scholarly journals Regulation of the formation of inositol phosphates by calcium, guanine nucleotides and ATP in digitonin-permeabilized bovine adrenal chromaffin cells

1991 ◽  
Vol 279 (2) ◽  
pp. 447-453 ◽  
Author(s):  
D A Eberhard ◽  
R W Holz
Keyword(s):  
Chromaffin Cells ◽  
Inositol Phosphates ◽  
Purinergic Receptor ◽  
Receptor Activation ◽  
Maximal Response ◽  
Intact Cells ◽  
Permeabilized Cells ◽  
Adrenal Chromaffin ◽  
Phosphatidylinositol 4 ◽  
Concentration Response Curve

Both micromolar Ca2+ and guanosine 5′-[gamma-thio]triphosphate (GTP[S]) stimulated the formation of inositol phosphates (InsPs) in digitonin-permeabilized chromaffin cells prelabelled with [3H]inositol. The production of InsPs was potentiated by ATP. Guanosine 5′-[beta-thio]diphosphate (GDP[S]) caused a GTP-reversible shift to higher concentrations in the Ca(2+)-concentration-response curve for the release of InsPs without changing the maximal response. GTP[S] caused a shift to lower concentrations of Ca2+ and also increased the maximal response. The effects of GTP[S] and Ca2+ were synergistic. Although as much as 80% of the InsPs were derived from phosphatidylinositol 4-phosphate (PtdInsP) or 4,5-bisphosphate (PtdInsP2), the amount of InsPs produced could be several times the total amount of PtdInsP and PtdInsP2 in the cells and was largely accounted for by a decrease in PtdIns. The levels of labelled PtdInsP and PtdInsP2 increased on stimulation with Ca2+, but decreased on stimulation with GTP[S] or the combination of Ca2+ and GTP[S]. Preincubation with Ca2+ and ATP amplified the subsequent GTP[S]-induced production of InsPs. ATP and its gamma-thio and beta gamma-imido analogues stimulated the formation of InsPs in intact cells. However, only ATP potentiated the responses to Ca2+ and GTP[S] in permeable cells. Our main conclusions are: (1) a GTP-binding protein participates in the Ca(2+)-induced production of InsPs by phospholipase C, and (2) ATP markedly potentiates the stimulated formation of InsPs, an effect with arises from its role in polyphosphoinositide synthesis and does not involve purinergic receptor activation in permeabilized cells. The data also suggest that the different effects of Ca2+ and GTP[S] on polyphosphoinositide synthesis probably contribute to the synergistic action of Ca2+ and GTP[S] on the generation of InsPs.

scholarly journals Inhibition of prostaglandin E2-induced phosphoinositide metabolism by phorbol ester in bovine adrenal chromaffin cells

1988 ◽  
Vol 255 (3) ◽  
pp. 957-962 ◽  
Author(s):  
H Yokohama ◽  
M Negishi ◽  
K Sugama ◽  
H Hayashi ◽  
S Ito ◽  
...  
Keyword(s):  
Prostaglandin E2 ◽  
Phorbol Ester ◽  
Chromaffin Cells ◽  
Inositol Phosphates ◽  
Phosphoinositide Metabolism ◽  
Adrenal Chromaffin Cells ◽  
Gtp Binding Protein ◽  
Intact Cells ◽  
Gtp Binding ◽  
Adrenal Chromaffin

In bovine adrenal chromaffin cells, prostaglandin E2 (PGE2) stimulates the formation of inositol phosphates and Ca2+ mobilization through its specific receptor [Yokohama, Tanaka, Ito, Negishi, Hayashi & Hayaishi (1988) J. Biol. Chem. 263, 1119-1122]. Here we show that PGE2-induced phosphoinositide metabolism was blocked by pretreatment with 12-O-tetradecanoylphorbol 13-acetate (TPA). Using intact cells, we also examined the inhibitory effect of TPA on the individual steps of the activation process of phosphoinositide metabolism. The inhibition was observed within 1 min and complete by 10 min after addition of 1 microM-TPA, and half-maximal inhibition by TPA occurred at 20 nM. TPA prevented Ca2+ mobilization induced by PGE2, but not by the Ca2+ ionophore ionomycin. The inactive phorbol ester 4 alpha-phorbol 12,13-didecanoate did not inhibit the formation of inositol phosphates and Ca2+ mobilization by PGE2. TPA treatment affected neither the high-affinity binding of [3H]PGE2 to intact cells and membrane fractions nor the ability of guanosine 5′-[gamma-thio]triphosphate to decrease the binding in membrane fractions. TPA also abolished phosphoinositide metabolism induced by muscarinic-receptor activation. NaF plus AlCl3 and ionomycin caused the accumulation of inositol phosphates, probably by directly activating a GTP-binding protein(s) and phospholipase C respectively; neither accumulation was inhibited by TPA treatment. These results suggest that protein kinase C serves as a feedback regulator for PGE2-induced phosphoinositide metabolism. The site of action of TPA appears to be distal to the coupling of the receptor to GTP-binding protein, but on a component(s) specific to the agonist-induced phosphoinositide metabolism.

scholarly journals Exocytosis from permeabilized bovine adrenal chromaffin cells is differently modulated by guanosine 5′-[γ-thio]triphosphate and guanosine 5′-[β γ-imido]triphosphate. Evidence for the involvement of various guanine nucleotide-binding proteins

1992 ◽  
Vol 284 (2) ◽  
pp. 321-326 ◽  
Author(s):  
G Ahnert-Hilger ◽  
U Wegenhorst ◽  
B Stecher ◽  
K Spicher ◽  
W Rosenthal ◽  
...  
Keyword(s):  
G Protein ◽  
Chromaffin Cells ◽  
Inhibitory Effect ◽  
Adrenal Chromaffin Cells ◽  
Prolonged Incubation ◽  
Permeabilized Cells ◽  
Alpha Subunits ◽  
Cytoplasmic Material ◽  
Streptolysin O ◽  
Adrenal Chromaffin

1. In bovine adrenal chromaffin cells made permeable either to molecules less than or equal to 3 kDa with alphatoxin or to proteins less than or equal to 150 kDa with streptolysin O, the GTP analogues guanosine 5′-[beta gamma-imido]triphosphate (p[NH]ppG) and guanosine 5′-[gamma-thio]triphosphate (GTP[S]) differently modulated Ca(2+)-stimulated exocytosis. 2. In alphatoxin-permeabilized cells, p[NH]ppG up to 20 microM activated Ca(2+)-stimulated exocytosis. Higher concentrations had little or no effect. At a free Ca2+ concentration of 5 microM, 7 microM-p[NH]ppG stimulated exocytosis 6-fold. Increasing the free Ca2+ concentration reduced the effect of p[NH]ppG. Pretreatment of the cells with pertussis toxin prevented the activation of the Ca(2+)-stimulated exocytosis by p[NH]ppG. 3. In streptolysin O-permeabilized cells, p[NH]ppG did not activate, but rather inhibited Ca(2+)-dependent catecholamine release under all conditions studied. In the soluble cytoplasmic material that escaped during permeabilization with streptolysin O, different G-protein alpha-subunits were detected using an appropriate antibody. Around 15% of the cellular alpha-subunits were detected in the supernatant of permeabilized control cells. p[NH]ppG or GTP[S] stimulated the release of alpha-subunits 2-fold, causing a loss of about 30% of the cellular G-protein alpha-subunits under these conditions. Two of the alpha-subunits in the supernatant belonged to the G(o) type, as revealed by an antibody specific for G(o) alpha. 4. GTP[S], when present alone during stimulation with Ca2+, activated exocytosis in a similar manner to p[NH]ppG. Upon prolonged incubation, GTP[S], in contrast to p[NH]ppG, inhibited Ca(2+)-induced exocytosis from cells permeabilized by either of the pore-forming toxins. This effect was resistant to pertussin toxin. 5. The p[NH]ppG-induced activation of Ca(2+)-stimulated release from alphatoxin-permeabilized chromaffin cells may be attributed to one of the heterotrimeric G-proteins lost during permeabilization with streptolysin O. The inhibitory effect of GTP[S] on exocytosis is apparently not mediated by G-protein alpha-subunits, but by another GTP-dependent process still occurring after permeabilization with streptolysin O.

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.

scholarly journals Phosphatidylinositol-4,5-Biphosphate (PI(4,5)P2) Is Required for Rapid Endocytosis in Chromaffin Cells

2020 ◽  
Vol 2020 ◽  
pp. 1-6
Author(s):  
Fouad Azizi
Keyword(s):  
Neurotransmitter Release ◽  
Chromaffin Cells ◽  
Inhibitory Effect ◽  
Adrenal Chromaffin Cells ◽  
Patch Clamp Recording ◽  
Vesicle Recycling ◽  
Patch Pipette ◽  
Cell Membrane Capacitance ◽  
Adrenal Chromaffin ◽  
Phosphatidylinositol 4

Objective. Phosphoinositides play a regulatory role in clathrin-mediated endocytosis. However, their involvement in clathrin-independent endocytosis termed rapid endocytosis (RE), which is the mode of vesicle recycling during neurotransmitter release by transient fusion (known as kiss-and-run), has not been investigated. Here, we used patch-clamp recording of whole-cell membrane capacitance in adrenal chromaffin cells (ACC) to monitor changes of RE kinetics in response to pharmacological alteration of phosphatidylinositol-4,5-biphosphate (PI(4,5)P2) level by phenylarsine oxide (PAO) or antibody against phosphatidylinositol 4-kinase (AbPI4K). Results. We found that PAO and AbPI4K significantly abrogated RE kinetics. Infusion of PI(4,5)P2 through the patch pipette potentiated RE kinetics and reversed PAO- and AbPI4K-induced blockade of RE. Similarly, the application of the bifunctional thiol dithiothreitol (DTT) to PAO-treated cells completely prevented the inhibitory effect of PAO on RE. These findings indicate that PI(4,5)P2 is implicated in the signaling (mechanistic) process of RE in ACC.

The interaction of arginine vasopressin and oxytocin with bovine adrenal medulla cells

1989 ◽  
Vol 121 (1) ◽  
pp. 133-139 ◽  
Author(s):  
A. H. Taylor ◽  
G. St J. Whitley ◽  
S. S. Nussey
Keyword(s):  
Arginine Vasopressin ◽  
Chromaffin Cells ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Binding Capacity ◽  
Camp Production ◽  
Low Concentrations ◽  
Inositol Phosphate Accumulation ◽  
Adrenal Chromaffin ◽  
Induced Changes

ABSTRACT Binding of [3H]arginine vasopressin (AVP) and [3H]oxytocin to primary monolayer cultures of bovine adrenal chromaffin cells was time-dependent, and the binding sites for each peptide were specific and saturable. Studies with the V1 AVP antagonist d(CH2)5Tyr(Me)2-AVP, the V2 agonist 1-deamino-8-d-AVP and the V2 antagonist d(CH2)5d-Leu2,Val4-AVP indicated that the AVP receptor was V1 in specificity. Scatchard plots showed that each ligand interacted with a single high-affinity, low-capacity binding site: oxytocin dissociation constant (Kd) 0·29 ± 0·02 nmol/l, maximum binding capacity (Bmax) 7·6 ± 0·2 fmol/106 cells (or 4500 ± 102 sites/cell) (n = 3); AVP Kd 0·09±0·02 nmol/l, Bmax 5·1±0·63 fmol/106 cells (or 3050 ± 318 sites/cell) (n = 3). Although forskolin in concentrations from 1 nmol/l to 1 mmol/l stimulated cyclic AMP (cAMP) production in isolated chromaffin cells, this did not result in detectable catecholamine release. Neither AVP nor oxytocin in concentrations between 10 pmol/l and 10 μmol/l stimulated cAMP production in these cells. Vasopressin in concentrations as low as 10 pmol/l stimulated a sixfold increase in total inositol phosphates; the dose–response curve was triphasic. Oxytocin had little effect on total inositol phosphate accumulation at low concentrations, but concentrations above micromolar stimulated total inositol phosphate production approximately fourfold. There was no measurable release of catecholamines in response to either peptide. The physiological consequences of these AVP-induced changes in inositol phosphate concentrations remain to be elucidated. Journal of Endocrinology (1989) 121, 133–139

Inositol 1,4,5-trisphosphate, inositide flux rates and pool sizes during smooth muscle relaxation

1992 ◽  
Vol 262 (1) ◽  
pp. L100-L104 ◽  
Author(s):  
C. B. Baron ◽  
J. N. Pompeo ◽  
R. F. Coburn
Keyword(s):  
Smooth Muscle ◽  
Inositol Phosphate ◽  
Muscle Relaxation ◽  
Total Content ◽  
Receptor Activation ◽  
Smooth Muscle Relaxation ◽  
Maximal Response ◽  
Inositol 1,4,5 Trisphosphate ◽  
Phosphatidylinositol 4 ◽  
Pool Sizes

Decreases in D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] content and changes in inositol phospholipid contents occurred during the time of atropine-induced relaxation of swine tracheal smooth muscle contracted with 55 microM carbachol. Decrease in Ins(1,4,5)P3 occurred in a pool which makes up 40% of the total content of this inositol phosphate and which has access to Ins(1,4,5)P3 5-phosphatase and/or 3-kinase. A 50% decrease in this pool occurred at 16 s after addition of atropine and within 6-10 s after inhibition of phospholipase C (PLC). The maximal fall in Ins(1,4,5)P3 occurred at a time when force had only decreased 30% of the maximal response. A phosphatidylinositol 4-phosphate (PIP) pool linked to muscarinic receptor-activation increased 160% after addition of atropine, the maximal response occurring at a time when relaxation was 80% complete. The mechanisms for this increase were the maintained formation of PIP and phosphatidylinositol 4,5-bisphosphate (PIP2) even though PIP2 hydrolysis was inhibited and the apparent chemical equilibrium between PIP and PIP2.

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