Biomedical subjects

T Balla

Publications and source records attributed to T Balla.

At least 73 records · Page 4Linked to original sources

PubMed1989

Phosphoinositide metabolism and hormone action.

Calcium-mobilizing hormones elicit diverse cellular responses by activating plasma membrane receptors coupled to guanine nucleotide regulatory proteins that promote activation of phospholipase C and hydrolysis of polyphosphoinositides. The cleavage products of phosphatidylinositol bisphosphate, inositol 1,4,5-trisphosphate and diacylglycerol, act as primary signals for calcium mobilization and activation of calmodulin-dependent enzymes and protein kinase C. The metabolism of inositol 1,4,5-trisphosphate to several higher inositol phosphates provides additional compounds with potential regulatory actions on calcium movement and cell responses, and the discovery of multiple isomers of protein kinase C reveals a mechanism for diversification and specificity of hormone action among target cells controlled by the same basic transduction system. In addition to its rapid activation by many hormones and neurotransmitters, the calcium-phospholipid signaling system is utilized by several growth factors and oncogenes, consistent with its pervasive role in the control of both immediate and long-term responses of cells to external messenger molecules.

Animals↗

PubMed1989

Angiotensin II and potassium activate different calcium entry mechanisms in rat adrenal glomerulosa cells.

Initial 45Ca uptake was measured in isolated rat glomerulosa cells. A small reduction in membrane potential produced by increasing the K+ concentration from 2 to 3.6 mmol/l stimulated 45Ca uptake by about 35%, while a bigger depolarization induced by 18.5 mmol K+/l increased the uptake by about 100%. Since Ca2+ influx was already activated at a calculated membrane potential below -70 mV, and was found to be sensitive to the dihydropyridine antagonist nifedipine (1 mumol/l), but insensitive to nickel ions (100 mumol/l), it does not meet the criteria established for T- or L-type voltage-dependent Ca2+ channels. Exposure of glomerulosa cells to angiotensin II (AII) for 10 min also enhanced the rate of 45Ca influx. The effect of AII was not sensitive to 1 mumol nifedipine/l, but was strongly inhibited by 5-(N-4-chlorobenzyl)-N-(2',4'-dimethyl)benzamil (CBDMB, 30 mumol/l), an inhibitor of the Na+/Ca2+ antiporter. These observations suggest that during the sustained phase of stimulation with AII, a CBDMB-sensitive mechanism, rather than dihydropyridine-sensitive calcium channels, is involved in Ca2+ uptake in rat glomerulosa cells. The bulk Ca2+ influx did not correlate with aldosterone production; however, the maintained activity of different Ca2+ entry mechanisms seems to be essential for AII-induced aldosterone production.

Aldosterone↗

PubMed1988

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

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

Animals↗

PubMed1988

Angiotensin II and guanine nucleotides stimulate formation of inositol 1,4,5-trisphosphate and its metabolites in permeabilized adrenal glomerulosa cells.

Angiotensin II (AII) interacts with specific receptors in the adrenal glomerulosa cell and stimulates the hydrolysis of plasma membrane phosphoinositides by phospholipase C, with production of inositol 1,4,5-trisphosphate (Ins-1,4,5-P3) and subsequent mobilization of intracellular Ca2+. In electrically permeabilized, [3H]inositol-labeled glomerulosa cells, AII stimulated Ins-1,4,5-P3 production within 15 s with half-maximal potency of 10(-9) M. The nonhydrolyzable GTP analog, guanosine 5'-O-thiotriphosphate (GTP gamma S), stimulated Ins-1,4,5-P3 formation in a dose-dependent manner with half-maximal effect at 10(-7) M. AII-activated Ins-1,4,5-P3 production was further increased by guanine nucleotides. The rate at which GTP gamma S-stimulated inositol polyphosphate production was consistently slower than that of AII. In adrenal membrane preparations, GTP gamma S-stimulated polyphosphoinositide hydrolysis was enhanced by Ca2+, with half-maximal activity at 300 nM free Ca2+. Ins-1,4,5-P3 formation was also increased by NaF, further indicating the involvement of a guanine nucleotide regulatory protein. In addition to Ins-1,4,5-P3 and its metabolites formed during degradation via the 4-monophosphate pathway, AII and GTP gamma S stimulated the formation of the phosphorylated metabolite inositol 1,3,4,5-tetrakisphosphate and inositol 1,3,4-trisphosphate in permeabilized cells. The absence of a significant rise in inositol 1-monophosphate indicated that phosphatidylinositol hydrolysis was not stimulated by AII or GTP gamma S. Pretreatment of glomerulosa cells with pertussis toxin for 12 h before permeabilization did not inhibit AII- or GTP gamma S-stimulated inositol polyphosphate formation. However, treatment with cholera toxin, forskolin, or 8-Br-cAMP for 12 h enhanced both basal and ligand-stimulated Ins-1,4,5-P3 production. These observations suggest that agonist binding to the AII receptor activates a polyphosphoinositide-specific phospholipase C in the adrenal glomerulosa cell, and that a distinctive guanine regulatory protein is involved in this mechanism.

Adrenal Cortex↗

PubMed1988

Characterization of inositol 1,4,5-trisphosphate receptors and calcium mobilization in a hepatic plasma membrane fraction.

The distribution of hepatic binding sites for the calcium-mobilizing second messenger, inositol 1,4,5-trisphosphate (IP3), was analyzed in subcellular fractions of the rat liver by binding studies with [32P]IP3 and compared with the Ca2+ release elicited by IP3 in each fraction. Three major subcellular fractions enriched in plasma membrane, mitochondria, and endoplasmic reticulum were characterized for their 5'-nucleotidase, glucose-6-phosphatase, succinate reductase, and angiotensin II binding activities. The fraction enriched in plasma membrane showed 7- and 20-fold increases in IP3 binding capacity over those enriched in endoplasmic reticulum and mitochondria, respectively, and contained a single class of high-affinity binding sites with Kd of 1.7 +/- 1.0 nM and concentration of 239 +/- 91 fmol/mg protein. IP3 binding reached equilibrium in 30 min at 0 degrees C, and the half-time of dissociation was about 15 min. The specificity of the IP3 binding sites was indicated by their markedly lower affinities for inositol 1-phosphate, phytic acid, fructose 1,6-bisphosphate, 2,3-bisphosphoglycerate, and inositol 1,3,4,5-tetrakisphosphate. The Ca2+-releasing activity of IP3 in the subcellular fractions was monitored with the fluorescent indicator, Fura-2. All three fractions showed ATP-dependent Ca2+ uptake and rapidly released Ca2+ in response in IP3. The fraction enriched in plasma membrane was the most active in this regard, releasing 174 +/- 67 pmol Ca2+/mg of protein compared to 45 +/- 10 and 48 +/- 7 pmol/mg protein for the fractions enriched in endoplasmic reticulum and mitochondria, respectively. These data suggest that the [32P]IP3 binding sites represent specific intracellular receptors through which IP3 mobilizes Ca2+ from a storage site associated (or co-purifying) with the plasma membrane of the rat liver. It is likely that a specialized vesicular system (to which IP3 can bind and trigger the release of Ca2+) is located in close proximity with the plasma membrane and is thus adjacent to the site at which IP3 is produced during stimulation of the hepatocyte by Ca2+-mobilizing hormones.

Animals↗

PubMed1988

Multiple pathways of inositol polyphosphate metabolism in angiotensin-stimulated adrenal glomerulosa cells.

The production and metabolism of inositol 1,4,5-trisphosphate (Ins-1,4,5-P3) and other inositol polyphosphates was studied in cultured bovine adrenal glomerulosa cells prelabeled for 24 h with [3H]inositol. During stimulation with angiotensin II, Ins-1,4,5-P3 increased to a peak of 15-fold above basal within 10 s, followed by a second phase of continuous increase over the next 30 min. Ins-1,4,5-P3 formed during agonist stimulation was rapidly metabolized by two distinct pathways. The more direct metabolic route was via degradation by sequential dephosphorylations to form inositol 1,4-bisphosphate and inositol 4-phosphate, and ultimately inositol. Lithium ions inhibited both the formation and dephosphorylation of inositol 4-monophosphate, which is a specific product of inositol polyphosphate metabolism. In addition, a cyclical metabolic sequence was initiated by the 3-phosphorylation of Ins-1,4,5-P3 to form inositol 1,3,4,5-tetrakisphosphate. The Ins-1,4,5-P3 3-kinase responsible for this reaction had a Km of 0.4 microM for Ins-1,4,5-P3 and a Vmax of 208 pmol/min/mg and was stimulated by increased Ca2+ concentrations in the micromolar range. Inositol 1,3,4,5-tetrakisphosphate was then dephosphorylated to inositol 1,3,4-trisphosphate, which in turn was either further degraded to inositol 3,4-bisphosphate or rephosphorylated to inositol 1,3,4,6-tetrakisphosphate. Lithium ions also inhibited the production of inositol 3,4-bisphosphate, explaining the large accumulation of inositol 1,3,4-trisphosphate in cells stimulated in the presence of lithium. Prolonged exposure to angiotensin II in the presence of Li+ caused a progressive decline in inositol polyphosphate formation without depletion of the lipid precursor, phosphatidyl-inositol 4,5-bisphosphate, suggesting that an accumulating product of polyphosphoinositide hydrolysis (possibly diacylglycerol) has an inhibitory effect on the phospholipase C-catalyzed breakdown process. These results indicate that, in addition to its breakdown by sequential dephosphorylations through Ins-1,4-P2 and Ins-4-P, Ins-1,4,5-P3 undergoes a complex series of phosphorylations and dephosphorylations to form at least two inositol tetrakisphosphates and their metabolites. These newly defined pathways may provide additional regulatory steps in the mechanism of cell activation by angiotensin II and other Ca2+-mobilizing hormones.

Adrenal Cortex↗

PubMed1988

Control of glomerulosa cell function by angiotensin II: transduction by G-proteins and inositol polyphosphates.

1. The receptor-activated mechanisms that mediate the steroidogenic actions of angiotensin II (AII) have been characterized in rat and bovine adrenal glomerulosa cells. In rat adrenal cells, the AII receptor is coupled to a guanine nucleotide inhibitory protein which reduces adenylate cyclase activity and cyclic AMP production. However, receptor-mediated stimulation of aldosterone production by AII is exerted through a separate pertussis-insensitive nucleotide regulatory protein that subserves coupling of activated receptors to phospholipase C. 2. In AII-stimulated glomerulosa cells, hydrolysis of phosphatidylinositol (4,5)-bisphosphate (PIP2) by phospholipase C yields diacylglycerol and inositol 1,4,5-trisphosphate (Ins-P3), which act as second messengers by activating calcium-calmodulin and calcium-phospholipid dependent protein kinase pathways. Ins-1,4,5-P3 is a potent stimulus of intracellular calcium mobilization, and is promptly inactivated by two major routes of metabolism. Direct degradation of Ins-1,4,5-P3 by a 5-phosphatase gives inositol 1,4-bisphosphate which in turn is metabolized to inositol-4-monophosphate. The latter product can be derived only from higher inositol phosphates, and thus serves as a specific marker of polyphosphoinositide breakdown in agonist-stimulated cells. In contrast, inositol-1-phosphate is largely derived from phosphatidylinositol hydrolysis, which is not increased during the initial phase of AII action. 3. Ins-1,4,5-P3 formed in AII-stimulated glomerulosa cells is also phosphorylated by a calcium-calmodulin dependent 3-kinase to form inositol 1,3,4,5-tetrakisphosphate (Ins-P4), which is rapidly dephosphorylated to the biologically inactive Ins-1,4,5-P3 isomer, Ins-1,3,4-trisphosphate. The latter metabolite, like Ins-1,4,5-P3, is both degraded to lower phosphates (Ins-3,4,P2 and Ins-1,3-P2) and phosphorylated to form a new tetrakisphosphate isomer (Ins-1,3,4,6-P4). Ins-1,4,5-P3 formed during AII action is bound with high affinity to specific intracellular receptors through which InsP3 causes calcium mobilization during the initiation of cellular responses to AII and other calcium-dependent ligands.

Angiotensin II↗

PubMed1988

Na+-H+ and Na+-Ca2+ exchange in glomerulosa cells: possible role in control of aldosterone production.

Sodium uptake by rat adrenal glomerulosa cells was stimulated by intracellular acidosis evoked by Na+-propionate. This process was inhibited by 5-(N,N-hexamethylene) amiloride (HMA), a known inhibitor of the Na+-H+ exchange. These experiments demonstrate the existence of the Na+-H+ exchange in glomerulosa cells. Although amiloride inhibited the angiotensin II- and adrenocorticotropic hormone (ACTH)-induced aldosterone response, HMA, a more specific inhibitor of Na+-H+ exchange, failed to do that. 45Ca2+ influx and efflux were dependent on intra- and extracellular Na+ concentrations. Amiloride analogues, known to inhibit Na+-Ca2+ exchange, reduced basal 45Ca influx. Although we could not reveal the activation of Na+-Ca2+ exchange by angiotensin II, inhibitors of Na+-Ca2+ exchange also inhibited the angiotensin- and ACTH-induced aldosterone response of glomerulosa cells. Our results suggest that Na+-Ca2+ exchange supports the maintenance of basal Ca2+ level in the cytoplasma of glomerulosa cells, and amiloride derivatives inhibit aldosterone production by reducing Ca2+ level below resting values.

Adrenal Glands↗

PubMed1988

Effect of angiotensin II and arginine vasopressin on aldosterone production and phosphoinositide turnover in rat adrenal glomerulosa cells: a comparative study.

We have previously shown that arginine vasopressin (AVP) stimulates the production of aldosterone in isolated superfused adrenal glomerulosa cells by a mechanism that involves an increased turnover of phosphoinositides. In the present study we compared the characteristics of AVP- and angiotensin II (AII)-induced changes in phosphoinositide turnover and aldosterone production in the rat. Selected concentrations of the two peptides, which were equipotent in terms of the magnitude of changes induced in phosphoinositide turnover, stimulated aldosterone production to the same extent only in the initial phase of the stimulation. A sustained aldosterone response was only observed in AII-stimulated cells. On the other hand, the AVP-induced increase in incorporation of [32P]phosphate into phosphatidylinositol and the stimulation of inositol phosphate production were maintained during incubation. Preincubation of the cells with AVP failed to modify the effects of AII on phosphoinositide breakdown or aldosterone production. These results indicate that desensitization at the level of the receptor or at a post-receptor site is not responsible for the transient character of AVP-induced aldosterone production. Delayed activation of an inhibitory mechanism by AVP can also be excluded. Additivity of the stimulation of the phosphoinositide turnover observed at submaximally, but not maximally, effective concentrations of AII indicates that the two agonists act on the same phosphoinositide pool. We suggest that the sustained steroidogenic effect of AII involves an as yet unidentified mechanism, which is absent when the cells are stimulated with AVP.

Adrenal Glands↗

PubMed1987

Formation of inositol 1,3,4,6-tetrakisphosphate during angiotensin II action in bovine adrenal glomerulosa cells.

Angiotensin II stimulates the formation of several inositol polyphosphates in cultured bovine adrenal glomerulosa cells prelabelled with [3H] inositol. Analysis by high performance anion exchange chromatography of the inositol-phosphate compounds revealed the existence of two additional inositol tetrakisphosphate (InsP4) isomers in proximity to Ins-1,3,4,5-P4, the known phosphorylation product of Ins-1,4,5-trisphosphate and precursor of Ins-1,3,4-trisphosphate. Both of these new compounds showed a slow increase after stimulation with angiotensin II. The structure of one of these new InsP4 isomers, which is a phosphorylation product of Ins-1,3,4-P3, was deduced by its resistance to periodate oxidation to be Ins-1,3,4,6-P4. The existence of multiple cycles of phosphorylation-dephosphorylation reactions for the processing of Ins-1,4,5-P4 may represent a new aspect of the inositol-lipid related signalling mechanism in agonist-activated target cells.

Adrenal Glands↗

PubMed1987

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

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

Adrenal Cortex↗

PubMed1987

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., Spät, 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.

2,3-Diphosphoglycerate↗

PubMed1987

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

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

Adrenal Cortex↗

PubMed1987

Angiotensin II receptors and mechanisms of action in adrenal glomerulosa cells.

The plasma-membrane receptors, coupling mechanisms, and effector enzymes that mediate target-cell activation by angiotensin II (AII) have been characterized in rat and bovine adrenal glomerulosa cells. The AII holoreceptor is a glycoprotein of Mr approximately 125,000 under non-denaturing conditions. Photoaffinity labeling of AII receptors with azido-AII derivatives has shown size heterogeneity among the AII binding sites between species and target tissues, with Mr values of 55,000 to 79,000. Such variations in molecular size probably reflect differences in carbohydrate content of the individual receptor sites. The adrenal AII receptor, like that in other tissues, is coupled to the inhibitory guanine nucleotide inhibitory protein (Ni). However, studies with pertussis toxin have shown that stimulation of aldosterone production by AII is not mediated by Ni but by a pertussis-insensitive nucleotide regulatory protein of unidentified nature. Although Ni is not involved in the stimulatory action of AII on steroidogenesis, it does mediate the inhibitory effects of high concentrations of AII upon aldosterone production. The actions of AII on adrenal cortical function are thus regulated by at least two guanine nucleotide regulatory proteins that are selectively activated by increasing AII concentrations. The principal effector enzyme in AII action is phospholipase C, which is rapidly stimulated in rat and bovine glomerulosa after AII receptor activation. AII-induced breakdown of phosphatidylinositol bisphosphate (PIP2) and phosphatidylinositol phosphate (PIP) leads to formation of inositol 1,4,5-trisphosphate (IP3) and inositol 1,4-bisphosphate (IP2). These are metabolized predominantly to inositol-4-monophosphate, which serves as a marker of polyphosphoinositide breakdown, whereas inositol-1-phosphate is largely derived from phosphatidylinositol hydrolysis. The AII-stimulated glomerulosa cell also produces inositol 1,3,4-trisphosphate, a biologically inactive IP3 isomer formed from Ins-1,4,5-trisphosphate via inositol tetrakisphosphate (IP4) during ligand activation in several calcium-dependent target cells. The Ins-1,4,5-P3 formed during AII action binds with high affinity to specific intracellular receptors that have been characterized in the bovine adrenal gland and other AII target tissues, and may represent the sites through which IP3 causes calcium mobilization during the initiation of cellular responses.

Adrenal Glands↗

PubMed1987

Inositol 1,4,5-trisphosphate binds to a specific receptor and releases microsomal calcium in the anterior pituitary gland.

The properties of inositol 1,4,5-trisphosphate (InsP3) receptor sites in the anterior pituitary were evaluated by binding studies with InsP3 labeled with 32P to high specific radioactivity. Specific binding of Ins[32P]P3 was demonstrable in pituitary membrane preparations and was linearly proportional to the amount of membrane added over the range 0.5-2 mg of protein. Kinetic studies showed that specific InsP3 binding was half-maximal in about 40 sec and reached a plateau after 15 min at 0 degree C. Addition of 1 microM unlabeled InsP3 was followed by rapid dissociation of the bound ligand, according to a single exponential function with half-time of about 1 min. Scatchard analysis of the binding data was consistent with a single set of high-affinity sites with Kd of 1.1 +/- 0.4 nM and maximal binding capacity of 28 +/- 15 fmol/mg of protein (n = 6). The specificity of Ins[32P]P3 binding to these sites was illustrated by the much weaker affinity for structural analogs such as inositol 1-phosphate, phytic acid, 2,3-bisphosphoglycerate, and fructose 1,6-bisphosphate. To assess the functional relevance of the InsP3 binding sites, the Ca2+-releasing activity of InsP3 was measured in pituitary membrane preparations. In the presence of oligomycin (2.5 micrograms/ml), Ca2+ movements were monitored with the fluorescent indicator fura-2 (free acid). Under these conditions, 1 mM ATP caused rapid uptake of Ca2+ by a vesicular component of the membrane fraction. Addition of InsP3 (50-2000 nM) caused a dose-dependent release of Ca2+ with a half-maximal effect at 240 nM. In the presence of 3% polyethylene glycol, GTP also stimulated calcium release. However, a nonhydrolyzable GTP analog, guanosine 5'-[gamma-thio]triphosphate, did not release calcium and completely blocked the effect of GTP. Under physiological conditions within the cytosol, the high-affinity InsP3 binding sites characterized in pituitary membranes could serve as the putative receptors through which InsP3 triggers Ca2+ mobilization in the anterior pituitary gland.

Animals↗

PubMed1986

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.

Adrenal Glands↗

PubMed1985

The effect of angiotensin II on arachidonate metabolism in adrenal glomerulosa cells.

The effect of angiotensin II on arachidonate metabolism was examined in rat adrenal glomerulosa cells. Incorporation of both [3H]arachidonate and [32P]phosphate into phosphatidylinositol (PI) were significantly stimulated by angiotensin II. These effects were abolished by lithium, a cation, which was found suitable to prevent increased synthesis of PI in our previous study (T. Balla et al., FEBS Letters 171, 179, 1984). On the other hand, the phospholipase A2 inhibitor mepacrine failed to inhibit the increased labelling of PI. These observations suggest that the increased 3H labelling of PI occurs via CDP-diacylglycerol, and not via enhanced deacylation-reacylation cycle. The validity of this assumption was further supported, since angiotensin II failed to stimulate the formation of lyso-PI, as examined by both [32P]phosphate incorporation and pulse-chase techniques. Angiotensin II decreased the incorporation of [3H]arachidonate into phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Considering that we did not find arachidonate release either from phospholipids or from other possible arachidonate sources this decrease may not be due to dilution of the tracer. Thus we assume that angiotensin II may induce a shift in phospholipid synthesis from PC and PE to phosphoinositides. These observations indicate that the enhanced hydrolysis and synthesis of PI in response to angiotensin II is not associated with increased phospholipase A2 activity in adrenal glomerulosa cells.

Adrenal Glands↗

PubMed1985

Angiotensin II and FCCP mobilizes calcium from different intracellular pools in adrenal glomerulosa cells; analysis of calcium fluxes.

The aim of the present study was to examine the effect of angiotensin II on the different pools of exchangeable Ca2+ in isolated rat adrenal glomerulosa cells. On the basis of steady state analysis of 45Ca exchange curves at least three kinetically distinct Ca2+ compartments are present in these cells. The most rapidly exchangeable compartment was regarded as Ca2+ loosely bound to the glycocalyx and the other compartments were considered to be intracellular Ca2+ pools. The effect of angiotensin II on different intracellular compartments was examined by adding the hormone at different phases of Ca2+ washout. Angiotensin increased the rate of 45Ca efflux within 1.5 min when added at the beginning of the washout. This effect, however, could not be detected when the hormone was added at the 30th min of washout, indicating that at least one hormone sensitive pool had lost most of its radioactivity by this time. In contrast to angiotensin II, the mitochondrial uncoupler FCCP mobilized almost the same quantity of 45Ca irrespective of the time of its addition during the washout. This latter finding suggests that this presumably mitochondrial Ca2+ pool has a slow rate of exchange and thus differs from the pool initially mobilized by angiotensin II. The initial Ca2+ mobilizing effect of angiotensin II was also observed in a Ca2+-free media which contained EGTA, indicating that this effect is not triggered by increased Ca2+ influx. In the present study we demonstrate in the intact glomerulosa cell that angiotensin II mobilizes Ca2+ from an intracellular Ca2+ store which appears to be distinct from the FCCP-sensitive store.

Adrenal Glands↗