Angiotensin-stimulated production of inositol trisphosphate isomers and rapid metabolism through inositol 4-monophosphate in adrenal glomerulosa cells.

The production and metabolism of inositol phosphates in rat adrenal glomerulosa cells prelabeled with [3H]inositol and stimulated with angiotensin II were analyzed by high-performance anion-exchange chromatography. Exposure to angiotensin II was accompanied by a rapid and substantial decrease in the phospholipid precursor, phosphatidylinositol (PtdIns) 4,5-bisphosphate with only a slight and transient increase in the level of the biologically active product, inositol 1,4,5-trisphosphate (Ins-1,4,5-P3), to a peak at about 5 sec. Inositol 1,3,4-trisphosphate (Ins-1,3,4-P3), the putative metabolite of Ins-1,4,5-P3, was also formed rapidly and maintained an elevated steady-state level during stimulation by angiotensin II. Inositol 1,4-bisphosphate (Ins-1,4-P2) exhibited a simultaneous and prominent increase that could not be accounted for solely by direct breakdown of PtdIns 4-phosphate, indicating that large amounts of Ins-1,4,5-P3 must also have been produced and metabolized. The rapid formation of a substantial amount of inositol 4-monophosphate (Ins-4-P), with no significant change in the level of inositol 1-monophosphate (Ins-1-P) during the first minute of stimulation, was a notable feature of the glomerulosa cell response to angiotensin II. These observations indicate that PtdIns-4,5-P2 catabolism in the angiotensin-stimulated glomerulosa cell initially proceeds via Ins-1,4,5-P3 through Ins-1,3,4-P3 and Ins-1,4-P2 to form Ins-4-P rather than Ins-1-P and that direct hydrolysis of PtdIns by phospholipase C does not occur during the initial phase of angiotensin action. In glomerulosa cells stimulated by angiotensin II in the presence of Li+, the progressive accumulation of both Ins-4-P, and after a short lag period, Ins-1-P indicated that dephosphorylation of both isomers was inhibited by Li+. The increase of Ins-P isomers in the presence of Li+ was associated with increased and progressive accumulation of Ins-1,4-P2 and Ins-1,3,4-P3 but not of Ins-1,4,5-P3. These data demonstrate that sustained and massive breakdown of PtdIns phosphates begins within seconds during cell activation by angiotensin II. The Ca2+-mobilizing metabolite, Ins-1,4,5-P3, is rapidly converted to Ins-1,3,4-P3 and degraded through Ins-1,4-P2 and Ins-4-P, in contrast to the previous view that conversion to Ins-1-P is the major route of PtdIns 4,5-bisphosphate metabolism.     

Pathway for inositol 1,3,4-trisphosphate and 1,4-bisphosphate metabolism.

We prepared [3H]inositol-,3-[32P]phosphate-and 4-[32P]phosphate-labeled inositol phosphate substrates to investigate the metabolism of inositol 1,3,4-trisphosphate and inositol 1,4-bisphosphate. In crude extracts of calf brain, inositol 1,3,4-trisphosphate is first converted to inositol 3,4-bisphosphate, then the inositol 3,4-bisphosphate intermediate is further converted to inositol 3-phosphate. Similarly, inositol 1,4-bisphosphate is converted to inositol 4-phosphate, and no inositol 1-phosphate is formed. We partially purified an enzyme that we tentatively name inositol polyphosphate 1-phosphatase. This cytosolic enzyme converts inositol 1,3,4-trisphosphate to inositol 3,4-bisphosphate and also converts inositol 1,4-bisphosphate to inositol 4-phosphate. The enzyme does not utilize inositol 1,3,4,5-tetrakisphosphate, inositol 1,4,5-trisphosphate, or inositol 1-phosphate as substrates. Thus we propose a new scheme for inositol phosphate metabolism. According to this pathway inositol 1,4,5-trisphosphate and inositol 1,4-bisphosphate are degraded to inositol 4-phosphate. Inositol 1-phosphate, which is the major inositol monophosphate formed in stimulated brain, is derived either from phospholipase C cleavage of phosphatidylinositol or from the degradation of inositol cyclic phosphates.     

Evidence for the existence of inositol tetrakisphosphate in mammalian heart. Effect of alpha 1-adrenoceptor stimulation

The time course of the effects of phenylephrine (10 mumol/l) on force of contraction and on inositol phosphates in electrically driven left auricles from rat hearts labeled with [3H]inositol was studied. All experiments were performed in the presence of propranolol (1 mumol/l) and LiCl (10 mmol/l). Products measured after separation with high-performance liquid chromatography were inositol 1-phosphate (1-IP1), inositol 1,4-bisphosphate (1,4-IP2), inositol 1,3,4-trisphosphate (1,3,4,-IP3), inositol 1,4,5-trisphosphate (1,4,5-IP3), and inositol 1,3,4,5-tetrakisphosphate (1,3,4,5-IP4). All inositol phosphates increased after stimulation with phenylephrine. 1,4,5-IP3 was the first compound to rise maximally within 30 seconds; this rise was followed by an increase in 1,3,4,5-IP4 and 1,4-IP2 beginning within 2 minutes. The increase in 1,3,4-IP3 and 1-IP1 was slower and did not reach steady state within 15 minutes. The positive inotropic effect of phenylephrine was maximal after 5 minutes. It is concluded that the increase in the presumed second messengers 1,4,5-IP3 and 1,3,4,5-IP4 coincides with the positive inotropic effect after alpha 1-adrenoceptor stimulation. Since the increase in 1,4,5-IP3 precedes the increase in force of contraction, 1,4,5-IP3 may initiate the positive inotropic effect of alpha 1-adrenoceptor agonists and 1,3,4,5-IP4 maintains the increase in force of contraction.     

Platelet-activating factor stimulates prolactin release from dispersed rat anterior pituitary cells in vitro

The biologically active phospholipid (platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) stimulated PRL release from dispersed rat anterior pituitary cells in culture. PAF-induced PRL release was dose dependent, with threshold stimulation at 1 nM and maximal stimulation at 100 nM. Stimulation occurred as early as 1 min of incubation and persisted for 2 h. The action of PAF on PRL release is consistent with a receptor-mediated mechanism based on the observations that the action of PAF is blocked by dopamine agonists and the PAF receptor antagonists L 652731 and SRI 63072. The structural analogs 1-O-alkyl-2-oleoyl-sn-glycero-3-phosphocholine and 1-O-alkyl-2-acetyl-sn-glycero-3-phosphoethanolamine, which lack the biological activity of PAF, are not able to stimulate PRL release over the dose range 0.2-2 microM. In addition, the PAF precursor lyso PAF and diacyl-sn-glycero-3-phosphocholine (phosphatidylcholine) were ineffective in stimulating PRL release. PAF induced the secretion of PRL and GH but not that of LH or TSH from hemipituitaries in short term incubations. PAF did not effect PRL release from GH3 cells. In conclusion, these data indicate that PAF stimulates PRL release from primary cultures of rat anterior pituitary cells in a dose-related, rapid, and specific manner.     

Thyrotropin stimulates the generation of inositol 1,4,5-trisphosphate in human thyroid cells

CONTEXT: Dual activation by TSH of the phospholipase C and cAMP cascades has been reported in human thyroid cells. In contrast, Singh et al. reported convincing data in FRTL-5 thyrocytes arguing against such an effect in this model. Their data in FRTL-5 cells indicated no increase in inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] in response to TSH. Therefore, the authors questioned results previously obtained on human cells by cruder methodology. OBJECTIVE: We investigated the formation of inositol phosphates by HPLC techniques in human thyroid slices to separate the inositol phosphate isomers. RESULTS: Ins(1,4,5)P3, inositol 1,3,4-trisphosphate, and inositol 1,3,4,5-tetrakisphosphate were increased after TSH stimulation. The effect of TSH in human thyroid cells was reproduced by recombinant TSH and prevented by antibodies blocking the TSH receptor. Thyroid-stimulating antibodies at concentrations eliciting a cAMP response equivalent to TSH failed to stimulate inositol phosphate generation. CONCLUSIONS: TSH, but not thyroid-stimulating antibodies, activates both cAMP and the phospholipase C cascade in human thyroid as now demonstrated by an increase in Ins(1,4,5)P3 and its inositol phosphate metabolites. Therefore, this effect cannot be extrapolated to the FRTL-5 cell line. The apparent discrepancy may be due to a difference between species (human vs. rat) or to the loss of the fresh tissue properties in a cell line. The dual effect of TSH in human cells, through cAMP on secretion of thyroid hormones and through the diacylglycerol, Ins(1,4,5)P3 Ca2+ pathway on thyroid hormone synthesis, implies the possible separation of these effects in thyroid disease.     

Prostaglandin F2 alpha stimulates inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate formation in bovine luteal cells.

The present studies were conducted to further evaluate inositol phosphate formation and metabolism in prostaglandin F2 alpha (PGF2 alpha)-stimulated bovine luteal cells. Corpora lutea were dispersed with collagenase, and luteal cells were prelabeled for 3 h with [3H]inositol. Inositol phosphates produced in response to PGF2 alpha were analyzed by ion exchange column chromatography and HPLC. Time-course experiments revealed that significant increases in inositol trisphosphate (InsP3) were apparent within 5 sec of incubation with PGF2 alpha. Increases in inositol bisphosphate (InsP2) were also apparent within 5 sec. InsP1 and InsP4 were observed after a short (5-sec) lag period. HPLC revealed that PGF2 alpha provoked rapid (5 sec) increases in inositol 1,4,5-trisphosphate (Ins 1,4,5-P3), which was rapidly converted to inositol 1,3,4,5-tetrakisphosphate (Ins 1,3,4,5-P4) and inositol 1,3,4-trisphosphate (Ins 1,3,4-P3). The primary inositol bisphosphate isomer present in PGF2 alpha-stimulated bovine luteal cells was inositol 1,4-bisphosphate (Ins 1,4-P2), with lesser amounts of Ins 1,3-P2. Inositol monophosphates were also increased. These findings were confirmed in studies in which the metabolism of purified [3H]Ins 1,4,5-P3 was followed temporally in saponin-permeabilized bovine luteal cells. Additional studies demonstrated the presence of an enzyme, InsP3-3-kinase, in the cytosolic fraction of bovine corpora lutea. InsP3-3-kinase phosphorylated Ins 1,4,5-P3 to form Ins 1,3,4,5-P4. The activity of InsP3-3-kinase was calcium dependent and was enhanced by calmodulin at low calcium concentrations. Calmidazolium, a calmodulin inhibitor, reduced InsP3-3-kinase activity in a concentration-dependent manner. These results demonstrate the presence of multiple polyphosphorylated inositol phosphates in PGF2 alpha-stimulated bovine luteal cells. The isomers were formed via the action of a specific calcium/calmodulin-regulated kinase (InsP3-3-kinase), which phosphorylated Ins 1,4,5-P3 during agonist-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate. These data suggest that the inositol tris/tetrakisphosphate pathway is an important sequelae to PGF2 alpha-stimulated inositol phospholipid hydrolysis, and that the pathway may be activated during agonist-mediated calcium mobilization.     

Inhibition by staurosporine of TRH-induced refractoriness of inositol phospholipid hydrolysis in rat anterior pituitaries

To clarify the mechanism(s) underlying the TRH-induced refractory state of the anterior pituitary, we evaluated rat pituitary inositol phospholipid hydrolysis in the presence of staurosporine. TRH caused a time- and dose-dependent accumulation of inositol phosphates in rat anterior pituitary slices. Pretreatment with 550 nmol TRH/l completely abolished the subsequent accumulation of inositol phosphates in response to 140 nmol TRH/l. TRH-stimulated accumulation of inositol phosphates did not occur after pretreatment with 0.2 mumol phorbol ester/l. Refractoriness of inositol phospholipid hydrolysis which was produced by pretreatment with TRH and phorbol ester was inhibited by staurosporine. The present data support the hypothesis that protein kinase C plays a profound role in TRH induction of the refractory state of inositol phospholipid hydrolysis in the anterior pituitary.     

Vasopressin activation of phosphatidylinositol metabolism in rat anterior pituitary in vitro and its modification by changes in the hypothalamo-pituitary-adrenal axis

The ability of vasopressin to stimulate the accumulation of 3H-labelled inositol phosphates was studied in vitro using prelabelled rat anterior pituitary quarters. [8-Arginine] vasopressin activates inositol lipid breakdown in this system in a time- and dose-dependent manner; vasopressin (3 X 10(-7) M) resulted in a 1.8-fold stimulation of inositol phosphate accumulation over control accumulation after 10 min. This response to vasopressin is inhibited by the specific V1 antagonist (CH2)5Tyr(Me)AVP. Both oxytocin and the selective V2 agonist DDAVP also show some agonist activity, but are considerably less potent than arginine vasopressin. Corticotrophin-releasing factor alone had no effect on inositol phosphate production, whilst a high dose given in conjunction with vasopressin resulted in a diminution of the response below that found with the same concentration of vasopressin alone. Anterior pituitaries from vasopressin-deficient Brattleboro rats also show a phosphatidylinositol response to vasopressin. Pituitaries from rats that had been adrenalectomized 4 days earlier showed no increase in inositol phosphate accumulation in response to vasopressin. Daily administration of dexamethasone (40 micrograms/day) reversed this effect of adrenalectomy. This reversal was not seen when dexamethasone (40 micrograms/ml) was added to the incubation medium of adrenalectomized rat pituitary quarters. These results confirm that the rat anterior pituitary contains functional vasopressin receptors capable of activating inositol phospholipid metabolism and that this biochemical response is modified by changes in the hypothalamo-pituitary-adrenal axis.     

Cholinergic stimulation of phosphoinositide hydrolysis in rat anterior pituitary

The production of inositol phosphates in response to carbachol was studied in rat anterior pituitary tissue prelabelled with [3H]inositol. Carbachol (10 microM) stimulated inositol mono-, bis- and trisphosphate production (IP1, IP2 and IP3) by 360 +/- 49, 338 +/- 49 and 503 +/- 49 (mean +/- SEM, P less than 0.001) percent respectively during a 30 min incubation. Mean basal production was 5.4 +/- 0.3, 4.1 +/- 0.5 and 0.9 +/- 0.3 expressed as a percent of total [3H]inositol lipid for IP, IP2 and IP3 respectively. Stimulated inositol phosphate production was dose dependent and detectable after 5 min. Atropine prevented this stimulation indicating mediation via muscarinic receptors. Removal of extracellular Ca2+ reduced both basal and stimulated total inositol phosphate production by 60% and 56% respectively but did not impair carbachol-induced phosphoinositide hydrolysis per se. Pretreatment of pituitary tissue with either somatostatin (5 micrograms/ml) or pertussis toxin (1 microgram/ml) had no effect on either basal or stimulated inositol phosphate production. These results demonstrate a cholinergic stimulation of phosphatidylinositol bisphosphate (PIP2) hydrolysis in the anterior pituitary which may be important in the action of cholinergic agonists on pituitary hormone secretion.     

Light-stimulated inositolphospholipid turnover in Samanea saman leaf pulvini

Leaflets of Samanea saman open and close rhythmically, driven by an endogenous circadian clock. Light has a rapid, direct effect on the movements and also rephases the rhythm. We investigated whether light signals might be mediated by increased inositolphospholipid turnover, a mechanism for signal transduction that is widely utilized in animal systems. Samanea motor organs (pulvini) labeled with [³H]inositol were irradiated briefly (5-30 sec) with white light, and membrane-localized phosphatidylinositol phosphates and their aqueous breakdown products, the inositol phosphates, were examined. After a 15-sec or longer light pulse, labeled phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate decreased and their labeled metabolic products inositol 1,4-bisphosphate and inositol 1,4,5-trisphosphate increased, changes characteristic of inositolphospholipid turnover. We conclude that inositolphospholipid turnover may act as a phototransduction mechanism in Samanea pulvini in a manner that is similar to that reported in animal systems.     

Action and formation of inositol bisphosphate and inositol trisphosphate in rat anterior pituitary cells

Inositol 4,5-bisphosphate and inositol 1,4,5-trisphosphate, administered exogenously at a concentration of 3 x 10(-5) mol/l increased LH release in superfused rat pituitary cells by 950 +/- 267% and 281 +/- 83%, respectively. This stimulatory effect was reversible and dose-dependent. Other inositol phosphates (inositol 1-monophosphate, inositol 1,4,5,6-tetrakisphosphate, inositol 1,3,4,5,6-pentakisphosphate and inositol 1,2,3,4,5,6-hexakisphosphate), tested in vitro, did not significantly influence LH release. In saponin-permeabilized cells, the rate of basal and stimulated LH release was twice that in non-permeabilized cells. Penetration of inositol bisphosphate and inositol trisphosphate into saponin-treated pituitary cells did not increase the secretory potency of these agents compared with their effect on non-permeabilized cells. The new findings document that inositol trisphosphate formation occurs within 5-45 s after GnRH (10(-7) mol/l) administration and seems to be involved in mediating the rapid, first phase of LH release, whereas inositol bisphosphate formation occurs after 3-15 min and is probably related to later phases of LH secretion. Our results suggest that inositol bisphosphate and inositol trisphosphate are important regulators of the release of luteinizing hormone and can exert their effects not only intracellularly, but also extracellularly.     

Bradykinin stimulates phosphoinositide metabolism and prolactin secretion in rat anterior pituitary cells

Bradykinin stimulated prolactin secretion from monolayer cultures of rat anterior pituitary cells, the stimulation being greater from the cells of male rats. This stimulated secretion was accompanied by a rise in total inositol phosphate accumulation, suggesting that the action of bradykinin is mediated by phosphoinositide hydrolysis. The increase in inositol phosphate accumulation was biphasic; a further sharp rise occurred when the concentration of bradykinin exceeded 1 mumol/l. This may indicate that bradykinin acts on other cell types in the pituitary gland. Bradykinin had no effect on growth hormone secretion from cells of normal pituitary glands, or on prolactin secretion and phosphoinositide metabolism in GH3 rat pituitary tumour cells. Bradykinin receptor antagonists (both B1 and B2) had no effect on either bradykinin-stimulated inositol phosphate accumulation or prolactin secretion. Kallikreins, the enzymes responsible for the generation of kinins, are known to be present in the adenohypophysis. Therefore, the results presented here would suggest that kinins may have a role as paracrine agents in the pituitary gland.     

Growth hormone-releasing factor does not stimulate phosphoinositides breakdown in primary cultures of rat and human pituitary cells

It has been reported that rat growth hormone releasing factor (rat GRF-43), similarly to the two human GRFs (GRF-40 and 44) stimulates adenylate cyclase activity in pituitary cells. Controversial findings have been presented by two different groups on the action of GRF on phosphoinositides (PI) metabolism, a phenomenon linked to Ca-- mediated intracellular mechanisms. In the work to be reported, we evaluated the accumulation of inositol phosphates induced by GRF exposure in primary cultures of rat and human pituitary cells. Addition of rat GRF-43 to rat pituitary cells at doses up to 1 microM had no effect on inositol phosphates accumulation, while already at a dose as low as 0.05 nM it increased growth hormone secretion in the incubation medium significantly. In the same cell system, TRH, a known activator of PI breakdown, significantly increased [3H]inositol phosphates. In primary cultures of human somatotrophs from acromegalic subjects as in rats, addition of hpGRF-40 and also of TRH did not elicit any modification in the accumulation of [3H]inositol phosphates. Consistent with in vivo findings, both peptides induced a significant release of GH in the medium. Our results show that the GH releasing effect of GRF does not involve the hydrolysis of phosphatidylinositol in normal rat as well as in tumoral human somatotrophs. In addition it appears that the anomalous response of TRH on adenomatous cells from acromegalic patients is differently mediated in respect to the action of the tripeptide on normal lactotrophs and thyrotrophs.     

Identification and characterization of insulin-like growth factor receptors on adult rat cardiac myocytes: linkage to inositol 1,4,5-trisphosphate formation.

Cultured cardiac myocytes from adult Sprague-Dawley rats express both insulin-like growth factor-I (IGF-I) receptors and insulin-like growth factor-II/mannose 6-phosphate (IGF-II/Man6P) receptors and respond to IGF-I with a dose-dependent accumulation of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and inositol 1,4-bisphosphate [Ins(1,4)P2]. Specific binding of [125I]IGF-I to isolated membranes from cultured cardiac myocytes amounted to 1-1.2%. Binding of [125I]IGF-I was inhibited by unlabeled IGF-I at nanomolar concentrations and insulin at much higher concentrations. These data suggest that IGF-I binds to its own receptor on rat cardiac myocytes. Competitive binding studies using isolated membranes from cardiac myocytes and [125I]IGF-II showed 2-4% specific binding. Binding of [125I]IGF-II was inhibited by IGF-II and much less potently by IGF-I and insulin. Immunoglobulin G (IgG) 3637 (an IgG directed against the IGF-II/Man6P receptor) partially inhibited binding of [125I]IGF-II whereas nonimmune IgG did not. Affinity cross-linking studies with [125I]IGF-II and cardiac myocyte membranes and subsequent analysis of the ligand-receptor complex using SDS-PAGE and autoradiography showed a radiolabeled band of approximately 250 kilodalton (kDa). The formation of the [125I]IGF-II-receptor complex was inhibited by incubation with IGF-II and IgG 3637 but not by insulin or nonimmune IgG. Western blotting of protein extracts from cultured cardiac myocytes was performed using IgG 3637 and an immunoperoxidase technique for the visualization of the IGF-II/Man6P receptor protein. A specific band at 220 kDa under nonreducing conditions was detected on the blots, providing further evidence for the expression of the IGF-II/Man6P receptor by cardiac myocytes. The effect of IGFs on the accumulation of inositol phosphates was measured by HPLC analysis of perchloric acid extracts from myo-[3H]inositol-labeled cultured cardiac myocytes. IGF-I (50 ng/ml) stimulated the accumulation both of Ins(1,4,5)P3 and Ins(1,4)P2 after 30 sec by 43% and 63%. IGF-II (up to 500 ng/ml) had no significant effect on inositol phosphate accumulation under the same conditions. However, in the presence of millimolar concentrations of Man6P, IGF-II (500 ng/ml) also increased Ins(1,4,5)P3 accumulation by 59%. We conclude that cardiac myocytes from adult rats express IGF receptors and respond to IGFs with the accumulation of Ins(1,4,5)P3 and Ins(1,4)P2. This effect seems to be mediated by an IGF-I receptor-specific pathway.     

The inositol trisphosphate pathway mediates platelet-activating-factor-induced pulmonary oedema.

Platelet-activating factor (PAF) is a pro-inflammatory lipid mediator that increases vascular permeability by simultaneous activation of two pathways, one dependent on the cyclooxygenase metabolite prostaglandin E2 and the other on the sphingomyelinase metabolite ceramide. The hypothesis that part of the PAF-induced oedema is mediated via the inositol 1,4,5-trisphosphate (IP3) pathway or Rho kinase pathway was investigated. Oedema formation was induced in isolated perfused rat lungs by injection of 5 nmol PAF into the pulmonary artery. Lungs were pre-treated with specific inhibitors: edelfosine (L108) to block phosphatidyl-inositol-specific phospholipase C, xestospongin to block the IP3 receptor, 5-iodonaphthalene-1-sulphonyl-homopiperazine (ML-7) to block myosin light chain kinase, and (+)-R-trans-4-(aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide (Y27632) to block Rho-associated protein kinase. Pre-treatment with L108 or xestospongin reduced PAF-induced oedema formation by 58 and 56%, respectively. The effect of L108 was additive to that of the cyclooxygenase inhibitor acetyl salicylic acid (88% oedema reduction). PAF-induced oedema formation was also reduced if extracellular calcium concentrations were lowered. Furthermore, treatment with ML-7 reduced oedema formation by 54%, whereas Y27632 was without effect. It is concluded that platelet-activating-factor-triggered oedema is mediated by activation of the inositol 1,4,5-trisphosphate pathway, influx of extracellular calcium and subsequent activation of a myosin light chain kinase-dependent and Rho-associated-protein-kinase-independent mechanism.     

Angiotensin peptides stimulate phosphoinositide breakdown and prolactin release in anterior pituitary cells in culture

We investigated the effects of angiotensin peptides on the breakdown of specific membrane phospholipids, the inositol lipids, in anterior pituitary cells in culture, measuring the water-soluble products (inositol phosphates) produced during the cleavage of phosphoinositides by phospholipase C. Both angiotensin II and angiotensin I in the presence of 10 mM LiCl potently increased, in a concentration-dependent manner, total [3H]inositol phosphate and PRL release in cultured rat anterior pituitary cells. The release of LH, TSH, or GH was not significantly enhanced by the peptides. The effect on inositol phosphate accumulation was significant at 0.01 nM, and maximal stimulation (approximately 5-fold increase) occurred at 10 nM, with an ED50 of about 0.3 nM. The stimulatory effects of both angiotensin II and angiotensin I were antagonized by the receptor antagonists saralasin and Sar1,Ile8-angiotensin II. Moreover, 1 microM captopril, an inhibitor of angiotensin-converting enzyme, antagonized the effects of 0.1 and 1 nM angiotensin I, suggesting that the effect of angiotensin I on phosphoinositide breakdown and PRL release is dependent on prior conversion of angiotensin I to angiotensin II. The effect of angiotensin II was very rapid. Fractionation of the water-soluble inositol phosphates showed that angiotensin II significantly increased inositol bisphosphate and inositol triphosphate at 10 sec, whereas inositol monophosphate was increased only after 40 sec. These data indicate that in the pituitary, and presumably in the lactotroph, the binding of angiotensin II to specific membrane receptors provokes increased polyphosphoinositide hydrolysis, leading to increased production of intracellular messengers, i.e. inositol triphosphate and 1,2-diacylglycerol, responsible for the stimulation of PRL release.     

The use of cells doubly labelled with [14C]inositol and [3H]inositol to search for a hormone-sensitive inositol lipid pool with atypically rapid metabolic turnover

Some, though not all, previous studies have suggested that the inositol lipid which is hydrolysed during transmembrane signalling in response to receptor activation might be drawn from a metabolically discrete and relatively small hormone-sensitive lipid pool that turns over more rapidly than the bulk of membrane inositol lipid. In order to seek evidence for the existence of this putative hormone-sensitive lipid pool, we have double-labelled cells by growing them for 3 days in a medium containing [14C]inositol and then supplying them with [3H]inositol for the final 2 h before stimulation. We anticipated that stimulation of these doubly labelled cells might provoke the formation, from the postulated hormone-sensitive pool, of small quantities of relatively 3H-enriched inositol phosphates, and that these could be harvested from cells (provided that the cytosolic inositol monophosphatase and inositol 1,4-bisphosphate/inositol 1,3,4-trisphosphate 1-phosphatase activities are first inhibited by Li+). Experiments of this type, using both vasopressin-stimulated WRK1 rat mammary tumour cells and 3T3 mouse fibroblasts stimulated by prostaglandin F2 alpha, have largely failed to demonstrate the formation of relatively 3H-enriched inositol phosphates. There was a tendency for phosphatidyl-inositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate to have slightly higher 3H: 14C ratios than phosphatidylinositol, but the 3H: 14C ratios of the inositol phosphates formed in stimulated cells were not substantially greater than the 3H: 14C ratios of the inositol lipids. We therefore conclude, at least for the two cell lines that we studied, that hormone-stimulated inositol lipid hydrolysis can call, either directly or indirectly, upon the majority of the inositol lipid complement of the stimulated cell.     

The effects of low density lipoprotein and high density lipoprotein on phosphoinositide hydrolysis in bovine aortic endothelial cells.

Low density lipoprotein (LDL) and high density lipoprotein (HDL3) were tested for their ability to induce inositol phospholipid turnover and inositol phosphate production in bovine aortic endothelial cells (BAEC). The production of inositol phosphates following hydrolysis of the phosphoinositides was demonstrated by two methods; release of [3H]inositol phosphates after labelling with [3H]myo-inositol and by a direct binding assay for inositol 1,4,5-trisphosphate (InsP3). Acute exposure to LDL induced InsP3 release at low concentrations of the lipoprotein within the physiological range of LDL in tissues. HDL3 did not cause any release of the inositol phosphates. Pre-incubation of BAEC with HDL3 suppressed bradykinin- and LDL-induced inositol phosphate production in BAEC in a concentration-dependent manner. It is concluded that LDL acutely stimulates phosphoinositide breakdown and that pre-incubation of cells with HDL3 inhibits this effect. The mechanism responsible for these effects remains to be elucidated.     

Mammalian inositol polyphosphate multikinase synthesizes inositol 1,4,5-trisphosphate and an inositol pyrophosphate

Using a consensus sequence in inositol phosphate kinase, we have identified and cloned a 44-kDa mammalian inositol phosphate kinase with broader catalytic capacities than any other member of the family and which we designate mammalian inositol phosphate multikinase (mIPMK). By phosphorylating inositol 4,5-bisphosphate, mIPMK provides an alternative biosynthesis for inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)]. mIPMK also can form the pyrophosphate disphosphoinositol tetrakisphosphate (PP-InsP(4)) from InsP(5). Additionally, mIPMK forms InsP(4) from Ins(1,4,5)P(3) and InsP(5) from Ins(1,3,4,5)P(4).     

The type II inositol 1,4,5-trisphosphate receptor can trigger Ca2+ waves in rat hepatocytes

BACKGROUND & AIMS: Ca2+ regulates cell functions through signaling patterns such as Ca2+ oscillations and Ca2+ waves. The type I inositol 1,4,5-trisphosphate receptor is thought to support Ca2+ oscillations, whereas the type III inositol 1,4,5-trisphosphate receptor is thought to initiate Ca2+ waves. The role of the type II inositol 1,4,5-trisphosphate receptor is less clear, because it behaves like the type III inositol 1,4,5-trisphosphate receptor at the single-channel level but can support Ca2+ oscillations in intact cells. Because the type II inositol 1,4,5-trisphosphate receptor is the predominant isoform in liver, we examined whether this isoform can trigger Ca2+ waves in hepatocytes. METHODS: The expression and distribution of inositol 1,4,5-trisphosphate receptor isoforms was examined in rat liver by immunoblot and confocal immunofluorescence. The effects of inositol 1,4,5-trisphosphate on Ca2+ signaling were examined in isolated rat hepatocyte couplets by using flash photolysis and time-lapse confocal microscopy. RESULTS: The type II inositol 1,4,5-trisphosphate receptor was concentrated near the canalicular pole in hepatocytes, whereas the type I inositol 1,4,5-trisphosphate receptor was found elsewhere. Stimulation of hepatocytes with vasopressin or directly with inositol 1,4,5-trisphosphate induced Ca2+ waves that began in the canalicular region and then spread to the rest of the cell. Inositol 1,4,5-Trisphosphate-induced Ca2+ signals also increased more rapidly in the canalicular region. Hepatocytes did not express the ryanodine receptor, and cyclic adenosine diphosphate-ribose had no effect on Ca2+ signaling in these cells. CONCLUSIONS: The type II inositol 1,4,5-trisphosphate receptor establishes a pericanalicular trigger zone from which Ca2+ waves originate in hepatocytes.