scholarly journals Cholera-toxin and corticotropin modulation of inositol phosphate accumulation induced by vasopressin and angiotensin II in rat glomerulosa cells

1988 ◽  
Vol 253 (3) ◽  
pp. 765-775 ◽  
Author(s):  
G Guillon ◽  
N Gallo-Payet ◽  
M N Balestre ◽  
C Lombard
Keyword(s):  
Angiotensin Ii ◽  
Cyclic Amp ◽  
Phospholipase C ◽  
Cholera Toxin ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Dependent Manner ◽  
Intact Cells ◽  
Adp Ribosylation ◽  
Glomerulosa Cells

Vasopressin (VP) and angiotensin II (AT II) stimulate the production of inositol phosphates (IP) in rat glomerulosa cells. Guanosine 5′-[gamma-thio]triphosphate (GTP[S]), but not VP or AT II, stimulates IP production in a myo-[3H]inositol-prelabelled glomerulosa-cell membrane preparation. In combination with GTP[S], these hormones potentiate the response to GTP[S], indicating the existence of a G-protein involved in the coupling of the VP and AT II receptor with the phospholipase C. ADP-ribosylation with pertussis toxin (IAP) revealed the specific labelling of a single molecule of 41 kDa. No significant inhibition of VP- or AT II-stimulated IP accumulation was detected in intact cells when the whole 41 kDa molecule was endogenously ADP-ribosylated by IAP treatment. On the contrary, when glomerulosa cells were infected with cholera toxin (CT), both the VP- and AT II-stimulated IP accumulations were inhibited in a dose-dependent manner. Yet these effects were partial even at high concentrations of CT, and could not be related to the ADP-ribosylation of ‘alpha s’ molecules. Similarly, when the cells were infected with 1 microgram of CT/ml, the specific binding of VP and AT II decreased by 50-60%. Such results may signify that the treatment primarily affects the densities of the hormone receptors. When glomerulosa cells were incubated for 15 h in the presence of 10 nM-corticotropin (ACTH), a condition in which the intracellular concentration of cyclic AMP was increased 3-fold, the maximum IP response to 0.1 microM-VP or -AT II was decreased by 50%. When similar experiments were carried out only after a 15 min incubation period with the same concentration of ACTH, the increase in cyclic AMP was more pronounced, but no inhibition of hormone-induced IP accumulation was observed. Altogether, these results may suggest that CT exerts its action on the VP- or AT II-sensitive phospholipase C systems via a prolonged increase in intracellular cyclic AMP.

scholarly journals Cholera toxin modulation of angiotensin II-stimulated inositol phosphate production in cultured vascular smooth muscle cells

1990 ◽  
Vol 265 (3) ◽  
pp. 799-807 ◽  
Author(s):  
L Socorro ◽  
R W Alexander ◽  
K K Griendling
Keyword(s):  
Smooth Muscle ◽  
Angiotensin Ii ◽  
Cyclic Amp ◽  
Vascular Smooth Muscle ◽  
Phospholipase C ◽  
Cholera Toxin ◽  
G Protein ◽  
Vascular Smooth Muscle Cells ◽  
Inositol Phosphate ◽  
Stimulation Of

Activation of phospholipase C by angiotensin II in vascular smooth muscle has been postulated to be mediated by an unidentified GTP-binding protein (G-protein). Using a permeabilized preparation of myo-[3H]inositol-labelled cultured vascular smooth muscle cells, we examined the ability of a non-hydrolysable analogue of GTP, guanosine 5′-[gamma-thio]triphosphate (GTP[S]), to stimulate inositol phosphate formation. GTP[S] (5 min exposure) stimulated inositol polyphosphate release by up to 3.8-fold in a dose-dependent manner, with an EC50 (concn. producing half-maximal stimulation) of approx. 50 microM. Inositol bisphosphate (IP2) and inositol trisphosphate (IP3) accumulations were also stimulated by NaF (5-20 mM). Furthermore, angiotensin II-induced inositol phosphate formation could be potentiated by a submaximal concentration of GTP[S] (10 microM), and this treatment appeared to interfere with the normal termination mechanism of the initial hormonal signal. The G-protein mediating angiotensin II-stimulated phospholipase C activation was insensitive to pertussis toxin at an exposure time and concentration which were sufficient to completely ADP-ribosylate all available substrate (100 ng/ml, 16 h). In contrast, a similar incubation with cholera toxin markedly inhibited angiotensin II-stimulated IP2 and IP3 release by 67 +/- 6% and 62 +/- 6% respectively. Cholera toxin appeared to inhibit angiotensin II stimulation of phospholipase C by a dual mechanism: it caused a 45% decrease in angiotensin II receptor number, and also inhibited G-protein transduction as assessed by GTP[S]-stimulated IP2 formation. This latter inhibition may be secondary to an increase in cyclic AMP, since it could be simulated by addition of dibutyryl cyclic AMP. Thus angiotensin II-stimulated inositol phosphate formation is cholera-toxin-sensitive, and is mediated by a pertussis-toxin-insensitive G-protein, which may be involved directly in termination of early signal generation.

scholarly journals Cyclic AMP agonists induce the phosphorylation of phospholipase C-τ and of a 76 kDa protein co-precipitated by anti-(phospholipase C-τ) monoclonal antibodies in BALB/c-3T3 cells. Relationship to inositol phosphate formation

1990 ◽  
Vol 272 (2) ◽  
pp. 297-303 ◽  
Author(s):  
N E Olashaw ◽  
S G Rhee ◽  
W J Pledger
Keyword(s):  
Monoclonal Antibodies ◽  
Growth Factor ◽  
Cyclic Amp ◽  
Phospholipase C ◽  
Cholera Toxin ◽  
Inositol Phosphate ◽  
Serine Phosphorylation ◽  
Intrinsic Activity ◽  
Phosphoinositide Metabolism ◽  
3T3 Cells

Previous studies have demonstrated enhanced phosphorylation of phospholipase C-tau (PLC-tau), a key regulatory enzyme in phosphoinositide metabolism, in cells treated with platelet-derived growth factor (PDGF) and epidermal growth factor, both of which act via specific receptor tyrosine kinases. Our studies on BALB/c-3T3 cells show that agents that promote cellular cyclic AMP accumulation also increase the phosphorylation, specifically the serine phosphorylation, of this enzyme. Increased phosphorylation of PLC-t (2-3-fold) was evident within 5-10 min of addition of isobutylmethylxanthine (IBMX) and either cholera toxin or forskolin to cells, and persisted for at least 3 h. Treatment of cells with cyclic AMP agonists also enhanced, with similar kinetics, the phosphorylation of a 76 kDa protein co-precipitated by anti-PLC-tau monoclonal antibodies. Brief exposure of cells to cholera toxin/IBMX or forskolin/IBMX decreased inositol phosphate formation induced by the GTP-binding protein (G-protein) activator aluminium fluoride by approx. 50%, but was without effect on PDGF-stimulated inositol phosphate formation. These findings suggest that PLC-tau, and perhaps the 76 kDa co-precipitated protein, are substrates of cyclic AMP-dependent protein kinase in BALB/c-3T3 cells: however, the lack of effect of cyclic AMP elevation on PDGF-stimulated inositol phosphate formation indicates that the intrinsic activity of PLC-tau is unaltered by cyclic AMP-mediated phosphorylation.

Angiotensin II-dependent proximal tubule sodium transport is mediated by cAMP modulation of phospholipase C

1994 ◽  
Vol 267 (5) ◽  
pp. C1239-C1245 ◽  
Author(s):  
J. R. Schelling ◽  
H. Singh ◽  
R. Marzec ◽  
S. L. Linas
Keyword(s):  
Angiotensin Ii ◽  
Proximal Tubule ◽  
Adenylyl Cyclase ◽  
Phospholipase C ◽  
Sodium Transport ◽  
Cultured Cells ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Ang Ii ◽  
Sodium Flux

Angiotensin II (ANG II) stimulates proximal tubule sodium transport by decreasing adenylyl cyclase activity. The role of ANG II-dependent phospholipase C is less certain. To determine the contribution of phospholipase C and adenylyl cyclase to apical (AP) ANG II-dependent sodium transport, unidirectional (AP to basolateral) 22Na flux was measured in rat proximal tubule cells cultured on permeable supports. AP ANG II (100 nM)-dependent sodium flux was prevented by preincubation with concentrations of the phospholipase C inhibitor U-73122 (1 microM) that blocked ANG II-dependent inositol phosphate formation. AP ANG II-dependent sodium flux was also abolished by preincubation with the intracellular calcium mobilization inhibitor 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester (TMB-8), further suggesting that ANG II-dependent sodium transport was mediated by inositol phosphates. Neither U-73122 nor TMB-8 prevented ANG II-dependent adenosine 3',5'-cyclic monophosphate (cAMP) decreases. Incubation with dibutyryl cAMP (10 microM) or forskolin (10 microM) prevented ANG II-dependent sodium flux as well as ANG II-dependent inositol phosphate formation. In conclusion, ANG II-dependent proximal tubule sodium transport in cultured cells was transduced by phospholipase C and adenylyl cyclase. The adenylyl cyclase effect on ANG II-dependent sodium transport was mediated by phospholipase C.

Effects of α-melanocyte-stimulating hormone on the cyclic AMP and phospholipid metabolism of rat adrenocortical cells

1986 ◽  
Vol 110 (3) ◽  
pp. 405-416 ◽  
Author(s):  
P. J. Hyatt ◽  
J. B. G. Bell ◽  
K. Bhatt ◽  
F. W. Chu ◽  
J. F. Tait ◽  
...  
Keyword(s):  
Angiotensin Ii ◽  
Cyclic Amp ◽  
Phosphatidic Acid ◽  
Inositol Phosphates ◽  
Phospholipid Metabolism ◽  
Zona Glomerulosa ◽  
Zona Fasciculata ◽  
Additional Increase ◽  
Glomerulosa Cells ◽  
Stimulation Of

ABSTRACT Results on the effects of peptides on the phospholipid metabolism and steroid and cyclic AMP (cAMP) outputs of rat adrenal capsular cells (96% zona glomerulosa, 4% zona fasciculata) were obtained in a series of three batch experiments. Their significance was examined by analysis of variance. Incorporation of [32P] into phosphatidylcholine, phosphatidic acid and phosphatidylinositol was measured. Production of [3H]inositol-1 monophosphate, inositol-1,4 bis-phosphate and inositol-1,4,5 tris-phosphate was estimated after prelabelling with [3H]inositol followed by 1 min incubation with a steroidogenic stimulus. Angiotensin II (0·25 nmol/l to 0·25 μmol/l) highly significantly (P < 0·01) stimulated aldosterone and corticosterone outputs, [32P] incorporation into phosphatidic acid and phosphatidylinositol (but not into phosphatidylcholine) and the production of the three [3H]inositol phosphates. Aldosterone and corticosterone outputs were stimulated by α-MSH (above 0·1 nmol/l). However, incorporation of [32P] was not significantly increased until 10 μmol α-MSH/l but, unlike with angiotensin II, incorporation into phosphatidylcholine was also then stimulated. Also, the production of the inositol phosphates was not increased significantly (P > 0·05) by any dose of α-MSH (10 nmol/l, 1 μmol/l and 0·1 mmol/l) used. Therefore, it can be concluded that α-MSH does not stimulate phospholipase C in rat zona glomerulosa cells. In further experiments, it was also found that there were significant increases in cAMP as well as in steroid outputs above 1 nmol α-MSH/l (highly significant above 10 nmol α-MSH/l). There were plateaux of the outputs of both steroids and cAMP from 0·1 to 1 μmol α-MSH/l. However, there were further increases in steroid and cAMP outputs of the capsular cells at higher doses. Concomitant results on the stimulation of corticosterone output by zona fasciculata–reticularis cells indicate that this additional increase was mostly due to the stimulation of the contaminating zona fasciculata cells. It was also confirmed that α-MSH preferentially stimulates steroidogenesis by the zona glomerulosa. However, under our conditions, α-MSH highly significantly increased the output of cAMP by both zona fasciculata and glomerulosa cells. J. Endocr. (1986) 110, 405–416

scholarly journals Thyroid-stimulating hormone stimulates increases in inositol phosphates as well as cyclic AMP in the FRTL-5 rat thyroid cell line

1987 ◽  
Vol 247 (3) ◽  
pp. 519-524 ◽  
Author(s):  
J B Field ◽  
P A Ealey ◽  
N J Marshall ◽  
S Cockcroft
Keyword(s):  
Cyclic Amp ◽  
Cell Line ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Thyroid Stimulating Hormone ◽  
Thyroid Cell ◽  
Dependent Manner ◽  
Thyroid Cell Line ◽  
Rat Thyroid ◽  
Stimulating Hormone

Studies were conducted to determine whether thyroid-stimulating hormone (TSH; thyrotropin), a hormone known to increase cytosol concentrations of cyclic AMP, also stimulates the formation of inositol phosphates in thyroid cells. TSH and noradrenaline both stimulated [3H]inositol phosphate formation in a concentration-dependent manner in the rat thyroid cell line, FRTL-5 cells, which had been prelabelled with [3H]inositol. The threshold concentration of TSH required to stimulate inositol phosphate formation was more than 20 munits/ml, which is approx. 10(3)-fold greater than that required for cyclic AMP accumulation and growth in these cells. We also demonstrate that membranes prepared from FRTL-5 cells possess a guanine nucleotide-activatable polyphosphoinositide phosphodiesterase, which suggests that activation of inositide metabolism in these cells may be coupled to receptors by the G-protein, Gp. Our findings suggest that two second-messenger systems exist to mediate the action of TSH in the thyroid.

scholarly journals Evidence that the epidermal growth factor receptor and non-tyrosine kinase hormone receptors stimulate phosphoinositide hydrolysis by independent pathways

1990 ◽  
Vol 270 (2) ◽  
pp. 337-344 ◽  
Author(s):  
J R Hepler ◽  
R A Jeffs ◽  
W R Huckle ◽  
H E Outlaw ◽  
S G Rhee ◽  
...  
Keyword(s):  
Angiotensin Ii ◽  
Phospholipase C ◽  
Tyrosine Phosphorylation ◽  
Hormone Receptors ◽  
Egf Receptor ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Phosphoinositide Hydrolysis ◽  
Inositol Phosphate Accumulation ◽  
Epidermal Growth

We have shown previously that exposure of a non-transformed continuous line of rat liver epithelial (WB) cells to epidermal growth factor (EGF), adrenaline, angiotensin II or [Arg8]vasopressin results in an accumulation of the inositol phosphates InsP1, InsP2 and InsP3 [Hepler, Earp & Harden (1988) J. Biol. Chem. 263, 7610-7619]. Studies were carried out with WB cells to determine whether the EGF receptor and other, non-tyrosine kinase, hormone receptors stimulate phosphoinositide hydrolysis by common, overlapping or separate pathways. The time courses for accumulation of inositol phosphates in response to angiotensin II and EGF were markedly different. Whereas angiotensin II stimulated a very rapid accumulation of inositol phosphates (maximal by 30 s), increases in the levels of inositol phosphates in response to EGF were measurable only following a 30 s lag period; maximal levels were attained by 7-8 min. Chelation of extracellular Ca2+ with EGTA did not modify this relative difference between angiotensin II and EGF in the time required to attain maximal phospholipase C activation. Under experimental conditions in which agonist-induced desensitization no longer occurred in these cells, the inositol phosphate responses to EGF and angiotensin II were additive, whereas those to angiotensin II and [Arg8]vasopressin were not additive. In crude WB lysates, angiotensin II, [Arg8]vasopressin and adrenaline each stimulated inositol phosphate formation in a guanine-nucleotide-dependent manner. In contrast, EGF failed to stimulate inositol phosphate formation in WB lysates in the presence or absence of guanosine 5′-[gamma-thio]triphosphate (GTP[S]), even though EGF retained the capacity to bind to and stimulate tyrosine phosphorylation of its own receptor. Pertussis toxin, at concentrations that fully ADP-ribosylate and functionally inactivate the inhibitory guanine-nucleotide regulatory protein of adenylate cyclase (Gi), had no effect on the capacity of EGF or hormones to stimulate inositol phosphate accumulation. In intact WB cells, the capacity of EGF, but not angiotensin II, to stimulate inositol phosphate accumulation was correlated with its capacity to stimulate tyrosine phosphorylation of the 148 kDa isoenzyme of phospholipase C. Taken together, these findings suggest that, whereas angiotensin II, [Arg8]vasopressin and alpha 1-adrenergic receptors are linked to activation of one or more phospholipase(s) C by an unidentified G-protein(s), the EGF receptor stimulates phosphoinositide hydrolysis by a different pathway, perhaps as a result of its capacity to stimulate tyrosine phosphorylation of phospholipase C-gamma.

scholarly journals Potentiation by cholera toxin of bradykinin-induced inositol phosphate production in the osteoblast-like cell line MC3T3-E1

1993 ◽  
Vol 292 (2) ◽  
pp. 401-408 ◽  
Author(s):  
Y Banno ◽  
T Sakai ◽  
T Kumada ◽  
Y Nozawa
Keyword(s):  
Cyclic Amp ◽  
Cholera Toxin ◽  
Cell Line ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Receptor Protein ◽  
Osteoblastic Cell ◽  
Osteoblastic Cell Line ◽  
Inositol Phosphate Production ◽  
Receptor Number

Cells of the osteoblastic cell line MC3T3-E1 were shown to contain at least three phosphatidylinositol-specific phospholipase C (PI-PLC) isoenzymes (PLC-beta, PLC-gamma and PLC-delta) by Western blotting analysis with various anti-PLC antibodies. Stimulation of inositol phosphate production in MC3T3-E1 cells by bradykinin (BK) occurred via a GTP-binding protein. Inositol phosphate formation on stimulation by BK was not affected by pretreatment with pertussis toxin, whereas it was potentiated by cholera toxin pretreatment. Elevation of cellular cyclic AMP levels by brief pretreatment with dibutyryl cyclic AMP or forskolin failed to enhance the BK-mediated generation of inositol phosphates, but long-term preincubation with these agents partially mimicked the action of the cholera toxin. Cholera toxin also caused an increase in BK receptor number. Cycloheximide, a protein biosynthesis inhibitor, prevented the potentiating actions of the cholera toxin and the cyclic AMP-elevating agents on BK-induced inositol phosphate production, and also inhibited the increase in BK receptor number. The specific binding of [3H]BK to the whole MC3T3-E1 cells in the presence or absence of cholera toxin was completely inhibited by the B2 BK receptor antagonist D-Arg[Hyp3,Thi5,8,D-Phe7]BK, but not by the B1 BK receptor agonist des-Arg9-BK. These data suggest that the activation of PI-PLC induced by cholera toxin in BK-stimulated MC3T3-E1 cells was caused by an enhancement of the synthesis of BK receptor protein(s), at least part of which was mediated by a sustained increase in the intracellular level of cyclic AMP.

scholarly journals Influence of bacterial toxins and forskolin upon vasopressin-induced inositol phosphate accumulation in WRK 1 cells

1989 ◽  
Vol 260 (3) ◽  
pp. 665-672 ◽  
Author(s):  
G Guillon ◽  
M N Balestre ◽  
C Lombard ◽  
F Rassendren ◽  
C J Kirk
Keyword(s):  
Cyclic Amp ◽  
Cholera Toxin ◽  
Pertussis Toxin ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Binding Capacity ◽  
Specific Binding ◽  
Phosphate Accumulation ◽  
Cyclic Amp Accumulation ◽  
Inositol Phosphate Accumulation

The accumulation of inositol phosphates in WRK 1 cells, stimulated with a range of vasopressin concentrations, was diminished by prior exposure to cholera toxin or forskolin, whilst that observed in the presence of maximal concentrations of the hormone was enhanced in pertussis-toxin-treated cells. In the presence of [32P]NAD+, both cholera toxin and pertussis toxin provoked the labelling of peptides with approximate Mrs of 45,000 and 41,000 respectively in the membranes of WRK 1 cells. Exposure to cholera toxin or forskolin for 15-18 h enhanced cyclic AMP accumulation in these cells. The concentrations of these agents which provoked half-maximal cyclic AMP accumulation were similar to those required to diminish receptor-mediated inositol phosphate accumulation by 50%. In contrast, half-maximal ADP-ribosylation of the 45,000Mr peptide needed 100-fold greater concentrations of the toxin than were effective in provoking half-maximal inhibition of inositol phosphate accumulation. Cholera toxin or forskolin also reduced the maximal specific binding, to intact WRK 1 cells, of both [3H][Arg8]vasopressin and the V1a antagonist [3H][beta-mercapto-beta,beta-cyclopentamethylenepropionic acid,O-methyl-Tyr2, Arg8]vasopressin. The kinetics for the loss of this binding capacity following cholera-toxin treatment were very similar to those describing the diminution of vasopressin-stimulated inositol phosphate accumulation in the same cells.

The properties of adrenal zona glomerulosa cells after purification by gravitational sedimentation

1974 ◽  
Vol 185 (1081) ◽  
pp. 375-407 ◽  
Keyword(s):  
Angiotensin Ii ◽  
Cyclic Amp ◽  
Microscopic Examination ◽  
Zona Glomerulosa ◽  
Maximal Response ◽  
Zona Fasciculata ◽  
Gravitational Sedimentation ◽  
Rat Adrenals ◽  
Glomerulosa Cells ◽  
Zona Glomerulosa Cells

The densities of latex spheres and biological cells can be reliably determined from their sedimentation rate in an albumin gradient under unit gravitational force. The densities of zona glomerulosa and fasciculata cells of rat adrenals were found to be 1.072 ± 0.004 and 1.040 ± 0.001 respectively. Purified zona glomerulosa cells of rat adrenals can be prepared by gravitational sedimentation of dispersed cells from capsule strippings of the gland, which originally contain 3 to10% zona fasciculata contamination. Electron and phase microscopic examination of the sedimented glomerulosa cells and their steroidogenic response to ACTH and cyclic AMP indicate that they are reasonably free of contamination from zona fasciculata cells. Electron microscopic examination of the purified glomerulosa cells indicates that most of them are reasonably normal in structure. Their basal production of corticosterone is decreased after sedimentation. However, their maximal response of corticosterone output to serotonin and potassium and their response to all potassium concentrations is not significantly altered, indicating normal function for the cells producing steroids. Their maximal responses to ACTH, valine angiotensin II and cyclic AMP are decreased, but, at the doses used, steroidogenesis by the zona fasciculata contamination in the unfractionated preparation would be stimulated by these substances. Purified zona glomerulosa cells have about the same maximal response of corticosterone output (about twofold) to potassium, valine and isoleucine angiotensin II, serotonin and ACTH. The maximal response of the purified zona glomerulosa cells to cyclic AMP is similar to that elicited by valine and isoleucine angiotensin II, potassium, serotonin or ACTH. This indicates that if these stimuli act by increasing cyclic AMP output, then the maximal response of corticosterone output (about twofold) is defined by the limited response of the biosynthetic pathways to cyclic AMP.

scholarly journals Calcium-sensitivity of inositol 1,4,5-trisphosphate metabolism in exocrine cells from the avian salt gland

1992 ◽  
Vol 282 (3) ◽  
pp. 703-710 ◽  
Author(s):  
J P Hildebrandt ◽  
T J Shuttleworth
Keyword(s):  
Substrate Concentration ◽  
Inositol Phosphates ◽  
Receptor Activation ◽  
Dependent Manner ◽  
Salt Glands ◽  
Concentration Dependent Manner ◽  
Intact Cells ◽  
Exocrine Cells ◽  
Tissue Homogenates

The generation of inositol phosphates upon muscarinic-receptor activation was studied in [3H]inositol-loaded exocrine cells from the nasal salt glands of the duck Anas platyrhynchos, and the metabolism of different inositol phosphates in vitro was studied in tissue homogenates, with particular reference to the possible interaction of changes in intracellular [Ca2+] ([Ca2+]i) with the metabolic processes. In intact cells, there was a rapid (within 15 s) generation of Ins(1,4,5)P3 and Ins(1,3,4,5)P4, followed by an accumulation of their breakdown products, Ins(1,3,4)P3 and inositol bis- and monophosphates. Ca(2+)-sensitivity of the Ins(1,4,5)P3 3-kinase was demonstrated in tissue homogenates, with the rate of phosphorylation increasing 2-fold at free Ca2+ concentrations greater than 1 microM. However, addition of calmodulin or the presence of the calmodulin inhibitor W-7 (up to 100 microM) had no effect. 3-Kinase activity increased proportionally with the initial Ins(1,4,5)P3 concentration up to 1 microM, but a 10-fold higher substrate concentration produced only a doubling in the phosphorylation rate. Ins(1,3,4,5)P4 was dephosphorylated to Ins(1,3,4)P3, which accumulated in the homogenate assays as well as in intact cells. Depending on its concentration, Ins(1,3,4)P3 was phosphorylated [in part to Ins(1,3,4,6)P4] or dephosphorylated. To investigate the Ca(2+)-sensitivity of the 3-kinase in intact cells, excess quin2 was used to buffer the receptor-mediated transient changes in [Ca2+]i in [3H]inositol-loaded cells. These experiments revealed that increasing [Ca2+]i from less than 100 to approx. 400 nM (i.e. within the physiological range) has no effect on the partitioning of Ins(1,4,5)P3 metabolism (phosphorylation versus dephosphorylation) and on the accumulation of Ins(1,4,5)P3 and Ins(1,3,4,5)P4. This indicates that activation of the 3-kinase by physiologically relevant Ca2+ concentrations may not play a major role in the generation of Ins(1,3,4,5)P4 signals upon receptor activation in these cells. The latter are mainly achieved by the receptor-mediated increase in Ins(1,4,5)P3 in the cell and its phosphorylation by the 3-kinase in a substrate-concentration-dependent manner.

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