scholarly journals Guanine nucleotide and NaF stimulation of phospholipase C activity in rat cerebral-cortical membranes. Studies on substrate specificity

1987 ◽  
Vol 244 (1) ◽  
pp. 35-40 ◽  
Author(s):  
I Litosch
Keyword(s):  
Substrate Specificity ◽  
Phospholipase C ◽  
Guanine Nucleotides ◽  
Guanine Nucleotide ◽  
Stimulatory Activity ◽  
Maximal Activation ◽  
Cortical Membranes ◽  
Phosphatidylinositol 4 ◽  
Hydrolysis Of ◽  
Stimulation Of

Guanyl-5′-yl imidodiphosphate (p[NH]ppG) stimulated a rapid phospholipase C-mediated breakdown of exogenously added phosphatidylinositol 4,5-bisphosphate (PIP2) in rat cerebral-cortical membranes, with half-maximal activation at approx. 33 microM. NaF stimulated phospholipase C activity, with half-maximal activation at 0.5 mM. Stimulation of phospholipase C activity by NaF exhibited pH optima at approx. 5.5 and 7.0, with the stimulatory activity at pH 7.0 greater than that at pH 5.5. With p[NH]ppG, only stimulation at pH 7.0 was observed. Neither p[NH]ppG nor NaF stimulated hydrolysis of added phosphatidylinositol (PI) or phosphatidylinositol 4-phosphate (PIP). Mg2+ (0.5 mM) potentiated p[NH]ppG-stimulated breakdown of PIP2. Ca2+ increased basal and p[NH]ppG-stimulated breakdown of PIP2. PI breakdown was stimulated only by high Ca2+ concentrations and was unaffected by p[NH]ppG at any Ca2+ concentration examined. These results indicate that, in cerebral-cortical membranes, activation of phospholipase C by guanine nucleotides or fluoride directly increases a phospholipase C activity which specifically hydrolyses PIP2.

scholarly journals Guanine nucleotides stimulate production of inositol trisphosphate in rat cortical membranes

1985 ◽  
Vol 232 (3) ◽  
pp. 799-804 ◽  
Author(s):  
R A Gonzales ◽  
F T Crews
Keyword(s):  
Inositol Phosphates ◽  
Adenine Nucleotides ◽  
Inositol Trisphosphate ◽  
Guanine Nucleotides ◽  
Guanine Nucleotide ◽  
Inositol Phospholipids ◽  
Cortical Membranes ◽  
Gamma S ◽  
Hydrolysis Of ◽  
Stimulation Of

The guanine nucleotides guanosine 5′[beta, gamma-imido]triphosphate (Gpp[NH]p), guanosine 5′-[γ-thio]-triphosphate (GTP gamma S), GMP, GDP and GTP stimulated the hydrolysis of inositol phospholipids by a phosphodiesterase in rat cerebral cortical membranes. Addition of 100 microM-Gpp[NH]p to prelabelled membranes caused a rapid accumulation of [3H)inositol phosphates (less than 30 s) for up to 2 min. GTP gamma S and Gpp [NH]p caused a concentration-dependent stimulation of phosphoinositide phosphodiesterase with a maximal stimulation of 2.5-3-fold over control at concentrations of 100 microM. GMP was as effective as the nonhydrolysable analogues, but much less potent (EC50 380 microM). GTP and GDP caused a 50% stimulation of the phospholipase C at 100 microM and at higher concentrations were inhibitory. The adenine nucleotides App[NH]p and ATP also caused small stimulatory effects (64% and 29%). The guanine nucleotide stimulation of inositide hydrolysis in cortical membranes was selective for inositol phospholipids over choline-containing phospholipids. Gpp[NH]p stimulated the production of inositol trisphosphate and inositol bisphosphate as well as inositol monophosphate, indicating that phosphoinositides are substrates for the phosphodiesterase. EGTA (33 microM) did not prevent the guanine nucleotide stimulation of inositide hydrolysis. Calcium addition by itself caused inositide phosphodiesterase activation from 3 to 100 microM which was additive with the Gpp[NH]p stimulation. These data suggest that guanine nucleotides may play a regulatory role in the modulation of the activity of phosphoinositide phosphodiesterase in rat cortical membranes.

scholarly journals Phospholipase C-mediated hydrolysis of phosphatidylcholine is activated by muscarinic agonists

1989 ◽  
Vol 263 (1) ◽  
pp. 115-120 ◽  
Author(s):  
M T Diaz-Meco ◽  
P Larrodera ◽  
M Lopez-Barahona ◽  
M E Cornet ◽  
P G Barreno ◽  
...  
Keyword(s):  
Growth Factors ◽  
Phospholipase C ◽  
Guanine Nucleotide ◽  
Muscarinic Agonists ◽  
Guanine Nucleotide Binding Protein ◽  
Permeabilized Cells ◽  
Guanine Nucleotide Binding ◽  
Oncogene Products ◽  
Hydrolysis Of ◽  
Stimulation Of

The phospholipase C-catalysed breakdown of inositol-containing phospholipids is an important source of diacylglycerol in cells stimulated by several agonists. However, recent experimental evidence suggests that major phospholipids such as phosphatidylcholine may also be substrates of the phosphodiesteratic hydrolysis activated by hormones, growth factors and oncogene products. We show here that stimulation of muscarinic agonists activates the release of phosphocholine, which, along with diacylglycerol, is a metabolic product of phospholipase C-mediated hydrolysis of phosphatidylcholine. Fluoroaluminates mimic this muscarinic effect, strongly suggesting that carbachol-activated release of phosphocholine may be mediated by a guanine-nucleotide-binding protein. Evidence for this was obtained from experiments using permeabilized cells in which non-hydrolysable analogues of GTP activated phosphocholine release synergistically with carbachol.

scholarly journals Interaction of cerebral-cortical membranes with exogenously added phosphatidylinositol 4,5-bisphosphate. Effects on measured phospholipase C activity

1989 ◽  
Vol 261 (2) ◽  
pp. 325-331 ◽  
Author(s):  
I Litosch
Keyword(s):  
Phospholipase C ◽  
Guanine Nucleotides ◽  
Cortical Membranes ◽  
Degree Of Association ◽  
Hydrolysis Of

Exogenously added phosphatidylinositol 4,5-bisphosphate (PtdInsP2) is rapidly associated with cerebral-cortical membranes. Substrate association with membranes was promoted by Mg2+, but inhibited by bivalent chelators. Once associated with the membrane, the PtdInsP2 was resistant to displacement by EDTA. The apparent phospholipase C activity was dependent on the degree of association of substrate with membranes. After preincubation of membranes with substrate, PtdInsP2 hydrolysis was independent of the incubation volume, indicating that substrate and membrane-associated phospholipase C were not independently diluted. Hydrolysis of the membrane-associated substrate was stimulated by Ca2+, guanosine 5′-[beta gamma-imido]triphosphate (p[NH]ppG), guanosine 5′[gamma-thio]triphosphate and carbachol in the presence of p[NH]ppG. Carbachol in the absence of guanine nucleotides, GDP, GTP, ATP and pyrophosphate was ineffective. These results demonstrate that exogenously added PtdInsP2 substrate is rapidly associated with membranes and hydrolysed by a phospholipase C whose activity is regulated by guanine nucleotides and agonist in the presence of guanine nucleotides. Use of exogenously added substrate for studies on the regulation of membrane phospholipase C requires consideration as to possible effects of incubation conditions on the partitioning of substrate into membranes.

scholarly journals Zymosan-induced release of inositol phosphates at resting cytosolic Ca2+ concentrations in macrophages

1987 ◽  
Vol 242 (2) ◽  
pp. 441-445 ◽  
Author(s):  
J Moscat ◽  
C Herrero ◽  
P Garcia-Barreno ◽  
A M Municio
Keyword(s):  
Phospholipase C ◽  
Inositol Phosphates ◽  
Binding Protein ◽  
Nucleotide Binding ◽  
Guanine Nucleotide ◽  
Guanine Nucleotide Binding Protein ◽  
Guanine Nucleotide Binding ◽  
The Relationship ◽  
Hydrolysis Of ◽  
Stimulation Of

Hydrolysis of polyphosphoinositides by phosphodiesterase has been demonstrated to be involved in the control of cytosolic Ca2+ concentrations. The stimulation of Ca2+ ionophores of the release of inositol phosphates in macrophages, and other cells, together with the Ca2+ requirements for zymosan-induced phospholipase C activation, make unclear the relationship between Ca2+ mobilization and polyphosphoinositide hydrolysis. The results in the present paper strongly suggest that, for zymosan-induced phospholipase C activation, a previous increase in cytosolic Ca2+ is not a required event. These results also show that zymosan-activated release of inositol phosphates may be mediated by a guanine-nucleotide-binding protein.

scholarly journals Guanine nucleotides mediate stimulatory and inhibitory effects on cerebral-cortical membrane phospholipase C activity

1989 ◽  
Vol 261 (1) ◽  
pp. 245-251 ◽  
Author(s):  
I Litosch
Keyword(s):  
Phospholipase C ◽  
Pertussis Toxin ◽  
Inhibitory Effect ◽  
Guanine Nucleotides ◽  
Regulatory Effect ◽  
Inhibitory Effects ◽  
Gtp Binding ◽  
Maximum Inhibition ◽  
Cortical Membranes ◽  
Stimulation Of

In cerebral-cortical membranes, hydrolysis-resistant guanine nucleotides exert a dual regulatory effect on phospholipase C activity. Nanomolar concentrations of guanosine 5′-[beta gamma-imido]triphosphate (p[NH]ppG) or guanosine 5′-[gamma-thio]triphosphate (GTP[S]) inhibited basal phospholipase C activity, with a maximum inhibition of 30% at 10 nM. Increasing the concentration of p[NH]ppG or GTP[S] to over 10 nM resulted in a reversal of the inhibitory effect and onset of stimulation of phospholipase C activity. These inhibitory effects were blocked by 100 microM-guanosine 5′-[beta-thio]diphosphate. GTP was relatively ineffective in producing either stimulation or inhibition of phospholipase C activity. Similarly, ATP, adenosine 5′-[beta gamma-imido]triphosphate and GDP were also ineffective. Expression of the dual effects of guanine nucleotides was affected by the Mg2+ concentration. At 0.3 mM-Mg2+, both the inhibitory and the stimulatory components of p[NH]ppG action were evident. At 2.5 mM-Mg2+, only p[NH]ppG stimulation was observed. Pertussis-toxin treatment blocked the p[NH]ppG-mediated inhibition of phospholipase C activity. These results demonstrate that non-hydrolysable guanine nucleotides exert both a stimulatory and an inhibitory effect on membrane phospholipase C activity. These effects may be mediated through distinct GTP-binding proteins.

Forskolin stimulation of adenylate cyclase in human thyroid membranes

1985 ◽  
Vol 108 (2) ◽  
pp. 200-205 ◽  
Author(s):  
Kikuo Kasai ◽  
Yoshinobu Suzuki ◽  
Masaki Hiraiwa ◽  
Hisamoto Kuroda ◽  
Tatsushi Emoto ◽  
...  
Keyword(s):  
Adenylate Cyclase ◽  
Nucleotide Binding ◽  
Guanine Nucleotides ◽  
Human Thyroid ◽  
Guanine Nucleotide ◽  
Guanine Nucleotide Binding ◽  
Stimulation Of

Abstract. Forskolin stimulates adenylate cyclase in human thyroid membranes approximately 7-fold with halfmaximal stimulation occurring at 5–10 μm. Guanine nucleotides are not required for stimulation of the enzyme by forskolin. Forskolin-stimulation is additive or greater than additive with that of TSH or Gpp(NH)p-(above 1 μm). Different from TSH- or Gpp(NH)p-stimulation of adenylate cyclase, uncoupling of the guanine nucleotide-binding regulatory component by increasing concentrations of MnCl2 did not result in uncoupling of forskolin stimulation. The finding indicates that forskolin may mainly act on the catalytic component of adenylate cyclase. From the present study, it is suggested that the diterpene forskolin stimulates adenylate cyclase in human thyroid membranes by a novel mechanism that differs from TSH- or Gpp(NH)p-stimulation, and that the diterpene may be a useful tool to investigate the metabolism of thyroid and its regulation in normal and pathological situations.

ATP stimulation of Na+/Ca2+exchange in cardiac sarcolemmal vesicles

1998 ◽  
Vol 274 (3) ◽  
pp. C724-C733 ◽  
Author(s):  
Graciela Berberián ◽  
Cecilia Hidalgo ◽  
Reinaldo Dipolo ◽  
Luis Beaugé
Keyword(s):  
Alkaline Phosphatase ◽  
Phospholipase C ◽  
Michaelis Constant ◽  
Apparent Affinity ◽  
Phosphatidylinositol 4 ◽  
Sarcolemmal Vesicles ◽  
Stimulation Of

In cardiac sarcolemmal vesicles, MgATP stimulates Na+/Ca2+exchange with the following characteristics: 1) increases 10-fold the apparent affinity for cytosolic Ca2+; 2) a Michaelis constant for ATP of ∼500 μM; 3) requires micromolar vanadate while millimolar concentrations are inhibitory; 4) not observed in the presence of 20 μM eosin alone but reinstated when vanadate is added; 5) mimicked by adenosine 5′- O-(3-thiotriphosphate), without the need for vanadate, but not by β,γ-methyleneadenosine 5′-triphosphate; and 6) not affected by unspecific protein alkaline phosphatase but abolished by a phosphatidylinositol-specific phospholipase C (PI-PLC). The PI-PLC effect is counteracted by phosphatidylinositol. In addition, in the absence of ATP,l-α-phosphatidylinositol 4,5-bisphosphate (PIP2) was able to stimulate the exchanger activity in vesicles pretreated with PI-PLC. This MgATP stimulation is not related to phosphorylation of the carrier, whereas phosphorylation appeared in the phosphoinositides, mainly PIP2, that coimmunoprecipitate with the exchanger. Vesicles incubated with MgATP and no Ca2+ show a marked synthesis ofl-α-phosphatidylinositol 4-monophosphate (PIP) with little production of PIP2; in the presence of 1 μM Ca2+, the net synthesis of PIP is smaller, whereas that of PIP2increases ninefold. These results indicate that PIP2 is involved in the MgATP stimulation of the cardiac Na+/Ca2+exchanger through a fast phosphorylation chain: a Ca2+-independent PIP formation followed by a Ca2+-dependent synthesis of PIP2.

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