scholarly journals Inhibition of phosphatidylinositol synthesis and the inactivation of calcium entry after prolonged exposure of the blowfly salivary gland to 5-hydroxytryptamine

1979 ◽  
Vol 178 (1) ◽  
pp. 59-69 ◽  
Author(s):  
M J Berridge ◽  
J N Fain
Keyword(s):  
Salivary Gland ◽  
Phosphatidic Acid ◽  
Salivary Glands ◽  
Calcium Transport ◽  
Prolonged Exposure ◽  
Calcium Entry ◽  
Ionophore A23187 ◽  
Hydrolysis Of ◽  
External Calcium ◽  
Stimulation Of

The incorporation of [32P]Pi into all salivary-gland phospholipids except phosphatidic acid was inhibited by 5-hydroxytryptamine. The accumulation of [32P]Pi into phosphatidic acid was actually enhanced by 5-hydroxytryptamine. There was an inhibition of labelled inositol incorporation into phosphatidylinositol by 5-hydroxytryptamine, which seems to be mediated by calcium because it was mimicked by the ionophore A23187, but was prevented if glands were stimulated with 5-hydroxytryptamine in the absence of external calcium. Inhibition of synthesis together with stimulation of breakdown will decrease the concentration of phosphatidylinositol, which could account for the inactivation of calcium transport observed at high 5-hydroxytryptamine concentrations. When salivary glands were stimulated with 1 micrometer-5-hydroxytryptamine, there was a rapid increase in the transfer of 45Ca2+ from the medium into the saliva, but with time this transport declined to a low value. If the glands were washed free of 5-hydroxytryptamine and incubated in the presence of 2mM-inositol for 1 h, the increase in calcium transport caused by 5-hydroxytryptamine was restored. There was little recovery in the absence of inositol. If glands were stimulated with 5-hydroxytryptamine in the absence of external calcium, a condition which prevents the inhibition of phosphatidylinositol synthesis, calcium transport in response to 5-hydroxytryptamine was greater than in glands preincubated with 5-hydroxytryptamine in the presence of calcium. The inactivation of calcium transport may result from a decrease in phosphatidylinositol concentration. These results support the hypothesis that the hydrolysis of phosphatidylinositol plays some role in either the opening or closing of calcium ‘gates’.

scholarly journals Relationship between hormonal activation of phosphatidylinositol hydrolysis, fluid secretion and calcium flux in the blowfly salivary gland

1979 ◽  
Vol 178 (1) ◽  
pp. 45-58 ◽  
Author(s):  
J N Fain ◽  
M J Berridge
Keyword(s):  
Salivary Gland ◽  
Fluid Secretion ◽  
Salivary Secretion ◽  
Calcium Flux ◽  
Ionophore A23187 ◽  
Bivalent Cation ◽  
Inositol 1 ◽  
Low Concentrations ◽  
Hormonal Activation ◽  
Stimulation Of

The addition of 5-hydroxytryptamine to the isolated blowfly salivary gland stimulates fluid secretion, transepithelial calcium transport and the breakdown of 32P- or 3H-labelled phosphatidylinositol The breakdown of [32P]phosphatidylcholine and [32P]-phosphatidylethanolamine was not stimulated by 5-hydroxytryptamine. In salivary glands incubated with myo-[2-3H]inositol for 1–3 h, more than 95% of the label retained by the tissue was in the form of phosphatidylinositol. The addition of 5-hydroxytryptamine resulted in an increase in the accumulation of label in intracellular inositol 1:2-cyclic phosphate, inositol 1-phosphate and free inositol along with an increase in the release of [3H]inositol to the medium and saliva. The release of [3H]inositol to the medium served as a sensitive indicator of phosphatidylinositol breakdown. The release of [3H]inositol was not increased by cyclic AMP or the bivalent-cation ionophore A23187 under conditions in which salivary secretion was accelerated. The stimulation of fluid secretion by low concentrations of 5-hydroxytryptamine was potentiated by 3-isobutyl-1-methylxanthine, which had no effect on inositol release. The stimulation of fluid secretion by 5-hydroxytryptamine was greatly reduced in calcium-free buffer, but the breakdown of phosphatidylinositol continued at the same rate in the absence of calcium. These results support the hypothesis that breakdown of phosphatidylinositol by 5-hydroxytryptamine is involved in the gating of calcium.

scholarly journals Influence of light and calcium on guanosine 5'-triphosphate in isolated frog rod outer segments.

1979 ◽  
Vol 74 (6) ◽  
pp. 649-669 ◽  
Author(s):  
M S Biernbaum ◽  
M D Bownds
Keyword(s):  
Light Intensity ◽  
Calcium Concentration ◽  
Control Mechanism ◽  
Continuous Light ◽  
Ionophore A23187 ◽  
Rod Outer Segments ◽  
Outer Segments ◽  
Induced Changes ◽  
Hydrolysis Of ◽  
External Calcium

Frog rod outer segments contain approximately 0.25 mol of GTP and 0.25 mol of ATP per mol of rhodopsin 3 min after their isolation from the retina. UTP and CTP are present at 10-fold and 100-fold lower levels, respectively. Concentrations of GTP and ATP decline in parallel over the next 4 min to reach relatively stable levels of 0.1 mol per mol of rhodopsin. Illumination reduces the concentration of endogenous GTP but not ATP. This light-induced decrease in GTP can be as large as 70% and has a half-time of 7 s. GTP is reduced to steady intermediate levels during extended illumination of intermediate intensity, but partially returns to its dark-adapted level after brief illumination. The magnitude of the decrease increases as a linear function of the logarithm of continuous light intensity at levels which bleach between 5 X 10(2) and 5 X 10(6) rhodopsin molecules/outer segment per second. This exceeds the range of intensities over which illumination causes decreases in the cyclic GMP content and permeability of isolated outer segments (Woodruff and Bownds. 1979. J. Gen. Physiol. 73:629-653). Thus, over 4 log units of light intensity, a sensitivity control mechanism functions to make extended illumination less effective in stimulating a GTP decrease. GTP levels in dark-adapted outer segments are sensitive to changes in calcium concentration in the suspending medium. If the external calcium concentration is reduced to 10(-8) M, GTP concentration is lowered to the same level caused by saturating illumination, and the GTP remaining is no longer light-sensitive. Lowering calcium concentration to intermediate levels between 10(-6) and 10(-8) M reduces GTP to stable intermediate levels, and the GTP remaining can be reduced by light. Restoration of millimolar calcium drives synthesis of GTP, but not of ATP, and GTP lability towards illumination is again observed. These calcium-induced changes in GTP are diminished by the addition of the divalent cation ionophore A23187. Lowering or raising magnesium levels does not influence the GTP concentration. These data raise the possibility that light activates either a calcium transport mechanism driven by the hydrolysis of GTP, or some other calcium-sensitive GTPase activity of unknown function. Known light-dependent reactions involving cyclic nucleotide transformations and rhodopsin phosphorylation appear to account for only a small fraction of the light-induced GTP decrease.

A comparison of bradykinin, angiotensin II and muscarinic stimulation of cultured bovine adrenal chromaffin cells

1989 ◽  
Vol 9 (2) ◽  
pp. 243-252 ◽  
Author(s):  
A. J. O'Sullivan ◽  
R. D. Burgoyne
Keyword(s):  
Angiotensin Ii ◽  
Chromaffin Cells ◽  
Calcium Concentration ◽  
Calcium Entry ◽  
Free Calcium ◽  
Adrenal Chromaffin Cells ◽  
Free Calcium Concentration ◽  
Adrenal Chromaffin ◽  
External Calcium ◽  
Stimulation Of

Bradykinin, angiotensin II and a mascarnic agonist, acetyl-B-methacholine (methacholine) were all found to elict catecholamine release from cultured bovine adrenal chromaffin cells. Bradykinin was the most potent of these secretagogues and methacholine the weakest, with angiotenin II intermediate in efficacy. All three secretagogues were much less effective than nicotinic stimulation. The three secretagogues all produced a rise in cytoplasmic free calcium concentration ([Ca2+]i), measured with the fluorescent indicator fura2, which was partially independent of external calcium. In the case of bradykinin the full rise in ([Ca2+]i) may involve a component of calcium entry in addition to release of calcium from an internal store. Secretion was also found to be partially independent of external calcium. The different efficacies of the three secretagogues in elicting secretion were correlated with the rise in ([Ca2+]i) produced. The differeing efficacies of the three secretagogues may be due to the extent of release of calcium from an intracellular store which itself is less effective in eliciting secretion than a rise in [Ca2+]i following calcium entry due to nicotine. Bradykinin also stimulates calcium entry, and this may increase the efficacy of the initial rise in [Ca2+]i. Treatment with pertussis toxin resulted in an enhancement of secretion in response to all of the secretagogues.

scholarly journals Forskolin as an activator of cyclic AMP accumulation and secretion in blowfly salivary glands

1982 ◽  
Vol 204 (1) ◽  
pp. 147-151 ◽  
Author(s):  
I Litosch ◽  
Y Saito ◽  
J N Fain
Keyword(s):  
Salivary Gland ◽  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Salivary Glands ◽  
Mammalian Cells ◽  
Salivary Secretion ◽  
Gland Secretion ◽  
Cyclic Amp Accumulation ◽  
Salivary Gland Secretion ◽  
Stimulation Of

Forskolin is a diterpene that activates adenylate cyclase in a variety of mammalian cells. In addition of forskolin to blowfly salivary glands increased cyclic AMP accumulation and salivary secretion. There was a small increase in transepithelial movement of labelled Ca2+. Forskolin did not induce breakdown of labelled phosphatidylinositol or inhibit the stimulation of phosphatidylinositol breakdown caused by 5-hydroxytryptamine. These data indicate that forskolin can mimic all the effects of 5-hydroxytryptamine on salivary-gland secretion that have been attributed to cyclic AMP.

ATP- and EGF-stimulated phosphatidylinositol synthesis by two different pathways, phospholipase D and diacylglycerol kinase, in A-431 epidermoid carcinoma cells

1996 ◽  
Vol 74 (2) ◽  
pp. 197-209 ◽  
Author(s):  
Kazuo Hosoi ◽  
Yoshimi Shioda ◽  
Takao Ueha ◽  
Toshiko Atsumi ◽  
Kenji Sugita ◽  
...  
Keyword(s):  
Phosphatidic Acid ◽  
Purinergic Receptors ◽  
Calcium Ionophore ◽  
Butyl Alcohol ◽  
Carcinoma Cells ◽  
Epidermoid Carcinoma ◽  
Ionophore A23187 ◽  
Acetoxymethyl Ester ◽  
Calmodulin Antagonist ◽  
Stimulation Of

The [3H]inositol incorporation into the membrane fraction of A-431 human epidermoid carcinoma cells was markedly increased by stimulation of the cells with either epidermal growth factor (EGF), ATP, bradykinin, or a calcium ionophore A23187 in the presence of 1 mM extracellular calcium ions; most incorporated [3H]inositol was found to have accumulated as phosphatidylinositol (PI). The EGF- and ATP-stimulated PI synthesis was inhibited by two protein kinase C inhibitors, staurosporine and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7), and an intracellular calcium chelator, 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl ester (BAPTA/AM), but not by the calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7). Pretreatment of cells with pertussis toxin (IAP, islet-activating protein) inhibited the PI synthesis, [Ca2+]i elevation, and inositol trisphosphate (IP3) production by ATP, suggesting that the phospholipase C (PLC) system coupled with IAP-sensitive G protein is involved in the ATP-stimulated PI synthesis. On the other hand, the ATP stimulation increased the release of [3H]choline and [32P]phosphatidic acid (PA) from radiolabeled cells, and such release was not inhibited by IAP. In the presence of n-butyl alcohol, which prevents the production of PA by generation of phosphatidylbutanol, the ATP-stimulated PI synthesis was reduced. Because n-butyl alcohol did not inhibit IP3 production and [Ca2+]i elevation, this fact suggests that the IAP-insensitive PLD system is involved in the ATP-stimulated PI synthesis. In A-431 cells, the stimulation of P2-purinergic receptors appears to activate the IAP-sensitive PLC system and IAP-insensitive PLD system, both of which are essential for the stimulation of PI synthesis. The present results imply the general prospect that ligand stimulation, which mobilizes second messengers and consumes their precursors, simultaneously provokes the pathway to synthesize and salvage the second messenger precursors as well.Key words: P2-purinergic receptors, phosphatidylinositol, EGF receptors, phosphatidic acid, A-431 cells.

scholarly journals Human platelet signaling defect characterized by impaired production of inositol-1,4,5-triphosphate and phosphatidic acid and diminished Pleckstrin phosphorylation: evidence for defective phospholipase C activation

Blood ◽  
1996 ◽  
Vol 88 (5) ◽  
pp. 1676-1683 ◽  
Author(s):  
X Yang ◽  
L Sun ◽  
S Ghosh ◽  
AK Rao
Keyword(s):  
Phosphatidic Acid ◽  
Phospholipase C ◽  
Platelet Activating Factor ◽  
Ionophore A23187 ◽  
Serotonin Secretion ◽  
Platelet Signaling ◽  
Intracellular Mediators ◽  
Gamma S ◽  
Hydrolysis Of ◽  
Pkc Activation

Signal transduction on platelet activation involves phosphoinositide- specific phospholipase C (PLC)-mediated hydrolysis of phosphatidylinositides and formation of inositol-1,4,5-triphosphate [I(1,4,5)P3], which mediates Ca2+ mobilization, and diacylglycerol (DG), which activates protein kinase C (PKC) to phosphorylate a 47-kD protein (Pleckstrin). We studied these events in two related patients previously reported (Blood 74:664, 1989) to have abnormal aggregation and 14C-serotonin secretion, and impaired intracellular Ca2+ mobilization in response to several agonists. Thrombin-induced I(1,4,5)P3 and phosphatidic acid formation were diminished. Pleckstrin phosphorylation was impaired on activation with thrombin, platelet- activating factor, and ionophore A23187, but was normal with PKC activator 1,2-dioctonyl-sn-glycerol (DiC8). Ca2+ mobilization induced by guanosine triphosphate (GTP) analog guanosine 5′-0-(3 thiotriphosphate) (GTP gamma S) was diminished. Pretreatment with either A23187 or DiC8 did not correct the impaired adenine diphosphate- induced secretion; however, upon stimulation with A23187 plus DiC8, pleckstrin phosphorylation and secretion were normal, indicating that both PKC activation and Ca2+ mobilization are essential for normal secretion. We conclude that these patients have a unique inherited platelet defect in formation of two key intracellular mediators [I(1,4,5)P3 and DG] and in the responses mediated by them due to a defect in postreceptor mechanisms of PLC activation.

Changes in Calcium Transport across Calliphora Salivary Glands Induced by 5-Hydroxytryptamine and Cyclic Nucleotides

1979 ◽  
Vol 78 (1) ◽  
pp. 137-148
Author(s):  
MICHAEL J. BERRIDGE ◽  
HERBERT LIPKE
Keyword(s):  
Cyclic Amp ◽  
Salivary Glands ◽  
Calcium Transport ◽  
Fluid Secretion ◽  
Ionophore A23187 ◽  
Active Calcium ◽  
University Of Massachusetts ◽  
Dependent Transport ◽  
The University ◽  
Internal Calcium

The efflux of 45Ca from prelabelled salivary glands was studied under a variety of conditions. 5-Hydroxytryptamine (5-HT) or cyclic AMP caused a large release of label most of which entered the saliva. The ionophore A23187 caused a similar release of calcium but EGTA had no effect. The presence of an active calcium pump on the lumenal surface was investigated further by studying calcium transport across the gland. This transport of calcium, which provides a measure of calcium entry into the cell, was very sensitive to 5-HT concentration but little affected by cyclic AMP. The small cyclic AMP-dependent transport was depressed by 8-bromo cyclic GMP with a parallel fall in the rate of fluid secretion. These studies provide more direct evidence that 5-HT acts to stimulate the entry of external calcium in addition to mobilizing internal calcium. Note: On sabbatical leave from the University of Massachusetts, Boston.

Phosphatidylcholine-directed phospholipase C: activation by complement C5b-9

1993 ◽  
Vol 265 (4) ◽  
pp. F551-F560 ◽  
Author(s):  
A. V. Cybulsky ◽  
M. D. Cyr
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Epithelial Cell ◽  
Phosphatidic Acid ◽  
Membranous Nephropathy ◽  
Ionophore A23187 ◽  
Additional Source ◽  
Glomerular Epithelial Cell ◽  
Kinase C ◽  
Hydrolysis Of

In rat membranous nephropathy, complement C5b-9 induces glomerular epithelial cell (GEC) injury and proteinuria. In cultured rat GEC, C5b-9 stimulates a phosphoinositide-directed phospholipase (PL) C and products of PLC downregulate C5b-9-mediated GEC injury. We now report that C5b-9-induced hydrolysis of phosphatidylcholine (PC) provides an additional source of 1,2-diacylglycerol (DAG). PC was labeled in intact GEC by brief incubation with 1-O-[alkyl-3H]2-lyso-PC. Assembly of C5b-9 stimulated an increase in PC-derived [3H]DAG (173 +/- 18% control), which was reduced in GEC depleted of protein kinase C (PKC) by prolonged preincubation with phorbol 12-myristate 13-acetate (PMA). Similar to C5b-9, [3H]DAG was released from PC after brief incubation of GEC with Ca2+ ionophore A23187 plus PMA. The increases in [3H]DAG induced by C5b-9 and A23187 plus PMA were paralleled by increases in DAG mass. C5b-9 also increased [3H]phosphatidic acid (PA; 182 +/- 37% control), but there was no significant interconversion of DAG and PA. Thus DAG probably originated via PLC. PC-directed PLC activity was also studied in GEC homogenates by release of [14C]DAG from exogenous 1-palmitoyl-2-[arachidonoyl-14C]PC. PLC activity was present at physiological Ca2+ concentration (200-1,200 nM), and PMA stimulated PLC activity in cell homogenates (in presence of ATP). These results demonstrate directly that PMA stimulates release of DAG from PC and are in keeping with the effect of PMA in [3H]lyso-PC-labeled GEC. Thus GEC contain a PC-directed PLC, whose activity is physiologically regulated and is present at nanomolar Ca2+ concentration. C5b-9 stimulates PC-directed PLC, leading to production of DAG. This DAG might trigger a mechanism for limiting injury during complement attack.

Stimulation of phospholipase D activity and phosphatidic acid production by norepinephrine in rat aorta

1993 ◽  
Vol 264 (3) ◽  
pp. C609-C616 ◽  
Author(s):  
A. W. Jones ◽  
S. D. Shukla ◽  
B. B. Geisbuhler
Keyword(s):  
Phosphatidic Acid ◽  
Phospholipase D ◽  
Time Course ◽  
Rat Aorta ◽  
Functional Responses ◽  
Phosphatidylinositol Bisphosphate ◽  
Phosphatidylinositol Phosphate ◽  
Transphosphatidylation Reaction ◽  
Hydrolysis Of ◽  
Stimulation Of

We sought to relate norepinephrine (NE) stimulation of phosphatidic acid (PA) production to functional responses of rat aorta and pathways for PA production. The time course for changes in PA was closely related to Ca-dependent tonic responses in 42K efflux and contraction. NE (30 microM for 1 min) increased PA and reduced phosphatidylcholine (PC) and phosphatidylinositol (PI) based on Pi analyses and 32P labeling of phospholipids. The 32P-to-Pi ratio in PA (0.8 +/- 0.2, n = 13) was similar to PC (0.8 +/- 0.1, n = 14) but was significantly lower (P < 0.001) than PI (4.6 +/- 0.5, n = 14). The 32P-to-Pi ratio in PA was also lower (P < 0.02) than phosphatidylinositol phosphate and phosphatidylinositol bisphosphate. NE also increased [3H]PA twofold (P < 0.05) when PC was selectively labeled with [3H]myristic acid. These observations are more consistent with PA being formed from the hydrolysis of PC by phospholipase D (PLD) than by the phosphorylation of diacylglycerol produced by the action of phospholipase C. PLD was assayed by the formation of phosphatidylethanol (PEt) via a transphosphatidylation reaction with ethanol (half-maximal stimulation at 0.4-0.5% vol/vol). The time course for PLD stimulation by NE was similar to PA, with significant increases (P < 0.002) during 10 s to 30 min exposure. Once formed, PEt was degraded slowly, with a half time > 3 h. It is concluded that NE stimulates PLD in rat aorta, which forms a significant amount of PA from the hydrolysis of PC.(ABSTRACT TRUNCATED AT 250 WORDS)

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