The effects of fatty acids on phosphoinositide synthesis and myo-inositol accumulation in exocrine pancreas

The effects of arachidonic acid (20:4) on phosphoinositide turnover were examined in rat pancreatic acinar cells prelabeled with myo-[3H]inositol. Arachidonic acid (50 microM) increased the accumulation of myo-[3H]inositol, but not that of [3H]inositol monophosphate, [3H]inositol bisphosphate, or [3H]inositol trisphosphate. By contrast, 10 microM carbamoylcholine increased the accumulation of all four compounds. A combination of arachidonic acid plus carbamoylcholine caused a selective and marked accumulation of myo-[3H]inositol, which was abolished by 10 mM LiCl. Arachidonic acid (10-100 microM) produced a concentration-dependent inhibition of myo-[3H]inositol incorporation into phosphoinositides and markedly depressed carbamoylcholine-induced increases in myo-[3H]inositol incorporation into inositol phospholipids. Several other unsaturated and saturated fatty acids failed to elicit a synergistic response with carbamoylcholine in stimulating myo-[3H]inositol accumulation and did not retard the incorporation of myo-[3H]inositol into phosphoinositides. The fact that eicosapentaenoic acid (20:5), but not arachidic acid (20:0), mimicked the depressant effect of arachidonate on phosphoinositide labeling suggests that the degree of unsaturation of the fatty acid, rather than chain length, is important for inhibition of phosphoinositide synthesis. The arachidonate-induced decrease in myo-[3H]inositol incorporation was accompanied by a reduction in the steady state level of [32P]phosphatidylinositol 4,5-bisphosphate. The mass of arachidonic acid liberated in response to carbamoylcholine was measured by gas chromatography-mass spectrometry, and the time course of stimulated arachidonate accumulation paralleled that of inositol phosphate accumulation and amylase release. These observations suggest that in exocrine pancreas, endogenous arachidonic acid serves as a negative feedback regulator of phosphoinositide turnover.     

Serotonin-induced alterations in inositol phospholipid metabolism in human platelets

When human platelets were incubated for 5 min with [32P]orthophosphate and then stimulated with serotonin, the 32P content of phosphatidylinositol (PI) increased within seconds, compared with the control. The 32P content of phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) only slightly increased during the first minute after addition of serotonin and became more apparent on prolonged stimulation. These changes were not caused by serotonin-induced change in the specific activity of ATP. Using inorganic phosphate determination for the chemical quantification of different inositol phospholipid pools, we found that the platelet PI content remained nearly constant; the amount of PIP increased while that of PIP2 decreased. When the platelets were first prelabeled for 80 min with [32P]orthophosphate, the changes in 32P-labeled inositol phospholipids after addition of serotonin were similar to their changes in mass. When the platelet inositol phospholipids were labeled with myo-[2-3H]inositol, serotonin induced an increase in [3H]inositol phosphates. From these data, it is concluded in addition to the earlier-reported effects on phospholipid metabolism (de Chaffoy de Courcelles, D. et al. (1985) J. Biol. Chem. 260, 7603-7608) that serotonin induces: a very rapid formation of PI; and alterations in inositol phospholipid interconversion that cannot be explained solely as a resynthesis process of PIP2.     

Thyrotropin-releasing hormone rapidly activates the phosphodiester hydrolysis of polyphosphoinositides in GH3 pituitary cells. Evidence for the role of a polyphosphoinositide-specific phospholipase C in hormone action

The early actions of thyrotropin-releasing hormone (TRH) have been studied in hormone-responsive clonal GH3 rat pituitary cells. Previous studies had demonstrated that TRH promotes a "phosphatidylinositol response" in which increased incorporation of [32P]orthophosphate into phosphatidylinositol and phosphatidic acid was observed within minutes of hormone addition. The studies described here were designed to establish whether increased labeling of phosphatidylinositol and phosphatidic acid resulted from prior hormone-induced breakdown of an inositol phosphatide. GH3 cells were prelabeled with [32P]orthophosphate or myo-[3H]inositol. Addition of TRH resulted in the rapid disappearance of labeled polyphosphoinositides, whereas levels of phosphatidylinositol and other phospholipids remained unchanged. TRH-promoted polyphosphoinositide breakdown was evident by 5 S and maximal by 15 s of hormone treatment. Concomitant appearance of inositol polyphosphates in [3H]inositol-labeled cells was observed. In addition, TRH rapidly stimulated diacylglycerol accumulation in either [3H]arachidonic- or [3H]oleic acid-labeled cultures. These results indicate that TRH rapidly causes activation of a polyphosphoinositide-hydrolyzing phospholipase C-type enzyme. The short latency of this hormone effect suggests a proximal role for polyphosphoinositide breakdown in the sequence of events by which TRH alters pituitary cell function.     

Differential effect of progesterone on the labeling of phosphatidylinositol with [3H]inositol and [32P]phosphate in the uterus of the estrogen-treated ovariectomized rat

In rat uterine mince incubated in vitro [3H]inositol was found to be incorporated into phosphatidylinositol (PI) predominantly via a pathway which could be markedly and dose dependently activated with Mn2+ (0.1-10 mM) and inhibited by Ca2+ (1-10 mM). These ions had no effect on the incorporation of [32P]phosphate (32P) into PI indicating a distinct inositol-exchange mechanism for the labeling of PI with [3H]inositol. Treatment of ovariectomized rats for 5 days with 2 micrograms estradiol dipropionate (EDP) increased about 3-fold (when measured in the presence of 1 mM Mn2+) and 4-5-fold (when measured in the presence of 1 mM Ca2+) the inositol-exchange activity in the rat uterus, and these effects were suppressed by 40 and 30% respectively by the concomitant administration of 2 mg progesterone (P). EDP alone or in combination with P increased to the same extent (by a factor of 2-3) the rate of labeling with 32P of phosphatidylcholine (PC), phosphatidylethanolamine (PE) and plasmenylethanolamine (PmE). The labeling rate of PI was increased 1.5-1.7-fold by treatment with EDP and this increase was selectively augmented further to about 2.5-fold by the simultaneous administration of P. Treatment with P alone had no significant effect on the incorporation of either labeled precursor. Steroid hormone treatments had no effect on the amount of these phospholipids in 100 mg uterine tissue, but they increased about 1.7-fold the rate of labeling of ATP with 32P. We conclude that P, when administered together with estradiol, regulates differentially the turnover of the inositol and phosphate moieties of PI with possible physiological consequences.     

Carbachol causes rapid phosphodiesteratic cleavage of phosphatidylinositol 4,5-bisphosphate and accumulation of inositol phosphates in rabbit iris smooth muscle; prazosin inhibits noradrenaline- and ionophore A23187-stimulated accumulation of inositol phosphates.

Rabbit iris smooth muscle was prelabelled with myo-[3H]inositol for 90 min and the effect of carbachol on the accumulation of inositol phosphates from phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], phosphatidylinositol 4-phosphate (PtdIns4P) and phosphatidylinositol (PtdIns) was monitored with anion-exchange chromatography. Carbachol stimulated the accumulation of inositol phosphates and this was blocked by atropine, a muscarinic antagonist, and it was unaffected by 2-deoxyglucose. The data presented demonstrate that, in the iris, carbachol (50 microM) stimulates the rapid breakdown of PtdIns(4,5)P2 into [3H]inositol trisphosphate (InsP3) and diacylglycerol, measured as phosphatidate, and that the accumulation of InsP3 precedes that of [3H]inositol bisphosphate (InsP2) and [3H]inositol phosphate (InsP). This conclusion is based on the following findings. Time course experiments with myo-[3H]inositol revealed that carbachol increased the accumulation of InsP3 by 12% in 15s and by 23% in 30s; in contrast, a significant increase in InsP release was not observed until about 2 min. Time-course experiments with 32P revealed a 10% loss of radioactivity from PtdIns(4,5)P2 and a corresponding 10% increase in phosphatidate labelling by carbachol in 15s; in contrast a significant increase in PtdIns labelling occurred in 5 min. Dose-response studies revealed that 5 microM-carbachol significantly increased (16%) the accumulation of InsP3 whereas a significant increase in accumulation of InsP2 and InsP was observed only at agonist concentrations greater than 10 microM. Studies on the involvement of Ca2+ in the agonist-stimulated breakdown of PtdIns(4,5)P2 in the iris revealed the following. Marked stimulation (58-78%) of inositol phosphates accumulation by carbachol in 10 min was observed in the absence of extracellular Ca2+. Like the stimulatory effect of noradrenaline, the ionophore A23187-stimulated accumulation of InsP3 was inhibited by prazosin, an alpha 1-adrenergic blocker, thus suggesting that the ionophore stimulation of PtdIns(4,5)P2 breakdown we reported previously [Akhtar & Abdel-Latif (1978) J. Pharmacol. Exp. Ther. 204, 655-688; Akhtar & Abdel-Latif (1980) Biochem. J. 192, 783-791] was secondary to the release of noradrenaline by the ionophore. The carbachol-stimulated accumulation of inositol phosphates was inhibited by EGTA (0.25 mM) and this inhibition was reversed by excess Ca2+ (1.5 mM), suggesting that EGTA treatment of the tissue chelates extracellular Ca2+ required for polyphosphoinositide phosphodiesterase activity. K+ depolarization, which causes influx of extracellular Ca2+ in smooth muscle, did not change the level of InsP3.(ABSTRACT TRUNCATED AT 400 WORDS)     

Platelet-activating factor stimulates metabolism of phosphoinositides via phospholipase A2 in primary cultured rat hepatocytes

Addition of platelet-activating factor (PAF) to cells doubly labeled with [14C]glycerol plus [3H]arachidonic acid resulted in a transient decrease of [14C]glycerol-labeled phosphatidylinositol (PI) and a transient increase of [14C]glycerol-labeled lysophosphatidylinositol (LPI). [3H]Arachidonate-labeled PI, on the other hand, decreased in a time-dependent manner. The radioactivity in phosphatidylethanolamine, phosphatidylcholine, sphingomyelin, and phosphatidylserine did not change significantly. The 3H/14C ratio decreased in PI in a time-dependent manner, suggesting the involvement of a phospholipase A2 activity. Although PAF also induced a gradual increase of diacylglycerol (DG), the increase of [14C]glycerol-labeled DG paralleled the loss of triacyl [14C]glycerol and the 3H/14C ratio of DG was 16 times smaller than that of PI. Thus, DG seemed not to be derived from PI. In myo- [3H]inositol-prelabeled cells, PAF induced a transient decrease of [3H]phosphatidylinositol-4,5-bis-phosphate (TPI) and [3H]phosphatidylinositol-4-phosphate (DPI) at 1 min. PAF stimulation of cultured hepatocytes prelabeled with 32Pi induced a transient decrease of [32P]polyphosphoinositides at 20 sec to 1 min. [32P]LPI appeared within 10 sec after stimulation and paralleled the loss of [32P]PI. [3H]Inositol triphosphate, [3H]inositol diphosphate, and [3H]inositol phosphate, which increased in a time-dependent manner upon stimulation with adrenaline, did not accumulate with the stimulation due to PAF. These observations indicate that PAF causes degradation of inositol phospholipids via phospholipase A2 and induces a subsequent resynthesis of these phospholipids.     

Lithium inhibits muscarinic-receptor-stimulated inositol tetrakisphosphate accumulation in rat cerebral cortex.

The effects of Li+ on carbachol-stimulated phosphoinositide metabolism were examined in rat cerebral-cortex slices labelled with myo-[2-3H]inositol. The muscarinic agonist carbachol evoked an enhanced steady-state accumulation of [3H]inositol monophosphate ([3H]InsP1), [3H]inositol bisphosphate ([3H]InsP2), [3H]inositol 1,3,4-trisphosphate ([3H]Ins(1,3,4)P3), [3H]inositol 1,4,5-trisphosphate ([3H]Ins(1,4,5)P3) and [3H]inositol tetrakisphosphate ([3H]InsP4). Li+ (5 mM), after a 10 min lag, severely attenuated carbachol-stimulated [3H]InsP4 accumulation while simultaneously potentiating accumulation of both [3H]InsP1 and [3H]InsP2 and, at least initially, of [3H]Ins(1,3,4)P3. These data are consistent with inhibition of inositol mono-, bis- and 1,3,4-tris-phosphate phosphatases to different degrees by Li+ in brain, but are not considered to be completely accounted for in this way. Potential direct and indirect mechanisms of the inhibitory action of Li+ on [3H]InsP4 accumulation are considered. The present results stress the complex action of Li+ on cerebral inositol metabolism and indicate that more complex mechanisms than are yet evident may regulate this process.     

Increased phospholipid synthesis in the stimulated rat and human pancreas

Stimulation of the exocrine pancreas is associated with marked changes in pancreatic phospholipid metabolism. It has been previously established that de novo synthesis of phospholipids constitutes part of this "phospholipid effect". This study has demonstrated that in vitro stimulation of the rat pancreas utilising bethanecol and pancreozymin results in increased incorporation of labelled glucose into phosphatidyl inositol and, to a lesser extent, other phospholipids, suggesting increased de novo synthesis of these compounds. However, secretin which is believed to act via a different intracellular pathway, did not exert such an effect. The relevance of this animal model is indicated by the demonstration of increased incorporation of labelled glucose into phospholipids of human pancreas stimulated in vitro by bethanecol or sincalide (the active carboxy terminal octapeptide of pancreozymin).     

An analysis of myo-[3H]inositol trisphosphates found in myo-[3H]inositol prelabelled avian erythrocytes.

Evidence is presented to show that acid extracts of avian erythrocytes prelabelled for 24-48 h with myo-[3H]inositol contain the following myo-[3H]inositol trisphosphates (expressed as a percentage of total myo-[3H]inositol trisphosphates extracted): 36% myo-[3H]inositol 1,4,5-trisphosphate; 33.7% myo-[3H]inositol 1,3,4-trisphosphate; 13% myo-[3H]inositol 3,4,5-trisphosphate; 9.7% myo-[3H]inositol 3,4,6-trisphosphate; 4.4% myo-[3H]inositol 1,4,6-trisphosphate and 3.3% myo-[3H]inositol 1,3,6-trisphosphate. The only phosphatidyl-myo-[3H]inositol bisphosphate that could be detected in [3H]Ins-prelabelled avian erythrocytes was phosphatidyl-myo-[3H]inositol 4,5-bisphosphate. Cellular myo-[3H]inositol 3,4,5-trisphosphate may be synthesized by dephosphorylation of myo-[3H]inositol 3,4,5,6-tetrakisphosphate. D- and L-myo-[3H]inositol 1,4,6-trisphosphate and D- and L-myo-[3H]inositol 1,3,6-trisphosphate may be dephosphorylation products of myo-[3H]inositol 1,3,4,6-tetrakisphosphate.     

Phytohemagglutinin induces rapid degradation of phosphatidylinositol 4,5-bisphosphate and transient accumulation of phosphatidic acid and diacylglycerol in a human T lymphoblastoid cell line, CCRF-CEM

The human T lymphoblastoid cell line designated CCRF-CEM responds to phytohemagglutinin with a 3.7-fold enhancement of the 32PO4 incorporation into phosphatidylinositol. In myo-[2-3H]inositol-prelabeled CCRF-CEM cells, phytohemagglutinin induced a 3.3-fold accumulation of myo-[2-3H]inositol phosphate during 15 min incubation at 37 degrees C in the presence of 5 mM LiCl. Since Li+ is a potent inhibitor of myo-inositol-1-phosphatase, the results indicate that phytohemagglutinin induces the hydrolysis of inositol lipids in CCRF-CEM cells. In 32PO4-prelabeled CCRF-CEM cells, phytohemagglutinin induced a breakdown of 28% of [32P]phosphatidylinositol 4,5-bisphosphate 40-60 s after the stimulation. The decrease of [32P]phosphatidylinositol 4,5-bisphosphate was found as early as 10 s after the stimulation. This decrease was followed by an increased 32P-labeling of phosphatidic acid. In [2-3H]glycerol-prelabeled CCRF-CEM cells, phytohemagglutinin induced a transient accumulation of [3H]phosphatidic acid and [3H]diacylglycerol. The amount of [3H]phosphatidic acid in the stimulated cells was 3.7-times the control value at 2 min after the stimulation, whereas the amount of [3H]diacylglycerol in the stimulated cells was 1.5-times the control value at 5 min after the stimulation. In [3H8]arachidonate-prelabeled CCRF-CEM cells, phytohemagglutinin induced a transient accumulation of [3H]phosphatidic acid; the amount was 2.5-times the control value at 2 min after the stimulation. Quinacrine (1 mM) caused 41% reduction in the amount of [3H]phosphatidic acid accumulated by the stimulation in [2-3H]glycerol-prelabeled cells. Stimulation in a Ca2+-free saline containing 1 mM EGTA caused 53% reduction in the amount of [3H]phosphatidic acid accumulated by the stimulation. The results presented in this paper indicate that a human T lymphoblastoid cell line, CCRF-CEM, responds to phytohemagglutinin with a rapid turnover of inositol lipids.     

Manganese Stimulates the Incorporation of [3H]Inositol into a Pool of Phosphatidylinositol in Brain That Is Not Coupled to Agonist-Induced Hydrolysis

Mn2+ greatly increases the incorporation of myo-[3H]inositol into phosphatidylinositol (PI) of brain and other tissues by stimulating the activity of a PI-myo-inositol exchange enzyme. This study examined the ability of norepinephrine (NE) and carbachol to stimulate the hydrolysis of [3H]PI formed in the absence and presence of Mn2+-stimulated [3H]inositol exchange. Rat cerebral cortical slices were incubated with myo-[3H]inositol for 60 min in an N-2-hydroxyethyl piperazine-N'-2-ethanesulfonic acid (HEPES) buffer without or with MnCl2 (1 mM). The tissue was washed and further incubated with unlabeled myo-inositol and LiCl (10 mM). Prelabeled slices were then incubated with NE (0.1 mM) or carbachol (1 mM) to induce agonist-stimulated [3H]PI hydrolysis. Mn2+ treatment resulted in eight- and sixfold increases in control levels of [3H]PI and [3H]inositol monophosphate [( 3H]IP), respectively. Both NE and carbachol stimulated [3H]IP formation in tissue prelabeled without or with manganese. However, the degree of stimulation (percentage of control values) was greatly attenuated in the presence of Mn2+. In the absence of Mn2+ treatment, NE decreased [3H]PI radioactivity in the tissue to 80% of control values. However, NE did not decrease [3H]PI radioactivity in the Mn2+-treated tissue. These data demonstrate that Mn2+ stimulates incorporation of myo-[3H]inositol into a pool of PI in brain that has a rapid turnover but is not coupled to agonist-induced hydrolysis.     

The incorporation of myo-inositol into phosphatidylinositol derivatives is stimulated during hormone-induced meiotic maturation of amphibian oocytes

The incorporation of myo-[3H]inositol into phosphatidylinositol and its phosphorylated derivatives was studied by microinjection of the radioactive precursor into Xenopus laevis oocytes. Induction of meiotic maturation of the oocytes by treatment with either progesterone one or insulin resulted in a significant increase in the incorporation of myo-[3H]inositol into the phospholipid fraction. This increase occurred 3-6 h after hormonal treatment, a time coincident with the start of the breakdown of the nuclear envelope, and requires protein synthesis. The effect of progesterone and insulin contrasts with the effect of acetylcholine, which acts through a muscarinic receptor causing the activation of phospholipase C, since the latter effector causes an increase in myo-[3H]inositol incorporation, which is more rapid and does not require protein synthesis. These results suggest that the meiotic maturation process is connected with changes in inositol metabolism in the amphibian oocyte.     

Effects of carbachol and pancreozymin (cholecystokinin-octapeptide) on polyphosphoinositide metabolism in the rat pancreas in vitro.

We studied the possibility that hydrolysis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] may be the initiating event for the increase in [32P]Pi incorporation into phosphatidic acid (PtdA) and phosphatidylinositol (PtdIns) during carbachol and pancreozymin (cholecystokinin-octapeptide) action in the rat pancreas. After prelabelling acini for 2h, [32P]Pi incorporation into PtdA, PtdIns(4,5)P2 and phosphatidylinositol 4-phosphate (PtdIns4P) had reached equilibrium. Subsequent addition of carbachol or pancreozymin caused 32P in PtdIns(4,5)P2 to decrease by 30-50% within 10-15 s, and this was followed by sequential increases in [32P]Pi incorporation into PtdA and PtdIns. Similar changes in 32P-labelling of PtdIns4P were not consistently observed. Confirmation that the decrease in 32P in chromatographically-purified PtdIns(4,5)P2 reflected an actual decrease in this substance was provided by the fact that similar results were obtained (a) when PtdIns(4,5)P2 was prelabelled with [2-3H]inositol, and (b) when PtdIns(4,5)P2 was measured as its specific product (glycerophosphoinositol bisphosphate) after methanolic alkaline hydrolysis and ion-exchange chromatography. The secretogogue-induced breakdown of PtdIns(4,5)P2 was not inhibited by Ca2+ deficiency (severe enough to inhibit amylase secretion and Ca2+-dependent hydrolysis of PtdIns), and ionophore A23187 treatment did not provoke PtdIns(4,5)P2 hydrolysis. The increase in the hydrolysis of PtdIns(4,5)P2 and the increase in [32P]Pi incorporation into PtdA commenced at the same concentration of carbachol in dose-response studies. Our findings suggest that the hydrolysis of PtdIns(4,5)P2 is an early event in the action of pancreatic secretogogues that mobilize Ca2+, and it is possible that this hydrolysis may initiate the Ca2+-independent labelling of PtdA and PtdIns. Ca2+ mobilization may follow these responses, and subsequently cause Ca2+-dependent hydrolysis of PtdIns and exocytosis.     

Relationship between hormonal activation of phosphatidylinositol hydrolysis, fluid secretion and calcium flux in the blowfly salivary gland.

The addition of 5-hydroxytryptamine to the isolated blowfly salivary gland stimulates fluid secretion, transepithelial calcium transport and the breakdown of 32P- or 3H-labelled phosphatidylinositol The breakdown of [32P]phosphatidylcholine and [32P]-phosphatidylethanolamine was not stimulated by 5-hydroxytryptamine. In salivary glands incubated with myo-[2-3H]inositol for 1--3 h, more than 95% of the label retained by the tissue was in the form of phosphatidylinositol. The addition of 5-hydroxytryptamine resulted in an increase in the accumulation of label in intracellular inositol 1:2-cyclic phosphate, inositol 1-phosphate and free inositol along with an increase in the release of [3H]inositol to the medium and saliva. The release of [3H]inositol to the medium served as a sensitive indicator of phosphatidylinositol breakdown. The release of [3H]inositol was not increased by cyclic AMP or the bivalent-cation ionophore A23187 under conditions in which salivary secretion was accelerated. The stimulation of fluid secretion by low concentrations of 5-hydroxytryptamine was potentiated by 3-isobutyl-1-methylxanthine, which had no effect on inositol release. The stimulation of fluid secretion by 5-hydroxytryptamine was greatly reduced in calcium-free buffer, but the breakdown of phosphatidylinositol continued at the same rate in the absence of calcium. These results support the hypothesis that breakdown of phosphatidylinositol by 5-hydroxytryptamine is involved in the gating of calcium.     

L-myo-inositol 1,4,5,6-tetrakisphosphate (3-hydroxy)kinase.

Homogenates of primary-cultured murine bone macrophages contain an enzyme capable of synthesizing myo-[3H]inositol pentakisphosphate from myo-[3H]inositol tetrakisphosphate fractions derived from myo-[3H]inositol-labelled mouse macrophages and chick erythrocytes. D-myo-inositol 1,3,4,5-tetrakis[32P]-phosphate present in the same incubations was not phosphorylated. Since the myo-[3H]inositol-labelled tetrakisphosphate fractions used as substrates consist of a mixture of L-myo-inositol 1,4,5,6-tetrakisphosphate (60-85%) and a periodate-resistant tetrakisphosphate(s) whose characteristics are consistent with those of D-myo-inositol 1,3,4,5-tetrakisphosphate (the preceding paper [Stephens, Hawkins, Carter, Chahwala, Morris, Whetton & Downes (1988) Biochem. J. 249, 271-282] ), these data suggest the existence of a kinase that phosphorylates L-myo-inositol 1,4,5,6-tetrakisphosphate to give a myo-inositol pentakisphosphate. A similar activity was identified in homogenates of rat cerebrum, liver, heart and parotid gland. D-myo-Inositol 1,3,4,5-tetrakis[32P]phosphate in the same incubations was not a substrate. The activity was almost entirely soluble in all the tissues investigated and was found at its greatest specific activity in brain cytosol. The activity was purified 120-fold from a rat brain homogenate by (NH4)2SO4 fractionation and anion-exchange chromatography. The activity was clearly distinct from D-myo-inositol 1,4,5-trisphosphate (3-hydroxy)kinase. Incubation of this partially purified preparation with L-myo-[3H]inositol 1,4,5,6-tetrakisphosphate from chick erythrocytes and [gamma-32P]ATP resulted in the formation of L-myo-[3H]-inositol [1-32P]1,3,4,5,6-pentakisphosphate. The enzyme is therefore identified as an L-myo-inositol 1,4,5,6-tetrakisphosphate (3-hydroxy)kinase.     

Sympathetic denervation impairs agonist-stimulated phosphatidylinositol metabolism in rat parotid glands.

Substance P, muscarinic and alpha-adrenoceptor agonists stimulated the incorporation of [3H]inositol into phosphatidylinositol in rat parotid gland slices. Surgical denervation of the sympathetic input to the rat parotid gland by superior cervical ganglionectomy produced marked reductions in these responses. The stimulated incorporation of radiolabelled precursors into phosphatidylinositol is a measure of its resynthesis after receptor-mediated breakdown of inositol phospholipids. We therefore examined the enzymic site of the lesion induced by sympathetic denervation using parotid gland slices labelled with either [3H]inositol or [32P]phosphate and stimulated with substance P. Receptor-activated phospholipase C attack upon [3H]inositol phospholipids was assayed by measuring the formation of [3H]inositol 1-phosphate in the presence of 10 mM-Li+ to inhibit further breakdown. It was not affected by denervation. Substance P elicited a rapid breakdown of phosphatidylinositol 4,5-bisphosphate and this response was reduced in the denervated gland. The second step in stimulated phosphatidylinositol turnover, phosphorylation of diacylglycerol to phosphatidate was not affected by denervation. Sympathetic denervation appears to induce a specific enzymic lesion in the parotid gland that impairs receptor-stimulated resynthesis of phosphatidylinositol from phosphatidate. This change in membrane lipid metabolism may be related to a number of the effects of sympathetic denervation, such as agonist supersensitivity, reduced gland cell proliferation and induction of new surface receptors.     

The conditions under which rat islets are labelled with [3H]inositol alter the subsequent responses of these islets to a high glucose concentration.

Isolated rat islets were incubated with myo-[2-3H]inositol for 2 h to label their phosphoinositide (PI) pools. Labelling was carried out under three separate conditions: in media containing low (2.75 mM) glucose, high (13.75 mM) glucose, or low (2.75 mM) glucose plus sulphated cholecystokinin (CCK-8S; 200 nM). After labelling, the islets were perifused and the insulin-secretory response to 20 mM-glucose was measured. PI hydrolysis in these same islets was assessed by measurements of both [3H]inositol efflux and the accumulation of labelled inositol phosphates. The following major observations were made. After prelabelling for 2 h in low glucose, perifusion with 20 mM-glucose resulted in a biphasic insulin-secretory response, an increase in [3H]inositol efflux and a parallel increase in the accumulation of labelled inositol phosphates. After prelabelling in high (13.75 mM) glucose, peak first-phase insulin secretion induced by 20 mM-glucose increased 2-2.5-fold, whereas the second phase of insulin release, as well as [3H]inositol efflux and inositol phosphate accumulation, were significantly decreased. The simultaneous infusion of the diacylglycerol kinase inhibitor 1-mono-oleoylglycerol (50 microM), along with 20 mM-glucose, restored the second-phase insulin-secretory response from these islets. After labelling in low (2.75 mM) glucose plus CCK-8S, the initial phases of the insulin-secretory and [3H]inositol-efflux responses to 20 mM-glucose were blunted and the sustained phases of both responses were markedly decreased. Inositol phosphate accumulation was also impaired. Labelling islets in high (13.75 mM) glucose or low (2.75 mM) glucose plus CCK-8S suppresses, in a parallel fashion, glucose-induced increases in PI hydrolysis and in second-phase insulin release. These findings suggest that desensitization of the insulin-secretory response is a consequence of impaired information flow in the inositol lipid cycle.     

Characterization of agonist-stimulated incorporation of myo-[3H]inositol into inositol phospholipids and [3H]inositol phosphate formation in tracheal smooth muscle.

The effects of the muscarinic agonist carbachol, histamine and bradykinin on incorporation of [3H]inositol into the phosphoinositides and the formation of [3H]InsPs were examined in bovine tracheal smooth-muscle (BTSM) slices labelled with [3H]inositol. These agonists result in substantial and dose-related increases in the incorporation of [3H]inositol into the phospholipids. Carbachol and histamine stimulated the incorporation of [3H]inositol into the phospholipids to the same degree, despite histamine being only 35% as effective as carbachol on [3H]InsP accumulation. Histamine and carbachol, at maximal concentrations, were non-additive with respect to both the stimulated incorporation of [3H]inositol and [3H]InsP formation. For carbachol this effect on incorporation was found to occur to a similar extent in PtdInsP and PtdInsP2 as well as PtdIns. The initial effect of carbachol on [3H]inositol incorporation was rapid (maximal by 10 min); however, with prolonged stimulation large secondary declines in PtdInsP and PtdInsP2 labelling were observed, with depletion of the much larger PtdIns pool only evident in the presence of Li+. Lowering buffer [Ca2+] increased the incorporation of [3H]inositol under basal conditions, but did not attenuate the subsequent agonist-stimulated incorporation effect. The large changes in specific radioactivity of the phosphoinositides, and consequently the [3H]InsP products, after carbachol stimulation resulted in the apparent failure of atropine to reverse the [3H]InsP response completely. Labelling muscle slices with [3H]inositol in the presence of carbachol or labelling for longer periods (greater than 6 h) prevented subsequent carbachol-stimulated effects on incorporation without significantly altering the dose-response relationship for carbachol-stimulated [3H]InsP formation and resulted in steady-state labelling conditions confirmed by the ability of atropine to reverse fully the [3H]InsP response to carbachol. This study demonstrates the profound effects of a number of agonists on [3H]inositol incorporation into the phospho- and polyphosphoinositides in BTSM with important consequent changes in the specific radioactivity of these lipids and the resulting [3H]InsP products. In addition, a selective depletion of PtdInsP and PtdInsP2 over PtdIns has been demonstrated with prolonged muscarinic-receptor stimulation.     

Rapid increase in inositol pentakisphosphate accumulation by nicotine in cultured adrenal chromaffin cells

When [3H]inositol-prelabeled cultured bovine adrenal chromaffin cells were stimulated with nicotine (10 microM), a large and transient increase in [3H]inositol pentakisphosphate (InsP5) accumulation was observed. The accumulation reached the maximum level at 15 s, then declined to the basal level at 2 min. Nicotine also induced [3H]inositol tetrakisphosphate (InsP4) and [3H]inositol hexakisphosphate (InsP6) accumulation with a slower time course and a lesser magnitude than [3H]InsP5. The peaks of [3H]InsP4, [3H]InsP5 and [3H]InsP6 coincided with those of 32P radioactivity, when cells were doubly labeled with [3H]inositol and inorganic 32P. These results suggest that inositol pentakisphosphate is rapidly increased by nicotine, a cholinergic agonist, in cultured adrenal chromaffin cells.     

The effects of different hexachlorocyclohexanes and cyclodienes on glucose uptake and inositol phospholipid synthesis in rat brain cortex.

The inositol lipids from rat brain miniprisms were deacylated and separated by anion-exchange chromatography in order to determine whether or not gamma-hexachlorocyclohexane (gamma-HCH, lindane) and related compounds affect the different phosphatidylinositols. The incorporation of myo-[2-3H]inositol into phosphatidylinositol, phosphatidylinositol monophosphate and phosphatidylinositol bisphosphate were inhibited by lindane and its delta-HCH isomer. The inhibitory effects on phosphatidylinositol synthesis are not prominent in alpha-HCH and they are not significant with the beta-HCH and cyclodienes. The results presented here indicate that the inhibitory effect of lindane and delta-HCH on the phosphatidylinositol metabolism was no exclusively due to an interference with glucose transport. Lindane-treated miniprisms showed decreased myo-[2-3H]inositol uptake and, proportionately, an even greater inhibition of inositol phospholipid synthesis. Cellular uptake can, therefore, not account for all of the lindane inhibition.