Stimulated Phospholipase C Activity Research Articles

Dandelion Root Extract Induces Intracellular Ca2+ Increases in HEK293 Cells.

Dandelion (Taraxacum officinale Weber ex F.H.Wigg.) has been used for centuries as an ethnomedical remedy. Nonetheless, the extensive use of different kinds of dandelion extracts and preparations is based on empirical findings. Some of the tissue-specific effects reported for diverse dandelion extracts may result from their action on intracellular signaling cascades. Therefore, the aim of this study was to evaluate the effects of an ethanolic dandelion root extract (DRE) on Ca2+ signaling in human embryonic kidney (HEK) 293 cells. The cytotoxicity of increasing doses of crude DRE was determined by the Calcein viability assay. Fura-2 and the fluorescence resonance energy transfer (FRET)-based probe ERD1 were used to measure cytoplasmic and intraluminal endoplasmic reticulum (ER) Ca2+ levels, respectively. Furthermore, a green fluorescent protein (GFP)-based probe was used to monitor phospholipase C (PLC) activation (pleckstrin homology [PH]–PLCδ–GFP). DRE (10–400 µg/mL) exposure, in the presence of external Ca2+, dose-dependently increased intracellular Ca2+ levels. The DRE-induced Ca2+ increase was significantly reduced in the absence of extracellular Ca2+. In addition, DRE caused a significant Ca2+ release from the ER of intact cells and a concomitant translocation of PH–PLCδ–GFP. In conclusion, DRE directly activates both the release of Ca2+ from internal stores and a significant Ca2+ influx at the plasma membrane. The resulting high Ca2+ levels within the cell seem to directly stimulate PLC activity.

Membrane Stretch‐Induced Activation of TRPM4 in Cerebral Artery Smooth Muscle Cells

In cerebral artery smooth muscle cells (SMCs), the melastatin transient receptor potential (TRP) channel TRPM4 is a crucial mediator of pressure‐induced membrane depolarization, and is activated by Ca2+ released from inositol trisphosphate (IP3) receptors located on the sarcoplasmic reticulum. In the current study, we examined the mechanosensitivity of TRPM4 channels in native SMCs. TRPM4 channel activity was recorded under perforated patch clamp conditions as the plasma membrane was stretched by applying suction to the patch pipette. Increases in cation current proportional to the magnitude of applied suction were recorded. Mechanosensitive currents were inhibited by the selective TRPM4 blocker 9‐phenenthral but were unaffected by the BKCa channel blocker paxilline. Phospholipase C (PLC) cleaves the membrane phospholipid phosphatidylinositol 4,5‐bisphosphate (PIP2) into IP3 and diacylglycerol (DAG). Prior studies suggest that mechanical stress can stimulate PLC activity. Therefore, we tested the hypothesis that PLC activity is necessary for plasma membrane stretch‐induced activation of TRPM4 in cerebral artery myocytes. We find that the PLC blocker U73122 inhibits stretch‐induced TRPM4 activation, suggesting that PLC plays an important role in activation of TRPM4 by mechanical stimulus in cerebral artery smooth muscle cells. RO1HL091905(SE); F31HL094145‐01(AG).

Differential Endothelin Receptor Expression and Function in Rat Myometrial Cells and Leiomyoma ELT3 Cells

Uterine leiomyoma are the most common benign tumors of the myometrium. We previously identified endothelin (ET)-1 as a proliferative and antiapoptotic factor in Eker rat-derived leiomyoma (ELT3) cells. A major role of ETB receptor in the prosurvival effect was revealed. Here we investigated, in ELT3 and myometrial cells, the respective contribution of ETA and ETB in the proliferative effect of ET-1. In myometrial cells, binding experiments show that ETA is almost exclusively expressed and stimulates phospholipase C (PLC) activity and ERK1/2 phosphorylation and proliferation. In ELT3 cells, ETB is expressed at about the same level as ETA, and the two receptors are differently coupled to Gi protein. The ETB agonist, sarafotoxin S6c, stimulates PLC activity 60% less than ET-1 but is as potent as ET-1 to increase ERK1/2 phosphorylation and induce proliferation. However, the ability of ETA to activate ERK1/2 is observed after ETB desensitization. Although ETA and ETB antagonists partially reduce ET-1 stimulated PLC activity, they are without effect on ET-1-induced ERK1/2 phosphorylation and proliferation. Only the simultaneous use of ETA and ETB antagonists reduces ET-1-triggered ERK1/2 activation. These unconventional properties of ETRs may reveal the existence of functional ETA-ETB heterodimers. Finally, treatment of ELT3 cells with ETB but not ETA-directed small interfering RNA reduces the proliferative effect of ET-1. All the data obtained in ELT3 cells strengthen the relation between ETB overexpression, which decreases the ETA to ETB ratio, and the ability of leiomyoma cells to highly proliferate and resist apoptosis.

Signaling by a Non-dissociated Complex of G Protein βγ and α Subunits Stimulated by a Receptor-independent Activator of G Protein Signaling, AGS8

Accumulating evidence suggests that heterotrimeric G protein activation may not require G protein subunit dissociation. Results presented here provide evidence for a subunit dissociation-independent mechanism for G protein activation by a receptor-independent activator of G protein signaling, AGS8. AGS8 is a member of the AGS group III family of AGS proteins thought to activate G protein signaling primarily through interactions with Gbetagamma subunits. Results are presented demonstrating that AGS8 binds to the effector and alpha subunit binding "hot spot" on Gbetagamma yet does not interfere with Galpha subunit binding to Gbetagamma or phospholipase C beta2 activation. AGS8 stimulates activation of phospholipase C beta2 by heterotrimeric Galphabetagamma and forms a quaternary complex with Galpha(i1), Gbeta(1)gamma(2), and phospholipase C beta2. AGS8 rescued phospholipase C beta binding and regulation by an inactive beta subunit with a mutation in the hot spot (beta(1)(W99A)gamma(2)) that normally prevents binding and activation of phospholipase C beta2. This demonstrates that, in the presence of AGS8, the hot spot is not used for Gbetagamma interactions with phospholipase C beta2. Mutation of an alternate binding site for phospholipase C beta2 in the amino-terminal coiled-coil region of Gbetagamma prevented AGS8-dependent phospholipase C binding and activation. These data implicate a mechanism for AGS8, and potentially other Gbetagamma binding proteins, for directing Gbetagamma signaling through alternative effector activation sites on Gbetagamma in the absence of subunit dissociation.

Shifting into Reverse

Dictyostelium discoideum are a widely used model system for the study of cellular motility. Like many mammalian cells, this single-celled organism exhibits both chemoattractive and chemorepulsive responses. It is well understood that chemoattraction involves polarization of membrane lipids with phosphatidylinositol-(3,4,5)-trisphosphate (PIP 3 ) accumulating at the front of the cell, which is oriented toward the area with higher concentration of chemoattractant, and the lipid phosphatase PTEN at the back of the cell, where lower concentration of the attractant is present; however, the mechanisms that control chemorepulsion are less well understood. Keizer-Gunnink et al . used analogs of cAMP, such as 8CPT-cAMP, to stimulate chemorepulsion of D. discoideum and monitored the accumulation of PIP 3 , phosphatidylinositol 3-kinase (PI3K), and PTEN by immunofluorescence techniques. In the presence of the chemorepulsive gradient, PIP3 and PI3K were enriched at the front of the cells (where concentrations of the repellent were lower), whereas PTEN was enriched at the back of the cell (where the repellent concentration was higher). Thus, the polarity of the cells with respect to the chemorepulsive gradient was opposite that of cells in a chemoattractive gradient. Cells in which two of three genes encoding PI3K or the gene encoding phospholipase C (PLC) were knocked out showed a chemoattractant response to cAMP but no response to the chemorepulsive 8CPT-cAMP. Exposure of the cells to cAMP stimulates PLC activity, which metabolizes phosphatidylinositol-(4,5)-diphosphate (PIP 2 ) to produce diacylglycerol and inositol trisphosphate, whereas 8CPT-cAMP leads to inhibition of PLC activity. Thus, in a chemoattractive gradient, the combination of PI3K and PLC activity at the front of the cell leads to a loss of PIP 2 to which PTEN binds, thereby causing PTEN to accumulate in the rear of the cell and PIP 3 to accumulate at the front of the cell. The authors propose that in a chemorepulsive gradient, PLC activity is inhibited, which allows PIP 2 to remain high in the side of the cell where the repellent concentration is highest, thereby recruiting PTEN to this side of the cell and allowing the cell to polarize PIP 3 in a direction away from the highest concentrations of repellent and thus move away. In the life of D. discoideum , both chemorepulsion and chemoattraction are involved in the response to starvation with a switch from a repulsive to an attractive behavior. After a short period of starvation (~1 hour), the cells migrate away from each other, and during this time they have the G protein that inhibits PLC. After longer periods of starvation (~5 hours), the cells secrete cAMP and produce the G protein that stimulates PLC, thereby allowing the cells to coalesce and form a multicellular structure I. Keizer-Gunnink, A. Kortholt, P. J. M. Van Haastert, Chemoattractants and chemorepellents act by inducing opposite polarity in phospholipase C and PI3-kinase signaling. J. Cell Biol. 177 , 579-585 (2007). [Abstract] [Full Text]

Chemokine-Directed Trafficking of Receptor Stimulus to Different G Proteins: Selective Inducible and Constitutive Signaling by Human Herpesvirus 6-Encoded Chemokine Receptor U51

The human herpes virus 6 (HHV-6)-encoded chemokine receptor U51 constitutively activates phospholipase C (PLC) and inhibits cAMP-responsive element (CRE)-mediated gene transcription via the activation of G(q/11) proteins. Yet, chemokines known to bind U51 differentially regulate U51 coupling to G proteins. CCL5/RANTES induced pertussis toxin (PTX)-insensitive increases in PLC activity and changes in intracellular free calcium concentration ([Ca2+]i), whereas both CCL2/MCP-1 and CCL11/eotaxin failed to stimulate PLC activity or increase [Ca2+]i. In contrast, all three chemokines counteracted the effects of U51 on CRE activity via the activation of PTX-sensitive G(i/o) proteins. For each of the tested chemokines, coexpression of U51 with a variety of G alpha subunits, however, revealed a distinct profile for preferred G-protein coupling, which could be shifted by modulation of the relative expression of G proteins. These findings are consistent with a chemokine-selective trafficking of receptor stimulus to distinct G proteins and suggest that the constitutive activity of U51 and the chemokine-induced signaling involve different active states of the receptor. By virtue of its ability to constitutively activate signaling pathways, its G-protein promiscuity, and the chemokine-directed trafficking of receptor stimulus, U51 can be considered a sensitive and versatile virally encoded signaling device, potentially of importance in HHV-6-related pathologies.

Pathways Used by Relaxin to Regulate Myometrial Phospholipase C

Relaxin exhibits pleiotropic effects on reproductive and nonreproductive tissues; the signaling mechanisms underlying these functions are still not well understood. Activation of protein kinase A and several other signal-regulated protein kinases results in the phosphorylation of phospholipase C (PLC)-beta3 and inhibit Galpha(q)-stimulated PLC activity. Therefore, PLCbeta3 may be targeted by both contractant and relaxant signaling pathways in myometrium and play a critical role in the balance between them. PHM1 cells express mRNA for relaxin receptor LGR7, and relaxin inhibits oxytocin-stimulated PLC activity in these cells. Thus, this model system may be useful in delineating signaling pathways used by relaxin. Here, we present evidence that relaxin stimulates phosphorylation of PLCbeta3 in PHM1 cells.

Amplification of Ca2+ Signaling by Diacylglycerol-mediated Inositol 1,4,5-Trisphosphate Production

Stimulation of various cell surface receptors leads to the production of inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) through phospholipase C (PLC) activation, and the IP3 and DAG in turn trigger Ca2+ release through IP3 receptors and protein kinase C activation, respectively. The amount of IP(3) produced is particularly critical to determining the spatio-temporally coordinated Ca(2+)-signaling patterns. In this paper, we report a novel signal cross-talk between DAG and the IP3-mediated Ca(2+)-signaling pathway. We found that a DAG derivative, 1-oleoyl-2-acyl-sn-glycerol (OAG), induces Ca2+ oscillation in various types of cells independently of protein kinase C activity and extracellular Ca2+. The OAG-induced Ca2+ oscillation was completely abolished by depletion of Ca2+ stores or inhibition of PLC and IP3 receptors, indicating that OAG stimulates IP3 production through PLC activation and thereby induces IP3-induced Ca2+ release. Furthermore, intracellular accumulation of endogenous DAG by a DAG-lipase inhibitor greatly increased the number of cells responding to agonist stimulation at low doses. These results suggest a novel physiological function of DAG, i.e. amplification of Ca2+ signaling by enhancing IP3 production via its positive feedback effect on PLC activity.

Zinc Homeostasis in C6 Glioma Cells: Phospholipase C Activity Regulates Cellular Zinc Export

Zinc homeostasis in mammalian cells is precisely regulated by cellular signal transduction mechanisms. The main result of this study is the finding that modulators of phospholipase C (PLC) activity affect cellular zinc export. Two different PLC inhibitors caused an increase of the total cellular zinc level whereas two different PLC activators caused a decrease. Furthermore, both the inhibition of cyclic nucleotide phosphodiesterases as well as the administration of 8-bromo-cAMP evoked a drop in the intracellular zinc level, indicating the involvement of cAMP in the control of cellular zinc export. It is concluded that the activity of PLC controls cellular zinc transport and that the effect of elevated zinc concentrations on PLC activity might be mediated by cAMP. However, modulation of other major signaling enzymes did not affect the cellular zinc homeostasis. These include activation and inhibition of guanylate cyclase, activation of protein kinase G, activation of protein kinase A, and activation or inhibition of protein kinase C. Furthermore there was no evidence for the existence of a zinc-sensing receptor in C6 glioma cells, which would stimulate PLC activity and evoke a mobilization of intracellular free-calcium levels.

Fish somatostatin sst3 receptor: comparison of radioligand and GTPγS binding, adenylate cyclase and phospholipase C activities reveals different agonist‐dependent pharmacological signatures

1 The fish somatostatin receptor 3 (fsst3) is one of the few somatostatin (SRIF) receptors cloned from a non-mammalian species so far. Here we extended our earlier characterization of this receptor by investigating the guanine nucleotide sensitivity of agonist radioligand binding at the fsst3 receptor recombinantly expressed in CCL39 (Chinese hamster lung fibroblast) cells. Further, we measured somatostatin (SRIF) and cortistatin (CST) analogues stimulated GTPgammaS binding, inhibition of forskolin-stimulated adenylate cyclase (FSAC) and stimulation of phospholipase C (PLC) activities. The present transductional data were then compared with previous radioligand binding and/or second messenger features determined for fsst3 and/or human SRIF receptors (hsst2, hsst3 and hsst5). 2 The GTP analogue guanylylimidodiphosphate (GppNHp) inhibited binding of [125I]CGP 23996 and [125I][Tyr3octreotide by 72 and 83% suggesting preferential labelling of G-protein-coupled fsst3 receptors. By contrast, [125I]LTT-SRIF28 and [125I][Tyr10]CST14 binding was rather GppNHp insensitive (42 and 35% inhibition) suggesting labelling of both coupled and non-coupled receptor states. These results might explain the apparent higher receptor densities determined in saturation experiments with [125I]LTT-SRIF28 and [125I][Tyr10]CST14 (4470 and 4030 fmol mg(-1)) compared with [125I]CGP 23996 and [125I][Tyr3]octreotide (3420 and 1520 fmol mg(-1)). 3 SRIF14 (10 microm)-stimulated specific [35S]GTPgammaS binding by three-fold; SRIF28 and octreotide displayed full agonism, whereas most other ligands displayed 60-80% intrinsic activity compared with SRIF14. SRIF14 and SRIF28 inhibited forskolin-stimulated AC (FSAC) activity by 60%; all tested ligands except BIM 23056 inhibited FSAC with comparable high intrinsic activities. SRIF14 stimulated PLC activity five- to six-fold, as determined by measuring total [3H] IP(x) accumulation; it was rather insensitive to pertussis toxin (PTX, 100 ng ml(-1), 21% inhibition), which suggests the G(q)-family proteins couple to PLC activity. SRIF14, SRIF28 and [Tyr10]CST14 showed full agonism at PLC, whereas all other ligands behaved as partial agonists (20-70% intrinsic activity). BIM 23056, which showed weak partial or no agonism, antagonized SRIF14-induced total [3H]-IP(x) production (pK(B) = 6.83), but failed to block competitively agonist-stimulated [35S]GTPgammaS binding or agonist-induced inhibition of FSAC activity. 4 Comparison of the pharmacological profiles of fsst3 receptors established in GTPgammaS binding, FSAC inhibition and PLC stimulation resulted in low correlations (r = 0.410-0.594). Both rank orders of potency and rank orders of relative efficacy varied in the three second messenger experiments. Significant, although variable correlations were obtained comparing GTPgammaS binding and inhibition of FSAC activity with previously reported affinity profiles of [125I]LTT-SRIF28, [125I][Tyr10]CST14, [125I]CGP 23996, [125I][Tyr3]octreotide (r = 0.75-0.83; 0.68-0.89). By contrast, the PLC stimulation and radioligand-binding profiles did not correlate. 5 Comparison of the functional data (GTPgammaS binding, FSAC inhibition, PLC stimulation) of fsst3 receptors with those of human sst2, sst3, sst5 receptors expressed in CCL39 cells resulted in highest correlation with the hsst5 receptor (r = 0.94, 0.97, 0.49) > hsst2 (0.80, 0.50, n.d.) > hsst3 (0.25, 0.19, 0.17). 6 In summary, fsst3 receptors expressed in CCL39 cells are involved in signalling cascades similar to those reported for mammalian SRIF receptors, suggesting SRIF receptors to be highly conserved in evolution. Binding and functional data showed highest similarity of fsst3 receptors with the human sst5 receptor subtype. Different affinities, receptor densities and GppNHp-sensitivities determined with the four radioligands (agonists) are assumed to results from ligand-specific states of the fsst3-ligand complex. The differences in the rank orders of potency and relative efficacy in the various signalling cascades may be explained by agonist-induced receptor trafficking.

The Tyrosine Phosphorylation and Cytoskeletal Translocation of Phospholipase C 1 in Bovine Adrenal Medullary Chromaffin Cells

The tyrosine phosphatase inhibitor BpV(phen) stimulated a concentration-dependent increase of phospholipase C (PLC) activity in bovine adrenal medullary chromaffin cells. This response was accompanied by an increase in PLCgamma1 tyrosine phosphorylation and its cytosketetal translocation. Insulin, at high concentrations, stimulated PLC activity to a similar extent as BpV(phen), a response that was also accompanied by an increase in PLCgamma1 translocation but not its tyrosine phosphorylation. BpV(phen) strongly enhanced the insulin-stimulated increase in PLC activity and caused a small rise in PLCgamma1 translocation above that seen with insulin alone. Despite the synergistic rise in activity PLCgamma1 tyrosine phosphorylation did not increase beyond that seen with BpV(phen) alone. These results indicate that PLCgamma1 activation in chromaffin cells may be more closely associated with its cytoskeletal translocation than its tyrosine phosphorylation although other factors may also be important for activation of enzyme activity.

Role of the C-domain in the biological activities of Clostridium perfringens alpha-toxin.

Clostridium perfringens alpha-toxin (370 residues) possesses hemolytic and lethal activities as well as the enzymatic activity of phospholipase C (PLC). In this study we examined the role of the C-domain (251-370 residues; CP251- 370) in biological activities of the toxin. The N-domain (1-250 residues; CP1- 250) of the alpha-toxin as well as the Bacillus cereus phospholipase C (BcPLC) possessed PLC activity, but did not bind to rabbit erythrocytes and lyse them. A hybrid protein (BC-CP251-370) consisting of BcPLC and CP251- 370 bound to the red cells and lysed them. Incubation of CP1-250 with CP251-370 completely complemented hemolytic and PLC activities. CP251-370 also conferred hemolytic activity on BcPLC. CP251-340 (251-340 residues) significantly stimulated PLC activity of CP1-250), but did not confer hemolytic activity on CP1-250. Kinetic analysis suggested that CP251-370 increased affinity toward the substrate of CP1-250. The results suggested that CP251-370 plays an important role in binding to erythrocytes and the hemolytic and enzymatic activities of CP1-250. Acrylodan-labeled CP251-370 variants (S263C and S365C) bound to liposomes and exhibited a marked blue shift, and in addition, an N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazolyl)ethylene diamine (NBD)-labeled CP251-370 (S365C) variant also bound to liposomes and the fluorescence intensity significantly increased, suggesting movement of CP251-370 to a hydrophobic environment. These observations suggest that interaction of CP251-370 of alpha-toxin with fatty acyl residues of phosphatidylcholine plays an important role in the biological activities of CP1-250.

Calcium-sensing receptor-mediated activation of phospholipase C-γ1 is downstream of phospholipase C-β and protein kinase C in MC3T3-E1 osteoblasts

Elevated extracellular calcium (Ca e) stimulates both chemotaxis and mitogenesis of MC3T3-E1 osteoblasts via a calcium-sensing receptor (CasR). Ca e-mediated chemotaxis of these bone-forming cells is dependent on phospholipase C (PLC) and blocked by the Gi-protein inhibitor pertussis toxin. In this study, we examine the signaling mechanisms by which the CasR stimulates PLC activity in MC3T3-E1 osteoblasts. We found that elevated Ca e stimulated PLC-γ1 tyrosine phosphorylation in a time-dependent and Ca e-concentration-dependent manner. The maximal increase in PLC-γ1 tyrosine phosphorylation was observed 3–5 min after increasing Ca e by 3.2 mmol/L from 1.8 mmol/L. Elevated Ca e also promoted a rapid increase in both inositol 1,4,5-trisphosphate [Ins(1,4,5)P 3], a second messenger formed by PLC-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate, and cytosolic free calcium ([Ca +2] i). The kinetics of the CasR-mediated increases in Ins(1,4,5)P 3 and [Ca +2] i and the sensitivity of the Ca e-stimulated elevation in [Ca +2] i to U73122 (a PLC inhibitor) together suggest that the osteoblast CasR is coupled via Gq to PLC-β. U73122 blocked the Ca e-promoted, but not PDGF-promoted, PLC-γ1 tyrosine phosphorylation, suggesting that the activation of PLC-β is upstream of PLC-γ1 activation. Inhibition of protein kinase C (PKC) disrupted Ca e-stimulated tyrosine phosphorylation of PLC-γ1. In addition, exposure to pertussis toxin or exogenous activation of protein kinase A (PKA) inhibited PLC-γ1 tyrosine phosphorylation in response to Ca e. The results indicate that: (a) the osteoblast CasR activates PLC-γ1 downstream of PLC-β in a PKC-dependent manner; (b) PKA is a negative regulator of Ca e-promoted PLC-γ1 phosphorylation; and (c) Gq and Gi are both involved in the CasR-mediated phosphorylation of PLC-γ1.

Activation of phospholipase C by cholecystokinin receptor subtypes with different G-protein-coupling specificities in hormone-secreting pancreatic cell lines

Phospholipase C (PLC) activity was investigated by stimulation of membrane preparations obtained from insulin (β-TC3)-, somatostatin (Rin 1027-B2)-, and glucagon (INR1-G9)-producing pancreatic cell lines using the non-hydrolyzable GTP analogue GTPγS alone, the C-terminal octapeptide cholecystokinin (CCK-8), or gastrin. All compounds caused a significant 2- to 4.4-fold stimulation of PLC activity in the different cell lines, which was diminished by the non-hydrolyzable GDP analogue GDPβS. CCK receptor subtypes were characterized by radioligand binding experiments. High-affinity binding sites for tritiated CCK A receptor antagonist L-364,718 ( K d = 0.24 nM) and tritiated CCK B receptor antagonist L-365,260 ( K d = 0.13 nM) were only present in Rin 1027-B2 cells. High-affinity binding sites for both ligands were not found in β-TC3 or INR1-G9 cells. Competition binding experiments with non-labeled CCK receptor antagonists CR 1505 (CCK A receptor-selective) and CR 2945 (CCK B receptor-selective), as well as microphysiometry experiments, resulted in the same receptor distribution. Reverse transcriptase–polymerase chain reaction confirmed the CCK receptor distribution pattern for Rin 1027-B2 cells, but in addition showed the existence of CCK B receptors in β-TC3 cells. Immunoblocking experiments with C-terminal antibodies against different G-protein α-subunits demonstrated inhibition of CCK-stimulated PLC activity in β-TC3 cells by G q/11α antiserum (70%), in Rin 1027-B2 cells by G q/11α antiserum (70%) and G i-3α antiserum (23%), and in INR1-G9 cells by G q/11α antiserum (60%) and G oα antiserum (45%). We conclude that CCK receptor subtypes with different G-protein-coupling specificities to PLC are present in the different hormone-secreting cells of the endocrine pancreas.

The role of cyclic nucleotides and calcium in the relaxation produced by amrinone in rat aorta

(1) The vasorelaxation produced by the phosphodiesterase 3 (PDE3) inhibitor, amrinone was investigated in isolated rat aorta denuded of endothelium. In the presence of extracellular Ca 2+, amrinone, milrinone and 3-isobutyl-1-methylxanthine (IBMX), relaxed endothelium-denuded rat aortic rings constricted with phenylephrine. While the actions of milrinone and IBMX were inhibited by the protein kinase G (PKG) inhibitor, Rp-8-Bromo guanosine-3′,5′ monophosphothioate (Rp-8-Br-cGMPS; 0.5 mM), that of amrinone was only slightly affected; whereas the protein kinase A (PKA) inhibitor, Rp-adenosine-3′,5′ cyclic monophosphothioate (Rp-cAMPS; 0.5 mM) had no effect on any agent. (2) Amrinone (100 μM) inhibited 45Ca 2+ influx through receptor- or store-operated Ca 2+ channels following stimulation with phenylephrine (1 μM) or thapsigargin (1 μM). In contrast, amrinone had no effect on KCl (120 mM)-stimulated Ca 2+ influx. (3) In the absence of extracellular Ca 2+, amrinone (30 μM) inhibited the constriction produced by phenylephrine, 5-hydroxytryptamine (5HT) and U46619, and this effect was not affected by Rp-cAMPS or Rp-8-Br-cGMPS. (4) The intracellular mechanism of action of amrinone may involve the phospholipase C (PLC)-inositol 1,4,5 trisphosphate (IP 3)-intracellular Ca 2+ signal transduction pathway. However, amrinone (100 μM) had no effect on either basal- or noradrenaline (100 μM)-stimulated PLC activity. Similarly, IP 3 stimulated a concentration-dependent release of Ca 2+ from rat brain microsomes that was not affected by amrinone (30 and 100 μM). (5) In conclusion, the vasorelaxant action of amrinone does not involve adenosine 3′,5′ cyclic monophosphate (cAMP) or involve guanosine 3′,5′ cyclic monophosphate (cGMP) but may include an inhibition of Ca 2+ influx through receptor- or store-operated Ca 2+ channels, although it does not directly affect intracellular Ca 2+ release.

Role of cAMP in the effects of K+ on the steroidogenesis of zona glomerulosa cells.

1. Investigations of the role of cAMP in the stimulation of the steroidogenesis of zona glomerulosa (ZG) cells by increased extracellular K+ concentration are reviewed. 2. Possible reasons for discrepancies in the results of different investigators on whether K+ increases the cAMP content or output of ZG tissue or dispersed cells are discussed. 3. The concentration of cAMP in the incubating media of ZG tissue or cells, rather than their cAMP content, seems to respond more sensitively to stimulation by extracellular K+, as was also found for adrenocorticotropic hormone stimulation of zona fasciculata-reticularis cells. 4. The addition of the phosphodiesterase inhibitor 1-methyl-3-isobutylxanthine (IBMX) to incubations with the aim of increasing the sensitivity of the response in cAMP to extracellular K+ in ZG cells may give rise to effects, probably nonspecific, which actually inhibit the measured response. 5. The immediate stimulation in the steroidogenesis of ZG cells with raised extracellular K+ is probably mostly due to the direct effect of increases in cytoplasmic Ca2+ (arising from increases in Ca2+ influx) on mitochondrial processes. However, increases in cAMP may prolong the stimulation of steroidogenesis by increased extracellular K+. This increased cAMP is probably due to stimulation of adenylyl cyclase activity. 6. It has been concluded that the increase in Ca2+ influx output after rises in the extracellular K+ concentration of ZG cells is responsible for most of the increase in cAMP. 7. According to one group of investigators, there is weak stimulation of phospholipase C (PLC) activity after increasing the extracellular K+ concentration of rat ZG cells. 8. If there is such a stimulation of PLC activity, it seems that the action of increased extracellular K+ can potentially involve all known mechanisms for the stimulation of steroidogenesis in endocrine cells. The common primary event is probably the increase in Ca2+ influx. The relative importance of these various potential mechanisms may depend on the particular in vitro conditions used.

The luteinizing hormone receptor activates phospholipase C via preferential coupling to Gi2.

Binding of lutropin/choriogonadotropin (LH/CG) to its cognate receptor results in the activation of adenylyl cyclase and phospholipase C. This divergent signaling of the LH receptor is based on the independent activation of distinct G protein subfamilies, i.e. , Gs, Gi, and potentially also Gq. To examine the selectivity of LH receptor coupling to phospholipase C beta-activating G proteins, we used an in vivo reconstitution system based on the coexpression of the LH receptor and different G proteins in baculovirus-infected insect cells. In this paper, we describe a refined expression strategy for the LH receptor in insect cells. The receptor protein was inserted into the cell membrane at an expression level of 0.8 pmol/mg of membrane protein. Sf9 cells expressing the LH receptor responded to hCG challenge with a concentration-dependent accumulation of intracellular cAMP (EC50 = 630 nM) but not of inositol phosphates, whereas stimulation of the histamine H1 receptor in Sf9 cells led to increased phospholipase C (PLC) activity. Immunoblotting experiments using G protein-specific antisera revealed the absence of quantitative amounts of alpha i in Sf9 cells, whereas alpha s and alpha q/11 were detected. We therefore attempted to restore the hCG-dependent PLC activation by infection of Sf9 cells with viruses encoding the LH receptor and different G protein alpha subunits. HCG stimulation of cells coexpressing the LH receptor and exogenous alpha i2 resulted in stimulation of PLC activity. In cells coinfected with an alpha i3-baculovirus, hCG challenge led to a minor activation of PLC, whereas no hCG-dependent PLC stimulation was observed in cells coexpressing alpha i1. Most notably, coinfection with baculoviruses encoding alpha q or alpha 11 did not reproduce the PLC activation by the LH receptor. Thus, the murine LH receptor activates adenylyl cyclase via Gs and PLC via selective coupling to Gi2.

RGS Proteins Determine Signaling Specificity of Gq-coupled Receptors

Regulators of G protein signaling (RGS) proteins accelerate GTP hydrolysis by Galpha subunits, thereby attenuating signaling. RGS4 is a GTPase-activating protein for Gi and Gq class alpha subunits. In the present study, we used knockouts of Gq class genes in mice to evaluate the potency and selectivity of RGS4 in modulating Ca2+ signaling transduced by different Gq-coupled receptors. RGS4 inhibited phospholipase C activity and Ca2+ signaling in a receptor-selective manner in both permeabilized cells and cells dialyzed with RGS4 through a patch pipette. Receptor-dependent inhibition of Ca2+ signaling by RGS4 was observed in acini prepared from the rat and mouse pancreas. The response of mouse pancreatic acini to carbachol was about 4- and 33-fold more sensitive to RGS4 than that of bombesin and cholecystokinin (CCK), respectively. RGS1 and RGS16 were also potent inhibitors of Gq-dependent Ca2+ signaling and acted in a receptor-selective manner. RGS1 showed approximately 1000-fold higher potency in inhibiting carbachol than CCK-dependent signaling. RGS16 was as effective as RGS1 in inhibiting carbachol-dependent signaling but only partially inhibited the response to CCK. By contrast, RGS2 inhibited the response to carbachol and CCK with equal potency. The same pattern of receptor-selective inhibition by RGS4 was observed in acinar cells from wild type and several single and double Gq class knockout mice. Thus, these receptors appear to couple Gq class alpha subunit isotypes equally. Difference in receptor selectivity of RGS proteins action indicates that regulatory specificity is conferred by interaction of RGS proteins with receptor complexes.

Pancreastatin activates β3 isoform of phospholipase C via G α11 protein stimulation in rat liver membranes

Pancreastatin (PST) receptors have been recently shown to mediate activation of phospholipase C (PLC) in rat liver membranes. There is evidence that the G protein that links pancreastatin receptor with PLC- β is pertussis toxin-insensitive and belongs to the G α q family. Here, we have employed blocking antisera to sort out the specific PLC- β isoform as well as the specific G α subunit activated by PST receptor in rat liver membranes. The presence of different PLC- β isoforms was checked by immunoblot analysis. Only PLC- β4 was not detected, whereas PLC- β1, β2 and β3 were abundant in rat liver membranes. However, only anti-PLC- β3 serum was able to block the PST receptor response. We also checked the expression of G α q and G α 11 in rat liver membranes by immunoblot. Even though both isoforms were present, only anti-G α 11 serum was able to block the PST receptor response. In order to check the specificity of the blocking antisera, we employed them to block the effect of ADP and thrombin stimulating PLC activity in platelet membranes, a system lacking G α 11. Anti-G α q but not anti-G α 11 sera were able to block the agonist stimulated PLC activity. These data suggest that PST receptor response is mediated by the activation of the β3 isoform of PLC via G α 11 protein stimulation in rat liver membranes.

Oxytocin- and aluminium fluoride-induced phospholipase C activity and prostaglandin F2 alpha secretion during the ovine luteolytic period.

A series of studies was conducted to characterize changes in components of the cell signalling cascade that mediates oxytocin-induced prostaglandin F2 alpha (PGF2 alpha) synthesis at the onset of luteolysis in sheep. In the first experiment, caruncular tissue was dissected from 20 ewes on days 12-15 of the oestrous cycle, and incubated for the measurement of phospholipase C (PLC) activity or secretion of PGF2 alpha. Activation of GTP-binding proteins with aluminium fluoride stimulated both inositol phosphate accumulation and PGF2 alpha secretion on all days examined. However, oxytocin did not stimulate PLC activity or PGF2 alpha accumulation until day 13. While the ability of oxytocin to stimulate PLC activity increased after day 13, oxytocin-induced PGF2 alpha secretion declined slightly from day 13 to 15, suggesting that cell signalling components downstream from PLC modulate the response to oxytocin after day 13. Oxytocin failed to stimulate PGF2 alpha synthesis on day 14 after oestrus. Secretion of endogenous luteal oxytocin may have rendered uterine tissues collected on day 14 refractory to oxytocin in vitro. Therefore, a second study was conducted in ovariectomized, steroid replaced ewes. Ovarian steroids were administered to mimic endogenous changes in progesterone and oestradiol. The temporal patterns of PGF2 alpha synthesis in response to oxytocin and pharmacological agents were similar to uterine tissues from cyclic ewes in the first experiment; however, the magnitude of the response was less. These data suggest that oxytocin receptors are absent or are not coupled to PLC until day 13 after oestrus.