scholarly journals Activation of muscarinic receptors in PC12 cells. Correlation between cytosolic Ca2+ rise and phosphoinositide hydrolysis

1986 ◽  
Vol 234 (3) ◽  
pp. 555-562 ◽  
Author(s):  
L M Vicentini ◽  
A Ambrosini ◽  
F Di Virgilio ◽  
J Meldolesi ◽  
T Pozzan
Keyword(s):  
Cyclic Amp ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Inositol Trisphosphate ◽  
Phosphodiesterase Inhibitors ◽  
Receptor Activation ◽  
Phosphoinositide Hydrolysis ◽  
Dibutyryl Cyclic Amp ◽  
Permeabilized Cells ◽  
Concentration Curves

The intracellular signals generated by carbachol activation of the muscarinic receptor [release of inositol phosphates as a consequence of phosphoinositide hydrolysis and rise of the cytosolic Ca2+ concentration ([Ca2+]i, measured by quin2)] were studied in intact PC12 pheochromocytoma cells that had been differentiated by treatment with nerve growth factor. When measured in parallel samples of the same cell preparation 30 s after receptor activation, the release of inositol trisphosphate and of its possible metabolites, inositol bis- and mono-phosphate, and the [Ca2+]i rise were found to occur with almost superimposable carbachol concentration curves. At the same time carbachol caused a decrease in the radioactivity of preloaded phosphatidylinositol 4,5-bisphosphate, the precursor of inositol trisphosphate. Neither the inositol phosphate nor the [Ca2+]i signal was modified by preincubation of the cells with either purified Bordetella pertussis toxin or forskolin, the direct activator of adenylate cyclase. Both signals were partially inhibited by dibutyryl cyclic AMP, especially when the nucleotide analogue was applied in combination with the phosphodiesterase inhibitors RO 201724 and theophylline. The latter drug alone profoundly inhibited the carbachol-induced [Ca2+]i rise, with only minimal effect on phosphoinositide hydrolysis. Because of the diverging results obtained with forskolin on the one hand, dibutyryl cyclic AMP and phosphodiesterase inhibitors on the other, the effects of the latter drugs are considered to be pharmacological, independent of the intracellular cyclic AMP concentration. Two further drugs tested, mepacrine and MY5445, inhibited phosphoinositide hydrolysis at the same time as the 45Ca2+ influx stimulated by carbachol. Taken together, our results concur with previous evidence obtained with permeabilized cells and cell fractions to indicate phosphatidylinositol 4,5-bisphosphate hydrolysis and [Ca2+]i rise as two successive events in the intracellular transduction cascade initiated by receptor activation. The strict correlation between the carbachol concentration curves for inositol trisphosphate generation and [Ca2+]i rise, and the inhibition by theophylline of the Ca2$ signal without major effects on inositol phosphate generation, satisfy important requirements of the abovementioned interpretation.

scholarly journals Evidence that muscarinic cholinergic receptors selectively interact with either the cyclic AMP or the inositol phosphate second-messenger response systems

1987 ◽  
Vol 247 (3) ◽  
pp. 793-796 ◽  
Author(s):  
J R Hepler ◽  
A R Hughes ◽  
T K Harden
Keyword(s):  
Cyclic Amp ◽  
Pertussis Toxin ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Second Messenger ◽  
Cholinergic Receptor ◽  
Receptor Activation ◽  
Phosphoinositide Hydrolysis ◽  
Response Systems ◽  
Muscarinic Cholinergic

The relative capacities of muscarinic cholinergic receptor (MR) and bradykinin (BK)-receptor activation to increase phosphoinositide hydrolysis and to increase cytosolic Ca2+ were compared in NG108-15 neuroblastoma x glioma and 1321N1 human astrocytoma cells. In 1321N1 cells, the muscarinic cholinergic agonist carbachol and BK each stimulated a concentration-dependent accumulation of inositol phosphates (K0.5 approximately 10 microM and approximately 10 nM respectively) and a rapid increase in cytosolic Ca2+ as determined by quin2 fluorescence. In NG108-15 cells, BK alone stimulated a pertussis-toxin-insensitive accumulation of inositol phosphates (K0.5 approximately 10 nM) under conditions in which pertussis toxin completely inhibited MR-mediated inhibition of adenylate cyclase. BK also stimulated a rapid increase in cytosolic Ca2+ in NG108-15 cells. In contrast, no MR-mediated increase in phosphoinositide hydrolysis or change in cytosolic Ca2+ concentration was observed in NG108-15 cells. These results support the idea that MR selectively interact with either the cyclic AMP or the inositol phosphate second-messenger systems.

Augmented phosphoinositide metabolism in aortas from genetically hypertensive rats

1990 ◽  
Vol 258 (1) ◽  
pp. H173-H178 ◽  
Author(s):  
M. B. Turla ◽  
R. C. Webb
Keyword(s):  
Phospholipase C ◽  
Vascular Reactivity ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Second Messengers ◽  
Receptor Activation ◽  
Phosphoinositide Hydrolysis ◽  
Phosphoinositide Metabolism ◽  
Hypertensive Rats ◽  
Wistar Kyoto

Recent studies suggest that serotonergic receptor activation is coupled to phospholipase C-mediated phosphoinositide hydrolysis, which results in the release of intracellular second messengers. The purpose of this study was to determine whether altered phosphoinositide metabolism is the basis for augmented vascular responsiveness to serotonin in genetic hypertension. Thoracic aortic segments isolated from stroke-prone spontaneously hypertensive rats (SHRSP) and Wistar-Kyoto normotensive rats (WKY) were labeled with myo-[3H]inositol and stimulated with serotonin in the presence of LiCl. Accumulation of [3H]inositol phosphates was then quantitated by column chromatography. Basal inositol phosphate accumulation and basal incorporation of myo-[3H]inositol into aortic cell membranes from SHRSP was not significantly different from WKY values. At 2.6 x 10(-7) to 2.6 x 10(-4) M serotonin, phosphoinositide metabolism was significantly augmented in aortae from SHRSP compared with WKY. Depolarization (100 mM KCl) did not increase phosphoinositide hydrolysis above basal levels in SHRSP or WKY. 2-Nitro-4-carboxyphenyl-N,N-diphenyl carbamate (NCDC), an inhibitor of phospholipase C, prevented the serotonin-induced phosphoinositide metabolism. NCDC also partially inhibited phasic contractions (responses in calcium-free solution) to serotonin in aortas from SHRSP and WKY. In conclusion, abnormal phosphoinositide metabolism may be one mechanism responsible for the characteristic increase in vascular reactivity to serotonin in hypertension.

scholarly journals Luteinizing hormone stimulates the formation of inositol trisphosphate and cyclic AMP in rat granulosa cells. Evidence for phospholipase C generated second messengers in the action of luteinizing hormone

1986 ◽  
Vol 238 (2) ◽  
pp. 597-604 ◽  
Author(s):  
J S Davis ◽  
L L Weakland ◽  
L A West ◽  
R V Farese
Keyword(s):  
Lipid Metabolism ◽  
Luteinizing Hormone ◽  
Cyclic Amp ◽  
Granulosa Cells ◽  
Cyclic Gmp ◽  
Time Course ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Second Messengers ◽  
Inositol Trisphosphate

The following studies were conducted to determine whether luteinizing hormone (LH), a hormone which increases cellular levels of cyclic AMP, also provokes increases in ‘second messengers’ derived from inositol lipid metabolism (i.e. inositol phosphates and diacylglycerol). Rat granulosa cells isolated from mature Graafian follicles were prelabelled for 3 h with myo-[2-3H]inositol. LH provoked rapid (5 min) and sustained (up to 60 min) increases in the levels of inositol mono-, bis, and trisphosphates (IP, IP2 and IP3, respectively). Time course studies revealed that IP3 was formed more rapidly than IP2 and IP following LH treatment. The response to LH was concentration-dependent with maximal increases at LH concentrations of 1 microgram/ml. LiCl (2-40 mM) enhanced the LH-provoked accumulation of all [3H]inositol phosphates, presumably by inhibiting the action of inositol phosphate phosphatases. The effectiveness of LH, however, was dependent on the concentration of lithium employed; maximal increases in IP were observed at 10 mM-LiCl, whereas maximal increases in IP2 and IP3 were observed at 20 mM- and 40 mM-LiCl, respectively. The stimulatory effects of LH on inositol phosphate and progesterone accumulation were also compared with changes in cyclic nucleotide levels. LH rapidly increased levels of inositol phosphates, progesterone and cyclic AMP, but transiently reduced levels of cyclic GMP. These results demonstrate that LH increases both cyclic AMP and inositol trisphosphate (and presumably diacylglycerol) in rat granulosa cells. Our findings suggest that two messenger systems exist to mediate the action of LH in granulosa cells.

scholarly journals Secretagogue-induced formation of inositol phosphates in rat exocrine pancreas. Implications for a messenger role for inositol trisphosphate

1984 ◽  
Vol 219 (2) ◽  
pp. 655-659 ◽  
Author(s):  
R P Rubin ◽  
P P Godfrey ◽  
D A Chapman ◽  
J W Putney
Keyword(s):  
Phospholipase C ◽  
Exocrine Pancreas ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Inositol Trisphosphate ◽  
Receptor Activation ◽  
Early Step ◽  
Pancreatic Acini ◽  
Rat Exocrine Pancreas ◽  
Stimulus Secretion Coupling

The formation of inositol phosphates in response to secretagogues was studied in rat pancreatic acini preincubated with [3H]inositol. Carbachol caused rapid increases in radioactive inositol phosphate, inositol bisphosphate and inositol trisphosphate . This effect was blocked by atropine, and also elicited by caerulein, but not by ionomycin or phorbol dibutyrate. Thus phospholipase C-mediated breakdown of polyphosphoinositides, with the resulting formation of inositol phosphates, may be an early step in the stimulus-secretion coupling pathway in exocrine pancreas. Inositol trisphosphate may function as a second messenger in the exocrine pancreas, coupling receptor activation to internal Ca2+ release.

Adrenergic stimulation of inositol phosphate accumulation in tracheal epithelium

1989 ◽  
Vol 66 (1) ◽  
pp. 504-508 ◽  
Author(s):  
T. Bainbridge ◽  
R. D. Feldman ◽  
M. J. Welsh
Keyword(s):  
Adrenergic Receptor ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Second Messengers ◽  
Receptor Activation ◽  
Adrenergic Stimulation ◽  
Cl Secretion ◽  
Phosphate Accumulation ◽  
Inositol Phosphate Accumulation ◽  
Stimulation Of

To determine whether inositol phosphates are important second messengers in the regulation of Cl- secretion by airway epithelia, we examined the relationship between inositol phosphate accumulation and Cl- secretion in response to adrenergic agonists. We found that epinephrine stimulated Cl- secretion and inositol phosphate accumulation with similar concentration dependence. Although isoproterenol stimulated Cl- secretion, there was no effect of beta-adrenergic receptor activation on inositol phosphate accumulation. In contrast, alpha 1-adrenergic receptor activation stimulated inositol phosphate accumulation but failed to induce Cl- secretion. Another Cl- secretagogue, prostaglandin E1, also failed to stimulate inositol phosphate accumulation. These data suggest that inositol phosphate accumulation is neither sufficient nor required for stimulation of Cl- secretion in cultured canine tracheal epithelial cells.

scholarly journals Effects of pertussis toxin on growth factor-stimulated inositol phosphate formation and DNA synthesis in Swiss 3T3 cells

1988 ◽  
Vol 249 (3) ◽  
pp. 917-920 ◽  
Author(s):  
C W Taylor ◽  
D M Blakeley ◽  
A N Corps ◽  
M J Berridge ◽  
K D Brown
Keyword(s):  
Growth Factor ◽  
Dna Synthesis ◽  
Pertussis Toxin ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Phosphoinositide Hydrolysis ◽  
Polypeptide Growth Factors ◽  
Swiss 3T3 ◽  
Swiss 3T3 Cell ◽  
Stimulation Of

We have compared the effects of pretreatment of Swiss 3T3 cell with pertussis toxin on the stimulation of DNA synthesis and phosphoinositide hydrolysis in response to a wide variety of mitogens. The toxin substantially inhibited the stimulation of DNA synthesis in response to a phorbol ester or various peptide and polypeptide growth factors irrespective of their ability to activate phosphoinositidase C. Production of inositol phosphates in response to platelet-derived growth factor, fibroblast growth factor and prostaglandin F2 alpha were unaffected by the toxin while bombesin- and vasopressin-stimulated formation of inositol phosphates were inhibited by only 27 and 23% respectively. These results argue against a major role for a pertussis toxin-sensitive G protein in coupling any of these mitogen receptors to activation of a phosphoinositidase C. Furthermore, the results suggest that the widespread inhibitory effects of pertussis toxin on mitogen-stimulated DNA synthesis may be unrelated to the toxin's limited actions on phosphoinositide hydrolysis.

scholarly journals Receptor-mediated gonadotropin action in the ovary. Regulatory role of cyclic nucleotide phosphodiesterase(s) in intracellular adenosine 3′:5′-cyclic monophosphate turnover and gonadotropin-stimulated progesterone production by rat ovarian cells

1979 ◽  
Vol 180 (1) ◽  
pp. 201-211 ◽  
Author(s):  
Salman Azhar ◽  
K. M. Jairam Menon
Keyword(s):  
Cyclic Amp ◽  
Kinase Activity ◽  
Phosphodiesterase Inhibitors ◽  
Protein Kinase Activity ◽  
Dibutyryl Cyclic Amp ◽  
Steroid Production ◽  
Progesterone Production ◽  
Ovarian Cells ◽  
Cyclic Amp Accumulation

The regulatory role of cyclic nucleotide phosphodiesterase(s) and cyclic AMP metabolism in relation to progesterone production by gonadotropins has been studied in isolated rat ovarian cells. Low concentrations of choriogonadotropin (0.4–5ng/ml) increased steroid production without any detectable increase in cyclic AMP, when experiments were carried out in the absence of phosphodiesterase inhibitors. The concentration of choriogonadotropin (10ng/ml) that stimulated progesterone synthesis maximally resulted in a minimal increase in cyclic AMP accumulation and choriogonadotropin binding. Choriogonadotropin at a concentration of 10ng/ml and higher, however, significantly stimulated protein kinase activity and reached a maximum between 250 and 1000ng of hormone/ml. Higher concentrations (50–2500ng/ml) of choriogonadotropin caused an increase in endogenous cyclic AMP, and this increase preceded the increase in steroid synthesis. Analysis of dose–response relationships of gonadotropin-stimulated cyclic AMP accumulation, progesterone production and protein kinase activity revealed a correlation between these responses over a wide concentration range when experiments were performed in the presence of 3-isobutyl-1-methylxanthine. The phosphodiesterase inhibitors papaverine, theophylline and 3-isobutyl-1-methylxanthine each stimulated steroid production in a dose-dependent manner. Incubation of ovarian cells with dibutyryl cyclic AMP or 8-bromo cyclic AMP mimicked the steroidogenic action of gonadotropins and this effect was dependent on both incubation time and nucleotide concentration. Maximum stimulation was obtained with 2mm-dibutyryl cyclic AMP and 8-bromo cyclic AMP, and this increase was close to that produced by a maximally stimulating dose of choriogonadotropin. Other 8-substituted derivatives such as 8-hydroxy cyclic AMP and 8-isopropylthio cyclic AMP, which were less susceptible to phosphodiesterase action, also effectively stimulated steroidogenesis. The uptake and metabolism of cyclic [3H]AMP in ovarian cells was also studied in relation to steroidogenesis. When ovarian cells were incubated for 2h in the presence of increasing concentrations of cyclic [3H]AMP, the radioactivity associated with the cells increased almost linearly up to 250μm-cyclic [3H]AMP concentration in the incubation medium. The 3H label in the cellular extract was recovered mainly in the forms ATP, ADP, AMP, adenosine and inosine, with cyclic AMP accounting for less than 1% of the total tissue radioactivity. Incubation of cyclic AMP in vitro with ovarian cells resulted in a rapid breakdown of the nucleotide in the medium. The degradation products in the medium have been identified as AMP, adenosine and inosine. The rapid degradation of cyclic AMP by phosphodiesterase(s) makes it difficult to correlate changes in cyclic AMP concentrations with steroidogenesis. These observations thus provide an explanation for the previously observed lack of cyclic AMP accumulation under conditions in which low doses of choriogonadotropin stimulated steroidogenesis without any detectable changes in cyclic AMP accumulation.

scholarly journals Rapid accumulation and sustained turnover of inositol phosphates in cerebral-cortex slices after muscarinic-receptor stimulation

1989 ◽  
Vol 260 (1) ◽  
pp. 237-241 ◽  
Author(s):  
I H Batty ◽  
S R Nahorski
Keyword(s):  
Cerebral Cortex ◽  
Muscarinic Receptor ◽  
Metabolic Flux ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Receptor Activation ◽  
Inositol Polyphosphate ◽  
Receptor Stimulation ◽  
Cortex Slices ◽  
Rapid Kinetics

The rapid kinetics of [3H]inositol phosphate accumulation and turnover were examined in rat cerebral-cortex slices after muscarinic-receptor stimulation. Markedly increased [3H]inositol polyphosphate concentrations were observed to precede significant stimulated accumulation of [3H]inositol monophosphate. New steady-state accumulations of several 3H-labelled products were achieved after 5-10 min of continued agonist stimulation, but were rapidly and effectively reversed by subsequent receptor blockade. The results show that muscarinic-receptor activation involves phosphoinositidase C-catalysed hydrolysis initially of polyphosphoinositides rather than of phosphatidylinositol. Furthermore, prolonged carbachol stimulation is shown not to cause receptor desensitization, but to allow persistent hydrolysis of [3H]phosphatidylinositol bisphosphate and permit sustained metabolic flux through the inositol tris-/tetrakis-phosphate pathway.

scholarly journals Ca2+-sensitive phosphoinositide hydrolysis is activated in synovial cells but not in articular chondrocytes

1999 ◽  
Vol 344 (2) ◽  
pp. 545-553 ◽  
Author(s):  
Ilaria CAPOZZI ◽  
Rossana TONON ◽  
Paola d'ANDREA
Keyword(s):  
Phospholipase C ◽  
Connexin 43 ◽  
Articular Chondrocytes ◽  
Inositol Phosphate ◽  
Receptor Activation ◽  
Phosphoinositide Hydrolysis ◽  
Functional Coupling ◽  
Synovial Cells ◽  
Dependent Manner ◽  
Concentration Dependent Manner

Cell-to-cell diffusion of second messengers across intercellular channels allows tissues to co-ordinate responses to extracellular stimuli. Intercellular diffusion of inositol 1,4,5-trisphosphate, locally produced by focal stimulations, sustains the propagation of intercellular Ca2+ waves, by stimulating the release of intracellular Ca2+ in neighbouring cells. We previously demonstrated that in cultured articular chondrocytes and HIG-82 synovial cells, studied with digitial fluorescence video imaging, mechanical stimulation of a single cell induced intercellular Ca2+ waves dependent on the presence of gap junctions. In the absence of extracellular Ca2+ the propagating distance of the wave decreased significantly in HIG-82 cells, but appeared unaffected in chondrocytes. We now show that both cells types express connexin 43 and a similar functional coupling, thus suggesting that the different Ca2+ sensitivity of intercellular waves is not due to major differences in gap junction constituent proteins. In HIG-82 synoviocytes, but not in chondrocytes, the Ca2+ ionophore ionomycin stimulated phosphoinositide hydrolysis in a concentration-dependent manner, an effect strictly dependent on the presence of extracellular Ca2+, suggesting the expression, in these cells, of a Ca2+-sensitive phospholipase C activity. Such an activity could be stimulated also by Ca2+ influx induced by P2Y receptor activation and considerably amplifies ATP-induced inositol phosphate (InsP) production. In contrast, Ca2+ influx did not affect considerably the response of chondrocytes to ATP stimulation. In HIG-82 cells, the combined application of ionomycin and ATP maximally stimulated InsP synthesis, suggesting the involvement of two independent mechanisms in inositol phosphate generation. These results suggest that in HIG-82 synovial cells the recruitment of a Ca2+-sensitive phospholipase C activity could amplify the cell response to a focally applied extracellular stimulus, thus providing a positive feedback mechanism for intercellular wave propagation.

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