Angiotensin II Effects on the Cytosolic Free Ca2+ Concentration in N1E-115 Neuroblastoma Cells: Kinetic Properties of the Ca2+ Transient Measured in Single Fura-2-Loaded Cells

The effect of angiotensin II on the cytosolic free Ca2+ concentration was measured in single mouse neuroblastoma N1E-115 cells loaded with fura-2. Angiotensin II induced a transient concentration-dependent increase in Ca2+ and also increased the production of inositol polyphosphates. The Ca2+ increase did not require extracellular Ca2+ and was unaffected by pretreatment with pertussis toxin. These data suggest that angiotensin II increased Ca2+ by an inositol trisphosphate-mediated release of intracellular Ca2+ following activation of phospholipase C via a pertussis toxin-insensitive guanine nucleotide binding protein. Similar results were obtained with bradykinin. The angiotensin II- or bradykinin-induced increase in Ca2+ occurred after a concentration-dependent latent period. Low concentrations of agonist elicited a small increase in Ca2+ following a variable lag that sometimes exceeded 1 min, whereas at maximally effective angiotensin II concentrations a larger, more rapid increase in Ca2+ occurred without a measurable delay. In some cells, oscillatory increases in Ca2+ were induced by angiotensin II and bradykinin. Possible mechanisms to explain the concentration dependency of the latent period and the oscillatory nature of the increases of Ca2+ are discussed. These results indicate that the mouse neuroblastoma N1E-115 cell represents a useful model for studying the signal response transduction mechanisms regulating the effects of angiotensin II in neuronal cells.     

Epidermal growth factor and angiotensin II stimulate formation of inositol 1,4,5- and inositol 1,3,4-trisphosphate in hepatocytes. Differential inhibition by pertussis toxin and phorbol 12-myristate 13-acetate

The ability of epidermal growth factor (EGF) and angiotensin II to stimulate production of inositol trisphosphate and mobilize intracellular Ca2+ in hepatocytes was compared using quin2 fluorescence to monitor changes in Ca2+ levels and high performance liquid chromatography to resolve the inositol trisphosphate (InsP3) isomers. Both EGF and angiotensin II stimulated an increase in free intracellular Ca2+ concentration ([Ca2+]i) as well as a rapid increase in the production of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3). Concentrations of angiotensin II which gave a rise in [Ca2+]i equivalent to that seen with maximal doses of EGF produced an equivalent increase in Ins(1,4,5)P3 formation. Both EGF and angiotensin II stimulated the formation of the Ins(1,3,4)P3 and inositol 1,3,4,5-tetrakisphosphate isomers. The formation of the Ins(1,3,4)P3 isomer lagged behind production of Ins(1,4,5)P3 but eventually reached higher levels in the cell. The initial rise in [Ca2+]i and InsP3 levels stimulated by EGF and angiotensin II was not affected by reducing the external Ca2+ concentration below 30 nM with an excess of [ethylenebis(oxyethylenenitrilo)] tetraacetic acid. Treatment of hepatocytes for 30-180 s with 1 micrograms/ml phorbol 12-myristate 13-acetate prior to the addition of EGF blocked the EGF-stimulated production of Ins(1,4,5)P3 and the increase in [Ca2+]i. Phorbol 12-myristate 13-acetate attenuated the production of Ins(1,4,5)P3 generated by angiotensin II over the concentration range of 10(-10) to 10(-8) M; however, the Ca2+ signal was only inhibited at the 10(-10) M dose of angiotensin II. Treatment of rats with pertussis toxin for 72 h prior to isolating hepatocytes blocked the ability of EGF to increase Ins(1,4,5)P3 and Ins(1,3,4)P3 but did not inhibit the ability of any concentration of angiotensin II to stimulate formation of InsP3 or inositol tetrakisphosphate. The observation that pertussis toxin selectively abolishes EGF-stimulated inositol lipid breakdown suggests that EGF and angiotensin II use different mechanisms to activate phospholipase C in hepatocytes.     

Increase in cytosolic calcium and phosphoinositide metabolism induced by angiotensin II and [Arg]vasopressin in vascular smooth muscle cells

Effects of angiotensin II and [Arg]vasopressin on cytosolic free Ca2+ concentration ([Ca2+]i) and phosphoinositide metabolism were studied in cultured aortic smooth muscle cells obtained from Wistar-Kyoto rats and their spontaneously hypertensive substrain. [Ca2+]i was measured using the fluorescent Ca2+ indicator quin2. No clear differences in basal [Ca2+]i were detected between cells derived from the two strains. High concentrations of angiotensin II (greater than or equal to 10 nM) and [Arg]vasopressin (greater than or equal to 100 nM) elicited large and rapid increases in [Ca2+]i, followed by a rapid return to control values. Low concentrations of these peptides (less than or equal to 1.0 nM) elicited small and slow increases in [Ca2+]i that persisted for minutes. These responses were blocked by specific antagonists for each of these peptides. Only high concentrations of angiotensin II caused [Ca2+]i increases in "Ca2+-free" medium, which suggested that high concentrations of angiotensin II could release Ca2+ from intracellular pools. A high concentration of angiotensin II and [Arg]vasopressin elicited progressive accumulations of inositol phosphates. Only high concentrations of angiotensin II caused inositol phosphate accumulation in Ca2+-free medium. Maximal accumulation of inositol phosphate elicited by angiotensin II and [Arg]vasopressin was found to be additive. A desensitization to the effects of both peptides on Ca2+ mobilization occurred despite the continued accumulation of inositol phosphates. These observations indicated that angiotensin II and [Arg]vasopressin interacted with independent receptors, both of which are linked to phosphoinositide breakdown and Ca2+ mobilization.     

The mechanism for synergism between phospholipase C- and adenylylcyclase-linked hormones in liver. Cyclic AMP-dependent kinase augments inositol trisphosphate-mediated Ca2+ mobilization without increasing the cellular levels of inositol polyphosphates.

The ability of cAMP-dependent hormones to modulate the actions of Ca2(+)-mobilizing hormones was studied in single fura-2-injected guinea pig hepatocytes. In 91% of cells the cAMP-linked hormone, isoproterenol, applied alone, did not alter cytosolic Ca2+ concentration. In 78% of cells which had been pre-exposed to a low concentration of angiotensin II, isoproterenol was able to increase cytosolic Ca2+. Isoproterenol did not, however, increase inositol 1,4,5-trisphosphate or inositol tetrakisphosphate on its own, or in the presence of angiotensin II. Isoproterenol was also able to raise cytosolic Ca2+ concentration in cells microinjected with inositol 2,4,5-trisphosphate or a photoactivatable derivative of inositol 1,4,5-trisphosphate. The elevation of cytosolic Ca2+ concentration induced by isoproterenol in angiotensin II-treated cells and cells injected with caged inositol 1,4,5-trisphosphate was blocked by heparin, implying that the effect was mediated by an inositol 1,4,5-trisphosphate receptor agonist. In permeabilized hepatocytes, inositol 1,4,5-trisphosphate-induced Ca2+ release was enhanced by 8-bromo-cAMP and the catalytic subunit of cAMP-dependent kinase. Cyclic AMP-dependent kinase shifted the dose-response curve for inositol 1,4,5-trisphosphate-mediated Ca2+ release to the left by a factor of 4 and increased the total amount of Ca2+ released by 25%. These results indicate that increased sensitivity of the intracellular Ca2+ releasing organelle to inositol 1,4,5-trisphosphate is responsible for synergism between phospholipase C- and adenylylcyclase-linked hormones in the liver.     

Phorbol ester and 1-oleoyl-2-acetylglycerol inhibit angiotensin activation of phospholipase C in cultured vascular smooth muscle cells

Angiotensin II acts on cultured rat aortic vascular smooth muscle cells (VSMC) to induce the rapid, phospholipase C-mediated generation of inositol trisphosphate from phosphatidylinositol 4,5-bisphosphate and mobilization of intracellular Ca2+. sn-1,2-Diacylglycerol, the other major product of inositol phospholipid breakdown, is known to activate protein kinase C, but its role in angiotensin II action on VSMC has not been defined. We report herein that, in cultured VSMC prelabeled with [3H]myoinositol, brief incubations (2-5 min) with 4 beta-phorbol 12-myristate 13-acetate (PMA) (1-100 nM) or 1-oleoyl-2-acetylglycerol (10-100 microM), two potent activators of protein kinase C, inhibit subsequent angiotensin II (100 nM)-induced increases in phosphatidylinositol 4,5-bisphosphate breakdown and inositol trisphosphate formation. In addition, pretreatment of VSMC with either PMA (IC50 approximately 1 nM) or 1-oleoyl-2-acetylglycerol (IC50 approximately 7.5 microM) also markedly inhibits angiotensin II (1 nM)-stimulated increases in cytosolic free Ca2+, as measured with the calcium-sensitive fluorescent indicator quin 2, or 45Ca2+ efflux. Neither PMA nor 1-oleoyl-2-acetylglycerol initiated phosphatidylinositol 4,5-bisphosphate breakdown or Ca2+ flux by itself. PMA treatment (10 nM, 5 min) did not influence the number or affinity of 125I-angiotensin II-binding sites in intact cells. These data suggest that one function of angiotensin II-generated sn-1,2-diacylglycerol in vascular smooth muscle may be to modulate, by protein kinase C-mediated mechanisms, angiotensin II receptor coupling to phospholipase C.     

Ca2(+)-mobilizing hormones stimulate Ca2+ efflux from hepatocytes

Treatment of hepatocytes with 2,5-di-(tert-butyl)-1,4-benzohydroquinone (tBuBHQ), a novel mobilizer of the inositol 1,4,5-trisphosphate-sensitive Ca2+ pool, produces a sustained elevation of [Ca2+]i (Kass, G. E. N., Duddy, S. K., and Orrenius, S. (1989) J. Biol. Chem. 264, 15192-15198). Exposure of hepatocytes to the Ca2(+)-mobilizing hormones, vasopressin, angiotensin II, or ATP following [Ca2+]i elevation by tBuBHQ produced a rapid return of [Ca2+]i to basal or near basal levels. Release of the inositol 1,4,5-trisphosphate-sensitive Ca2+ pool by tBuBHQ following pretreatment with vasopressin or angiotensin II resulted in a [Ca2+]i transient and not the sustained [Ca2+]i elevation observed in the absence of the Ca2(+)-mobilizing hormones. The G-protein activator, NaF plus AlCl3, mimicked both effects of the Ca2(+)-mobilizing hormones on [Ca2+]i. The mechanism for Ca2+ removal from the cytosol by Ca2(+)-mobilizing hormones did not involve cyclic nucleotides nor did it require protein kinase C activation or cyclo- and lipoxygenase-dependent metabolites of arachidonic acid. Furthermore, the hormone-mediated decrease in [Ca2+]i did not involve the pertussis toxin-sensitive Gi-protein. Removal of the tBuBHQ-mobilized Ca2+ from the cytosol of hepatocytes by Ca2(+)-mobilizing hormones was mediated by stimulation of a Ca2+ efflux pathway. Thus, in addition to initiating [Ca2+]i transients by releasing Ca2+ from the inositol 1,4,5-trisphosphate-sensitive Ca2+ store and stimulating Ca2+ influx, Ca2(+)-mobilizing hormones also regulate the termination of the [Ca2+]i transient by stimulating a Ca2+ efflux pathway.     

Multiple transduction mechanisms of dopamine, somatostatin and angiotensin II receptors in anterior pituitary cells

The concept of multifactorial pituitary control is now well established. As in other cell systems, integration of complex messages involves dynamic interactions of receptors and coupling mechanisms. Regulation of adenohypophyseal secretions has been shown to involve cyclic AMP production, the modulation of phosphatidylinositol phosphate breakdown and Ca2+ mobilization. Dopamine, somatostatin and angiotensin II receptors are negatively coupled to adenylate cyclase in anterior pituitary cells. In the case of angiotensin, this effect on adenylate cyclase appears paradoxical since the peptide markedly stimulates prolactin secretion. In fact, angiotensin II also markedly stimulates inositol phosphate production and this effect could account for the stimulated hormone secretion. In addition, dopamine could inhibit inositol phosphate production stimulated by angiotensin II and thyrotropin-releasing hormone. Dopamine and somatostatin also directly modulate voltage-dependent calcium channels, perhaps through a direct coupling with potassium channels. On the other hand, steroids modulate the sensitivity of adenohypophyseal cells to neurohormones by different mechanisms. In the case of somatostatin, it increases the number of specific binding sites, while in the case of dopamine estradiol affects the transduction mechanisms of D2 dopamine receptors. In conclusion, dopamine and somatostatin receptors appear coupled to various transduction mechanisms through pertussis-sensitive G proteins in anterior pituitary cells.     

Platelet-activating factor-induced homologous and heterologous desensitization in cultured vascular smooth muscle cells

Intracellular free Ca2+ concentrations were monitored in vascular smooth muscle cells (VSMC) using the Ca2+-sensitive dye fura II. Superfusion of VSMC with platelet-activating factor (S-PAF; 1-100 nM) increased cytosolic Ca2+ in a dose-dependent manner. The response was transient and returned to base line even though the agonist was still present. A second, higher dose of PAF did not elicit a response. The inactive optical isomer, R-PAF, was ineffective suggesting that the S-PAF response is specific and receptor-mediated. Pretreatment of VSMC with PAF attenuated angiotensin II-stimulated Ca2+ mobilization but not vasopressin-stimulated Ca2+ mobilization. Treatment of VSMC with PAF (10 nM) stimulated inositol trisphosphate and inositol tetrakisphosphate formation above control by 260 +/- 15% and 195 +/- 11%, respectively. Diacylglycerol levels also rose during PAF stimulation and remained increased over 15 min. Pretreatment of VSMCs with phorbol-12,13-myristate acetate (10 nM) for 30 min abolished both the PAF- and angiotensin II-induced increases in cytosolic Ca2+, but not the vasopressin-induced increase. Pretreatment of VSMC with dioctanoylglycerol (10 microM) abolished the S-PAF-, angiotensin II-, and vasopressin-induced elevation in cytosolic Ca2+. We propose that this desensitization is possibly mediated by diacylglycerol formed in response to PAF.     

Tumour promotor 12-O-tetradecanoylphorbol 13-acetate inhibits angiotensin II-induced inositol phosphate production and cytosolic Ca2+ rise in rat renal mesangial cells

Preincubation of rat renal mesangial cells with 12-O-tetradecanoylphorbol 13-acetate (TPA) strongly inhibited the increases of inositol phosphates and of free cytosolic Ca2+ induced by angiotensin II (10(-7) M). TPA had no significant effect on the basal values of inositol phosphates and of free cytosolic Ca2+. Inhibition appeared already after 1 min and was maximal after 5 min. These effects occur without significant changes on angiotensin II binding in intact cells. The concentration of TPA needed (10(-9)-10(-7) M) was in the range believed to cause specifically an activation of protein kinase C. Furthermore the biologically inactive phorbol ester 4 alpha-phorbol 12,13-didecanoate was without effect. From the entirety of these results it is likely that protein kinase C inhibits angiotensin II activation of phospholipase C at a stage distal to receptor occupancy.     

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.     

A mammalian capacitative calcium entry channel homologous to Drosophila TRP and TRPL.

Intracellular Ca2+ signalling evoked by Ca2+ mobilizing agonists, like angiotensin II in the adrenal gland, involves the activation of inositol(1,4,5)trisphosphate(InsP3)-mediated Ca2+ release from internal stores followed by activation of a Ca2+ influx termed capacitative calcium entry. Here we report the amino acid sequence of a functional capacitative Ca2+ entry (CCE) channel that supports inward Ca2+ currents in the range of the cell resting potential. The expressed CCE channel opens upon depletion of Ca2+ stores by InsP3 or thapsigargin, suggesting that the newly identified channel supports the CCE coupled to InsP3 signalling.     

ACTH-induced inhibition of the action of angiotensin II in bovine zona glomerulosa cells. A modulatory effect of cyclic AMP on the angiotensin II receptor.

Both angiotensin II and adrenocorticotropic hormone (ACTH) are well known to play a crucial role on the regulation of aldosterone production in adrenal glomerulosa cells. Recent observations suggest that the steroidogenic action of ACTH is mediated via the cAMP messenger system, whereas angiotensin II acts mainly through the phosphoinositide pathway. However, there have been no reports concerning the interaction between the cAMP messenger system activated by ACTH and the Ca2+ messenger system induced by angiotensin II. Both ACTH and angiotensin II simultaneously act on adrenal cells for regulating steroidogenesis under physiological conditions. Thus the present experiments were performed to examine the effect of ACTH on the action of angiotensin II by measuring angiotensin II receptor activity, cytosolic Ca2+ movement, and aldosterone production. The major findings of the present study are that short-term exposure to a high dose of ACTH (10(-7) M) inhibited 125I-angiotensin II binding to bovine adrenal glomerulosa cells, decreased the initial spike phase of [Ca2+]i induced by angiotensin II, and inhibition of angiotensin II-induced aldosterone production. Low dose of ACTH (10(-10) M), which did not increase cAMP formation, did not affect angiotensin II receptor activity. These studies have shown that angiotensin II receptors of bovine adrenal glomerulosa cells can be down-regulated by 1 mM dibutyryl cyclic AMP, as well as by effectors which are able to activate cAMP formation (10(-7) M ACTH and 10(-5) M forskolin). The rapid decrease in angiotensin II receptors induced by 10(-7)M ACTH was associated with a decreased steroidogenic responsiveness and a decreased rise in the [Ca2+]i response induced by angiotensin II. These studies show that the cAMP-dependent processes activated by ACTH have the capacity to interfere with signal transduction mechanisms initiated by receptors for angiotensin II.     

Diversity in responses from endogenous and expressed mammalian receptors which cause chloride ion efflux from ovarian follicles of Xenopus laevis

Inositol phosphates are produced in ovarian follicles of Xenopus laevis on activation of endogenous acetylcholine receptors, which also stimulates Ca2+ release and efflux of Cl- ions detected electrophysiologically. Inositol phosphates were not detectable on activation of endogenous angiotensin II receptors which did, however, stimulate both a dose-dependent Ca2+ efflux and a depolarizing current very similar in maximum size and other characteristics to those caused by acetylcholine action. In contrast, activation of exogenous receptors for angiotensin II expressed by microinjected mRNA extracted from bovine adrenal did form measurable inositol phosphates. Also, the endogenous electrophysiological responses to angiotensin II and acetylcholine desensitize homologously but fail to cross-desensitize (Lacy, McIntosh, and McIntosh, 1989, Biochem. Biophys. Res. Commun, 159, 658-663). It appears that endogenous ovarian angiotensin II receptors in Xenopus activate a different transduction mechanism from endogenous acetylcholine receptors and expressed mammalian adrenal angiotensin II receptors and/or may be sited in the electrically connected follicular cells rather than in the oocyte itself.     

Inositol phosphate release and steroidogenesis in rat adrenal glomerulosa cells. Comparison of the effects of endothelin, angiotensin II and vasopressin.

Endothelin has steroidogenic activity in adrenal glomerulosa cells, as do two other vasoconstrictor peptides, angiotensin II and vasopressin. The steroidogenic activities of angiotensin II and vasopressin are probably mediated via the phosphatidylinositol-turnover pathway and associated changes in cytosolic Ca2+ concentration. Endothelin caused a steroidogenic response, which was small compared with that to angiotensin II and quantitatively similar to the vasopressin response. Cytosolic free Ca2+ responses were similarly higher to angiotensin II than to either of the other two peptides. However, total inositol phosphate responses to endothelin and angiotensin II were similar when these were measured over 20 min, and were quantitatively greater than the vasopressin response. A detailed study has been made of the phosphatidylinositol-turnover response to endothelin in comparison with responses to angiotensin II and vasopressin. Each of the three peptides produced a rapid and transient rise in Ins(1,4,5)P3 (max. 5-15 s), followed by a slow sustained rise. Ins(1,4,5)P3 was metabolized by both dephosphorylation and phosphorylation pathways, but the relative importance of the two metabolic pathways was different under stimulation by each of the three peptides. These findings show that adrenal glomerulosa cells can distinguish between the stimulation of phosphatidylinositol turnover by three different effectors. These differences in the pathway may be associated with the observed different steroidogenic and Ca2+ responses to the three peptides.     

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

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

Angiotensin II causes phosphatidylinositol turnover and increases 1,2-diacylglycerol mass but is not mitogenic in rat liver T51B cells.

Proliferation in rat liver T51B cells has previously been shown to be initiated by the tyrosine-kinase activator epidermal growth factor. We have found that T51B cells also contain angiotensin II receptors, and, as the transforming mas oncogene has been identified as a functional angiotensin receptor [Jackson, Blair, Marshall, Goedert & Hanley (1988) Nature (London) 335, 437-440], we have investigated the possibility that angiotensin II might also regulate proliferation of T51B cells. Angiotensin II at concentrations up to 10 microM did not promote DNA synthesis, even in the presence of the co-mitogens serum (1%) or 12-O-tetradecanoylphorbol 13-acetate (TPA) (50 ng/ml). The addition of 1 microM angiotensin II to myo-[3H]inositol-radiolabelled T51B cells did however result in a rapid accumulation of multiple inositol phosphates as well as in an increase in intracellular Ca2+, demonstrating the coupling of the angiotensin receptor in these cells to a polyphosphoinositide-hydrolysing phospholipase C. The increases in both inositol phosphates and intracellular Ca2+ were lower in cells pretreated for 10 min with 50 ng of TPA/ml and potentiated by a 24 h pretreatment with TPA. In addition, angiotensin II increased 1,2-diacylglycerol levels. These results demonstrate that, although angiotensin II is capable of increasing phosphoinositide-derived second messengers in T51B cells, these responses are not sufficient to trigger DNA synthesis.     

The phosphatidylinositide-Ca2+ hypothesis does not apply to the steroidogenic action of corticotropin.

The hypothesis that ACTH (corticotropin) stimulates steroidogenesis by a mechanism that involves breakdown of polyphosphoinositides and increase in intracellular Ca2+ (called here the 'phosphatidylinositide-Ca2+ mechanism') was tested in Y-1 adrenal-tumour cells and in bovine fasciculata cells, by using incorporation of 32P and myo-[3H]inositol to study phospholipid metabolism, and quin-2 and fura 2 to measure intracellular Ca2+. As a positive control, we repeated experiments showing that angiotensin II stimulates glomerulosa cells by way of the phosphatidylinositide-Ca2+ mechanism, by using the same methods. With Y-1 and fasciculata cells, no change was observed in the incorporation of either of the labelled precursors into any phosphatidylinositide or into any of three major phosphoinositols, i.e. inositol phosphate, bisphosphate and trisphosphate. Moreover, no change in mass of any of these compounds was seen. No change was observed in the concentration of intracellular Ca2+ in Y-1 or fasciculata cells on addition of ACTH, by using either quin-2 or fura 2. By contrast, decreased incorporation of 32P into phosphatidylinositol bisphosphate and an increase in intracellular Ca2+ were seen when glomerulosa cells were treated with angiotensin II. It is concluded that the phosphatidylinositide-Ca2+ mechanism is not used by Y-1 adrenal or bovine fasciculata cells in the steroidogenic response to ACTH unless the mechanism is radically different from that seen with all other hormones so far tested in which this mechanism occurs.     

Potentiation of angiotensin II-stimulated phosphoinositide hydrolysis, calcium mobilization and contraction of renal mesangial cells upon down-regulation of protein kinase C

Long-term pretreatment of rat mesangial cells with 12-O-tetradecanoylphorbol 13-acetate (TPA) down-regulated protein kinase C activity and potentiated the angiotensin II-induced inositol trisphosphate (InsP3) formation. This increased response to angiotensin II occurred without a significant change in the receptor number or Kd value of angiotensin II binding to the cells. The biologically inactive phorbol ester 4 alpha-phorbol 12,13-didecanoate was without effect on angiotensin II-stimulated InsP3 generation. Long-term pretreatment with TPA also increased the angiotensin II-induced mobilization of Ca2+ and the subsequent contraction of mesangial cells.     

Intracellular angiotensin II activates rat myometrium

Angiotensin II is a modulator of myometrial activity; both AT(1) and AT(2) receptors are expressed in myometrium. Since in other tissues angiotensin II has been reported to activate intracellular receptors, we assessed the effects of intracellular administration of angiotensin II via microinjection on myometrium, using calcium imaging. Intracellular injection of angiotensin II increased cytosolic Ca(2+) concentration ([Ca(2+)](i)) in myometrial cells in a dose-dependent manner. The effect was abolished by the AT(1) receptor antagonist losartan but not by the AT(2) receptor antagonist PD-123319. Disruption of the endo-lysosomal system, but not that of Golgi apparatus, prevented the angiotensin II-induced increase in [Ca(2+)](i). Blockade of AT(1) receptor internalization had no effect, whereas blockade of microautophagy abolished the increase in [Ca(2+)](i) produced by intracellular injection of angiotensin II; this indicates that microautophagy is a critical step in transporting the peptide into the endo-lysosomes lumenum. The response to angiotensin II was slightly reduced in Ca(2+)-free saline, indicating a major involvement of Ca(2+) release from internal stores. Blockade of inositol 1,4,5-trisphosphate (IP(3)) receptors with heparin and xestospongin C or inhibition of phospholipase C (PLC) with U-73122 abolished the response to angiotensin II, supporting the involvement of PLC-IP(3) pathway. Angiotensin II-induced increase in [Ca(2+)](i) was slightly reduced by antagonism of ryanodine receptors. Taken together, our results indicate for the first time that in myometrial cells, intracellular angiotensin II activates AT(1)-like receptors on lysosomes and activates PLC-IP(3)-dependent Ca(2+) release from endoplasmic reticulum; the response is further augmented by a Ca(2+)-induced Ca(2+) release mechanism via ryanodine receptors activation.     

Epidermal growth factor activates store-operated Ca2+ channels through an inositol 1,4,5-trisphosphate-independent pathway in human glomerular mesangial cells

One of the fastest cellular responses following activation of epidermal growth factor receptor is an increase in intracellular Ca2+ concentration. This event is attributed to a transient Ca2+ release from internal stores and Ca2+ entry from extracellular compartment. Store-operated Ca2+ channels are defined the channels activated in response to store depletion. In the present study, we determined whether epidermal growth factor activated store-operated Ca2+ channels and further, whether depletion of internal Ca2+ stores was required for the epidermal growth factor-induced Ca2+ entry in human glomerular mesangial cells. We found that 100 nm epidermal growth factor activated a Ca2+-permeable channel that had identical biophysical and pharmacological properties to channels activated by 1 microm thapsigargin in human glomerular mesangial cells or A431 cells. The epidermal growth factor-induced Ca2+ currents were completely abolished by a selective phospho-lipase C inhibitor, U73122. However, xestospongin C, a specific inositol 1,4,5-trisphosphate receptor inhibitor, did not affect the membrane currents elicited by epidermal growth factor despite a slight reduction in background currents. Following emptying of internal Ca2+ stores by thapsigargin, epidermal growth factor still potentiated the Ca2+ currents as determined by the whole-cell patch configuration. Furthermore, epidermal growth factor failed to trigger measurable Ca2+ release from endoplasmic reticulum. However, another physiological agent linked to phospholipase C and inositol 1,4,5-trisphosphate cascade, angiotensin II, produced a striking Ca2+ transient. These results indicate that epidermal growth factor activates store-operated Ca2+ channels through an inositol 1,4,5-trisphosphate-independent, but phospholipase C-dependent, pathway in human glomerular mesangial cells.