)-trisphosphate in permeabilized adrenal glomerulosa cells is calcium-sensitive and ATP-dependent

The metabolism of [3H]inositol (1,4,5)-trisphosphate was followed in permeabilized bovine adrenal glomerulosa cells. At low Ca++ concentration (pCa = 7.2), more than 90% of [3H]inositol (1,4,5)-trisphosphate had disappeared within 2 min, while two other metabolites, [3H]inositol (1,3,4)-trisphosphate and [3H]inositol (1,3,4,5)-tetrakisphosphate appeared progressively. At higher Ca++ concentrations (pCa = 5.7 and 4.8), the formation of these two metabolites was markedly increased, but completely abolished if the medium was ATP-depleted. The peak levels for the generation of [3H]inositol (1,3,4,5)-tetrakisphosphate (1 min) preceded those of [3H]inositol (1,3,4)-trisphosphate and were closely correlated. These results suggest that, in adrenal glomerulosa cells, the isomer inositol (1,3,4)-trisphosphate is generated from inositol (1,4,5)-trisphosphate via a calcium-sensitive and ATP-dependent phosphorylation/dephosphorylation pathway involving the formation of inositol (1,3,4,5)-tetrakisphosphate.     

Inositol trisphosphate formation and calcium mobilization in Swiss 3T3 cells in response to platelet-derived growth factor.

Swiss 3T3 cells incubated for 60 h with [3H]inositol incorporated radioactivity into phosphatidylinositol (PI) and the two polyphosphoinositides phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2). On stimulation with platelet-derived growth factor (PDGF) there were significant increases in the levels of inositol 1-phosphate (IP1), inositol 1,4-bisphosphate (IP2) and inositol 1,4,5-trisphosphate (IP3). The effect of PDGF and IP3 on Ca2+ mobilization was studied in both intact cells and in 'leaky' cells that had been permeabilized with saponin. In intact cells, PDGF stimulated the efflux of 45Ca2+, whereas IP3 had no effect. Conversely, IP3 stimulated 45Ca2+ efflux from 'leaky' cells, which were insensitive to PDGF. 'Leaky' cells, which accumulated 45Ca2+ to a steady state within 20 min, were found to release approx. 40% of the label within 1 min after addition of 10 microM-IP3. This stimulation of 45Ca2+ release by IP3 was reversible and was also dose-dependent, with a half-maximal effect at approx. 0.3 microM. It seems likely that an important action of PDGF on Swiss 3T3 cells is to stimulate the hydrolysis of PIP2 to form IP3 and diacylglycerol, both of which may function as second messengers. Our results indicate that IP3 mobilizes intracellular Ca2+, and we propose that diacylglycerol may act through C-kinase to activate the Na+/H+ antiport. By generating two second messengers, PDGF can simultaneously elevate the intracellular level of Ca2+ and alkalinize the cytoplasm by lowering the level of H+.     

Thrombin and phorbol ester stimulate inositol 1,3,4,5-tetrakisphosphate 3-phosphomonoesterase in human platelets

Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), which mobilizes intracellular Ca2+, is metabolized either by dephosphorylation to inositol 1,4-bisphosphate(Ins-(1,4)P2) or by phosphorylation to inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4). It has been shown in vitro that Ins(1,3,4,5)P4 is also dephosphorylated by a 5-phosphomonoesterase to inositol 1,3,4-trisphosphate. However, we have found that exogenous Ins(1,3,4,5)P4 is dephosphorylated to predominantly Ins(1,4,5)P3 in saponin-permeabilized platelets in the presence of KCl (40-160 mM). This inositol polyphosphate 3-phosphomonoesterase activity is independent of Ca2+ (0.1-100 microM), and it was also observed when the ionic strength of the incubation medium was increased with Na+. The action of KCl appears to be due to activation of a 3-phosphomonoesterase as well as an inhibition of the 5-phosphomonoesterase, because the dephosphorylation of Ins(1,4,5)P3 to Ins(1,4)P2 was completely inhibited by KCl. The 3-phosphomonoesterase may be regulated by a protein kinase C, since both thrombin and phorbol dibutyrate increase 3-phosphomonoesterase activity and this is inhibited by staurosporine. The formation of Ins(1,4,5)P3 from Ins(1,3,4,5)P4 reported here provides an additional pathway for the formation of the Ca2+-mobilizing second messenger in stimulated cells.     

Breakdown of polyphosphoinositides and not phosphatidylinositol accounts for muscarinic agonist-stimulated inositol phospholipid metabolism in rat parotid glands.

The molecular mechanisms underlying the ability of muscarinic agonists to enhance the metabolism of inositol phospholipids were studied using rat parotid gland slices prelabelled with tracer quantities of [3H]inositol and then washed with 10 mM unlabelled inositol. Carbachol treatment caused rapid and marked increases in the levels of radioactive inositol 1-phosphate, inositol 1,4-bisphosphate, inositol 1,4,5-trisphosphate and an accumulation of label in the free inositol pool. There were much less marked changes in the levels of [3H]phosphatidylinositol, [3H]phosphatidylinositol 4-phosphate and [3H]phosphatidylinositol 4,5-bisphosphate. At 5 s after stimulation with carbachol there were large increases in [3H]inositol 1,4-bisphosphate and [3H]inositol 1,4,5-trisphosphate, but not in [3H]inositol 1-phosphate. After stimulation with carbachol for 10 min the levels of radioactive inositol 1,4-bisphosphate and inositol 1,4,5-trisphosphate greatly exceeded the starting level of radioactivity in phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate respectively. When carbachol treatment was followed by addition of sufficient atropine to block all the muscarinic receptors the radioactive inositol phosphates rapidly returned towards control levels. The carbachol-evoked changes in radioactive inositol phosphate and phospholipid levels were blocked in the presence of 2,4-dinitrophenol (an uncoupler of oxidative phosphorylation). The results suggest that muscarinic agonists stimulate a polyphosphoinositide-specific phospholipase C and that these lipids are continuously replenished from the labelled phosphatidylinositol pool. [3H]Inositol 1-phosphate in the stimulated glands probably arises via hydrolysis of inositol 1,4-bisphosphate and not directly from phosphatidylinositol.     

The inositol trisphosphate phosphomonoesterase of the human erythrocyte membrane.

Human erythrocyte ghosts exhibit an inositol trisphosphate phosphomonoesterase activity that rapidly converts inositol 1,4,5-trisphosphate into inositol 1,4-bisphosphate and Pi. Degradation of the released inositol 1,4-bisphosphate is not observed. This activity is dependent on Mg2+ (or Mn2+) and it is not activated by Ca2+. Optimum activity is around pH 7 and activity is abolished by heat denaturation. The Km for inositol trisphosphate is approx. 25 microM. 2,3-bisphosphoglycerate is a competitive inhibitor, with a Ki of approx. 0.35 mM. Glycerophosphoinositol 4,5-bisphosphate is attacked at about one-eighth of the rate for inositol trisphosphate, but glycerophosphoinositol 4-phosphate is not a substrate. Incubation of 32P-labelled erythrocyte membranes with Mg2+ causes little breakdown of phosphatidylinositol 4,5-bisphosphate, the parent compound from which both glycerophosphoinositol 4,5-bisphosphate and inositol 1,4,5-trisphosphate are derived. On the basis of its substrate specificity and the inhibition by 2,3-bisphosphoglycerate, we suggest that this enzyme is selective for the 5-phosphate in those water-soluble phosphate esters of inositol that possess the vicinal pair of 4,5-phosphates but that it may also interact less strongly with other water-soluble compounds that have pairs of vicinal phosphates.     

Angiotensin II-induced increase in inositol 1,4,5-trisphosphate in cultured rat mesangial cells: evidence by refined high performance liquid chromatography

Angiotensin II-induced change in inositol phosphates were studied in cultured rat mesangial cells prelabeled with [3H]myo-inositol. By using anion-exchange high performance liquid chromatography, we could analyzed the change in inositol mono-, bis-, and tris-phosphate more rapidly and easily with higher resolution than the previously reported methods. Angiotensin II rapidly increased inositol 1,4,5-trisphosphate and inositol 1,4-bisphosphate within 15 sec, followed by an increase in inositol 1-monophosphate at 30 sec. Angiotensin II-induced increases in inositol phosphates were dose-dependent and completely blocked by saralasin. These results indicate that angiotensin II induces the production of inositol phosphates including inositol 1,4,5-trisphosphate, an intracellular Ca2+-releasing factor, in cultured rat mesangial cells.     

Separation of multiple isomers of inositol phosphates formed in GH3 cells.

A high-performance-liquid-chromatography (h.p.l.c.) separation was developed, which resolves isomers of inositol monophosphate (IP), inositol bisphosphate (IP2), and inositol trisphosphate (IP3) in a single run. In GH3 cells labelled with [3H]inositol, treated with Li+ and thyrotropin-releasing hormone (TRH), radiolabelled components identified as inositol 1-phosphate (I1P), inositol 2-phosphate (I2P), inositol 4-phosphate (I4P), inositol 1,4-bisphosphate [I(1,4)P2], inositol 1,3,4-trisphosphate [I(1,3,4)P3] and inositol 1,4,5-trisphosphate [I(1,4,5)P3] are present, as are multiple unidentified IP2 peaks. After TRH stimulation, both I1P and I4P increase, the increase in I4P preceding that of I1P; I(1,4)P2 and an unknown IP2 increase; and both I(1,3,4)P3 and I(1,4,5)P3 increase, the increase in I(1,4,5)P3 being rapid and transient, whereas the increase in I(1,3,4)P3 is slower and more sustained. The most rapidly appearing inositol phosphates produced after TRH stimulation are I(1,4)P2 and I(1,4,5)P3.     

2-Aminoethoxydiphenyl borate affects the inositol 1,4,5-trisphosphate receptor, the intracellular Ca2+ pump and the non-specific Ca2+ leak from the non-mitochondrial Ca2+ stores in permeabilized A7r5 cells

2-Aminoethoxydiphenyl borate (2APB) is a membrane-permeable blocker of the inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release in bi-directional Ca2+ -flux conditions. We have now studied the effects of 2APB on the 45Ca2+ uptake into, and on the basal and IP(3)-stimulated unidirectional 45Ca2+ efflux from the non-mitochondrial Ca2+ stores in permeabilized A7r5 smooth-muscle cells. 2APB inhibited the IP3 -induced Ca2+ release, with a half maximal inhibition at 36 microM 2APB, without affecting [3H]IP3 binding to the receptor. This inhibition did not depend on the IP3, ATP or free Ca2+ concentration. The Ca2+ pumps of the non-mitochondrial Ca2+ stores were half-maximally inhibited at 91microM 2APB. Higher concentrations of 2APB increased the non-specific leak of Ca2+ from the stores. We conclude that 2APB can not be considered as a selective blocker of the IP3 -induced Ca2+ release. Our results can explain the various effects of 2APB observed in intact cells.     

Separation of inositol phosphates and glycerophosphoinositol phosphates by high-performance liquid chromatography

We developed a HPLC method which separates the following nine inositol-containing compounds of biological interest: inositol, inositol 1-monophosphate, inositol 2- or 4-monophosphate, inositol 1,2-cyclic phosphate, inositol 1,4-bisphosphate, inositol 1,4,5-trisphosphate, glycerophosphoinositol, glycerophosphoinositol 4-monophosphate, and glycerophosphoinositol 4,5-bisphosphate. The method shows good resolution and sufficient recovery (70-80%) for each compound. By applying this method to human platelets prelabeled with [3H]inositol and stimulated with thrombin, we found an early increase of inositol 1,4-bisphosphate and inositol 1,4,5-trisphosphate. Accumulation of glycerophosphoinositol, inositol 1-monophosphate, and an inositol monophosphate which cochromatographs with inositol 2- and inositol 4-monophosphate occurs later. The method is simple, and--after removal of salts from the incubation buffer--can be directly applied to the measurement of aqueous soluble [3H]inositol-labeled compounds in biological samples.     

Recovery of hepatocytes from attack by the pore former amphotericin B.

Factors underlying the transience of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] accumulation following muscarinic stimulation of RINm5F cells were examined. Transience was not due to a protein kinase C-mediated stimulation of Ins(1,4,5)P3 dephosphorylation, since pretreatment of cells with tetradecanoyl-phorbol acetate (TPA) did not alter the rate of this conversion. However, preincubation with TPA did inhibit carbamoylcholine-stimulated Ins(1,4,5)P3 formation. In permeabilized cells, the conversion of Ins(1,4,5)P3 to inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] was slightly enhanced in the presence of TPA or cyclic AMP, but much more markedly by raising the Ca2+ concentration from 10(-7) M to 10(-6) or 10(-5) M. In intact cells the most rapid rate of accumulation of Ins(1,4,5)P3 and Ins(1,3,4,5)P4 occurred in the first 2 s following stimulation, whereas the levels of inositol 1,4-bisphosphate were not increased until after 5 s. This suggests that Ins(1,4,5)P3 kinase is chiefly responsible for the early disposal of Ins(1,4,5)P3 following cellular stimulation. The results are consistent with the proposal that the transient accumulation of Ins(1,4,5)P3 is due both to its enhanced metabolism via the Ca2+-calmodulin-sensitive Ins(1,4,5)P3 kinase, as well as a down-regulation of phosphatidylinositol 4,5-bisphosphate hydrolysis.     

Modulation of agonist-induced inositol phosphate metabolism by cyclic adenosine 3',5'-monophosphate in adrenal glomerulosa cells

Activation of the cAMP messenger system was found to cause specific changes in angiotensin-II (All)-induced inositol phosphate production and metabolism in bovine adrenal glomerulosa cells. Pretreatment of [3H]inositol-labeled glomerulosa cells with 8-bromo-cAMP (8Br-cAMP) caused both short and long term changes in the inositol phosphate response to stimulation by All. Exposure to 8Br-cAMP initially caused dose-dependent enhancement (ED50 = 0.7 microM) of the stimulatory action of All (50 nM; 10 min) on the formation of D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and its immediate metabolites. This effect of 8Br-cAMP was also observed in permeabilized [3H]inositol-labeled glomerulosa cells in which degradation of Ins(1,4,5)P3 was inhibited, consistent with increased activity of phospholipase-C. Continued exposure to 8Br-cAMP for 5-16 h caused selective enhancement of the All-induced increases in D-myo-inositol 1,3,4,6-tetrakisphosphate [Ins(1,3,4,6)P4] and myo-inositol 1,4,5,6-tetrakisphosphate. The long term effect of 8Br-cAMP on the 6-phosphorylated InsP4 isomers, but not the initial enhancement of Ins(1,4,5)P3 formation, was inhibited by cycloheximide. The characteristic biphasic kinetics of All-induced Ins(1,4,5)P3 formation were also changed by prolonged treatment with 8Br-cAMP to a monophasic response in which Ins(1,4,5)P3 increased rapidly and remained elevated during All stimulation. In permeabilized glomerulosa cells treated with 8Br-cAMP for 16 h, the conversion of D-myo-inositol 1,3,4-trisphosphate [Ins(1,3,4)P3] to Ins(1,3,4,6)P4 was consistently increased, whereas dephosphorylation of Ins(1,4,5)P3 to D-myo-inositol 1,4-bisphosphate and of D-myo-inositol 1,3,4,5-tetrakisphosphate to Ins(1,3,4)P3, was reduced.(ABSTRACT TRUNCATED AT 250 WORDS)