Release Of Inositol Phosphates Research Articles

The Impact of Mash pH and Exogenous Phosphorolytic Enzymes on the Release of Inositol Phosphates, Fermentable Sugars and Polypeptide Profile in the Technology of Buckwheat Beer


 The first - already published - stage of the project, which conducted optimization of the temperature mashing program, yielded promising results in the form of an increase in the pool of bioactive myo-inositol by 50% in buckwheat wort supplemented with phosphorolytic enzymes. In this study, it was possible to enhance this effect by adjusting the pH of the mash close to the optimal for phytases, which resulted in a nearly 3-fold increase in myo-inositol concentration, while anti-nutritional phytate was reduced by nearly 75% with a mash pH value at 5.0 and supplementation with a cocktail of commercial phosphorolytic enzymes: Finase P and Finase AP. Changes in the polypeptide profile observed in subsequent wort at a reduced pH of the mash indicate the dominant position of polypeptides in the middle range of molecular weights (10-45 kDa). Ion chromatography coupled with amperometric and conductometric detection modes was used to observe the profile of the key components and the changes that occur in fermentation broths at different pH ranges.

Impacts of dietary calcium, phytate, and phytase on inositol hexakisphosphate degradation and inositol phosphate release in different segments of digestive tract of broilers

A total of 720 straight-run Heritage 56 M × fast feathering Cobb 500F broiler chickens was fed from 11 to 13 d of age to determine the impacts of dietary calcium (Ca), phytate phosphorus (PP), and phytase concentrations on inositol phosphate (IP3–6) profile in different digestive tract (GI) segments. The experiment was a 2 × 2 × 3 randomized block design with 2 Ca (0.7 and 1.0%) and 2 PP (0.23 and 0.34%) concentrations and 3 doses of Buttiauxella sp. phytase (0, 500, and 1,000 FTU/kg). The experiment was replicated in time (block) with 3 replicates per treatment (Trt) of 10 birds per block. Concentrations of IP3–6 in the crop, proventriculus (Prov) plus (+) gizzard (Giz), and distal ileum, as well as the ileal IP6 and P disappearance were determined at 13 d of age. The detrimental impact of Ca on IP6 and P disappearance was observed only in the ileum, where 11% reduction in both IP6 and P disappearance was seen when Ca increased from 0.7 to 1.0% (P < 0.05). Higher IP5 and IP6 concentrations were seen in both the crop and Prov+Giz at 0.34% PP as compared to birds fed to 0.23% PP diets, regardless of Ca or phytase (P < 0.05), whereas IP3 and IP4 concentrations were not affected by PP (P > 0.05). Inclusion of phytase, at both 500 and 1,000 FTU/kg, resulted in lower IP6 and the accumulation of lower IP ester (IP3–5) concentrations in all GI segments (P < 0.05). Improved IP6 and P disappearance was seen as a result of phytase inclusion, despite the degree of improvement affected by PP (P < 0.05). On average, 5.5 and 6.7 times improvement in IP6 was observed with 500 and 1,000 FTU phytase/kg inclusion, respectively, resulting in 41 and 64% greater P digestibility, respectively. In conclusion, phytase can effectively degrade IP6 to lower esters and increase P utilization. However, the efficacy of phytase can be affected by diet Ca and PP concentrations.

Cloning and Expression of a Novel Phytase Gene (phyMS) from &amp;lt;em&amp;gt;Mycobacterium smegmatis&amp;lt;/em&amp;gt;

Phytase, also known as phytate-degrading enzyme, catalyzes the hydrolysis of phytate (inositol hexakisphosphate) with sequential release of phosphate and lower inositol phosphate. We report here a new plasmid construct designated as pMSuia from pBAD-TOPO that harbors a 1.1 kb phytase gene (phyMS) from Mycobacterium smegmatis, and expression as well as characterization of the purified recombinant M. smegmatis phytase. DNA sequencing analysis and multiple alignment exercise indicated that the M. smegmatis phytase is different from both known acid and alkaline phytases. The active ~45 kDa recombinant enzyme was expressed and confirmed by enzyme assay and Western blot analyses. Ni-NTA affinity purified recombinant M. smegmatis phytase exhibited specific activity of 233.51 U/mg, optimal pH of 3 and 7 and optimal temperature of 60°C. The purified enzyme retains almost 30% of the initial activity after incubation at 90°C for 60 min. The enzyme showed broad substrate specificity with Km and Vmax of the recombinant enzyme for sodium phytate substrate of 0.105 ± 0.016 mM and 26.93 ± 1.21 mM min-1, respectively.

Albuterol Isomers Modulate Platelet-Activating Factor Synthesis and Receptor Signaling in Human Bronchial Smooth Muscle Cells

Background: Racemic albuterol is a 50:50 mixture of the (R)- and (S)-enantiomers of albuterol. Its clinical efficacy resides in the (R)-enantiomer (levalbuterol). Studies have shown that (S)-albuterol induces human bronchial smooth muscle cell (HBSMC) proliferation via a pathway linked to platelet-activating factor (PAF), but the underlying mechanism by which (S)-albuterol augments PAF effects is not clear. In this study, we compared effect of levalbuterol and (S)-albuterol on PAF receptor (PAFr)-mediated signaling and PAF metabolism by HBSMCs after incubation with the albuterol isomers. Methods: PAF binding and inositol phosphate (IP₃) release were studied on adherent cultured cells. PAFr protein expression was measured by Western blotting, PAF synthesis and catabolism were measured in membrane and cytosolic proteins of cells incubated with albuterol isomers. Results: Compared to control conditions, (S)-albuterol increased PAF binding by 70% after 30 min of preincubation and by 150% after 24 h of preincubation. Levalbuterol had no effect on PAF binding under both conditions. (S)-albuterol also augmented PAF stimulation of IP₃ release, while levalbuterol and the racemic mixture had no effect. WEB 2170, a PAFr antagonist, inhibited the ability of (S)-albuterol to increase PAF binding or stimulate IP₃ release. (S)-albuterol stimulated PAFr protein expression. With PAF metabolism, (S)-albuterol treatment augmented PAF synthesis, but significantly inhibited PAF catabolism. Conclusions: Our data suggest that one mechanism by which (S)-albuterol stimulates HBSMC proliferation involves upregulation of PAFr-mediated effects including increased PAF synthesis and decreased PAF catabolism.

Mapping of the binding sites for the OX 1 orexin receptor antagonist, SB-334867, using orexin/hypocretin receptor chimaeras

The binding sites for agonists and antagonist of orexin receptors are not know, hampering progressive drug design approaches. In the current study, we utilized chimaeric orexin receptor approach to map the receptor areas contributing to the selectivity of the classical antagonist, SB-334867, for OX 1 receptors. Altogether ten chimaeras between OX 1 and OX 2 orexin receptors were utilized. The receptors were transiently expressed in HEK-293 cells. The ability ( K B) of SB-334867 to inhibit orexin-A-induced inositol phosphate release (phospholipase C activity) was measured. The results, in synthesis, suggest that there are several possible interactions contributing to the high affinity binding, all of which are not required simultaneously. This is indicated by the fact that most of the chimaeras display affinity (at least somewhat) higher than OX 2. As previously shown for the agonist distinction, the second quarter of the receptor, from the C-terminal part of the transmembrane helix 2 to the transmembrane helix 4 seems to be most central also for SB-334867 binding, but also the third quarter, from the transmembrane helix 4 to the transmembrane helix 6 is able to contribute (and compensate for loss of other sites). A previous study has suggested that amino acids conserved between OX 1 and OX 2 receptors would somehow confer selectivity for subtype-selective antagonists. In contrast to previous findings, our results indicate that the amino acids distinct between the receptor subtypes are in key position.

Phospholipase C-η2 is activated by elevated intracellular Ca 2+ levels

Phospholipase C-η2 (PLCη2) is a novel enzyme whose activity in a cellular context is largely uncharacterised. In this study the activity of PLCη2 was examined via [ 3H]inositol phosphate release in COS7 cells expressing the enzyme. PLCη2 activity increased approximately 5-fold in response to monensin, a Na +/H + antiporter. This was significantly inhibited by CGP-37157 which implies that the effect of monensin was due, at least in part, to mitochondrial Na +/Ca 2+-exchange. Direct activation of PLCη2 by < 1 μM Ca 2+ was confirmed in permeabilised transfected cells. The roles of the PH and C2 domains in controlling PLCη2 activity via membrane association were also investigated. A PH domain-lacking mutant exhibited no detectable activity in response to monensin or Ca 2+ due to an inability to associate with the cell membrane. Within the C2 domain, mutation of D920 to alanine at the predicted Ca 2+-binding site dramatically reduced enzyme activity highlighting an important regulatory role for this domain. Mutation of D861 to asparagine also influenced activity, most likely due to altered lipid selectivity. Of the C2 mutations investigated, none altered sensitivity to Ca 2+. This suggests that the C2 domain is not responsible for Ca 2+ activation. Collectively, this work highlights an important new component of the Ca 2+ signalling toolkit and given its sensitivity to Ca 2+, this enzyme is likely to facilitate the amplification of intracellular Ca 2+ transients and/or crosstalk between Ca 2+-storing compartments in vivo.

Prolonged hypoxia modulates platelet activating factor receptor-mediated responses by fetal ovine pulmonary vascular smooth muscle cells

Hypoxia augments PAF receptor (PAFr) binding and PAFr protein expression in venous SMC (SMC-PV). We compared effect of acute and prolonged hypoxia (pO 2 < 40 torr) on PAFr-mediated responses in arterial SMC (SMC-PA) and SMC-PV. Cells were studied for 30 min (acute) or for 48 h (prolonged) hypoxia and compared to normoxic (pO 2 ~ 100 torr) conditions. PAF binding was quantified in fmol/10 6 cells (mean ± SEM). PAF binding in normoxia were SMC-PA, 5.2 ± 0.2 and in SMC-PV, 19.3 ± 1.1; values in acute hypoxia were SMC-PA, 7.7 ± 0.4 and in SMC-PV, 27.8 ± 1.7. Prolonged hypoxia produced 6-fold increase in binding in SMC-PA, but only 2-fold increase in SMC-PV, but binding in SMC-PV was still higher. Acute hypoxia augmented inositol phosphate release by 50% and 40% in SMC-PA and SMC-PV, respectively. During normoxia, PAFr mRNA expression by both cell types was similar, but expression in hypoxia by SMC-PA was greater. In SMC-PA, hypoxia and PAF augmented intracellular calcium flux. Re-exposure of cells to 30 min normoxia after 48 h hypoxia decreased binding by 45–60%, suggesting immediate down-regulation of hypoxia-induced PAFr-mediated effects. We speculate that re-oxygenation immediately reverses hypoxia effect probably due to oxygen tension-dependent reversibility of PAFr activation and suggest that exposure of the neonate to prolonged state of hypoxia will vilify oxygen exchange capacity of the neonatal lungs.

Heterogeneity in platelet activating factor (PAF) receptor binding in ovine fetal intrapulmonary arterial (PA) and venous (PV) smooth muscle cells (SMC)

We have reported previously that hypoxia augments PAF receptor (PAF-r) binding and protein expression in fetal ovine venous smooth muscle cells (SMC-PV). We compared the effects of hypoxia (pO2 ~30 torr) on PAF-r binding in SMC from arteries (SMC-PA) and SMC-PV. Cells were incubated for 48 h in normoxia (pO2 ~100 torr) or hypoxia. PAF-r binding was quantified in fmol/106 cells; means ± SEM, n=5. In normoxia, PAF binding in SMC-PA was 7.7±0.4, which increased to 37.4±2.1 after hypoxia, a 6-fold increase. In SMC-PV, the values in normoxia and hypoxia were 19.33 ± 1.08 and 54.1±3.0, 2-fold increase in hypoxia. Though the increase in binding with hypoxia increased more in SMC-PA, binding in SMC-PV was still greater. Hypoxia augmented inositol phosphate (IP3) release by 28%–40% respectively; the levels of IP3 in SMC-PV during hypoxia were 12-fold higher than in SMC-PA. Re-exposure of hypoxic cells to normoxia for 30 min resulted in decreased binding by 45–60%, suggesting immediate down-regulation of PAF binding by normoxia. These data show that in fetal ovine SMC, PAF-r mediated responses are greater in veins than in arteries and that hypoxia significantly augments this response. However, re-oxygenation immediately reverses this effect of hypoxia. We speculate that this is due to an oxygen tension-dependent reversibility of auto-phosphorylation-dephosphorylation of PAF receptors. Grant NHLBI HL-077819.

Ric-8 Enhances G Protein βγ-Dependent Signaling in Response to βγ-Binding Peptides in Intact Cells

Peptides derived from a random-peptide phage display screen with purified Gbeta(1)gamma(2) subunits as the target promote the dissociation of G protein heterotrimers in vitro and activate G protein signaling in intact cells. In vitro, one of these peptides (SIRKALNILGYPDYD; SIRK) promotes subunit dissociation by binding directly to Gbetagamma subunits and accelerating the dissociation of GalphaGDP without catalyzing nucleotide exchange. The experiments described here were designed to test whether the mechanism of SIRK action in vitro is in fact the mechanism of action in intact cells. We created a mutant of Gbeta(1) subunits (beta(1)W332A) that does not bind SIRK in vitro. Transfection of Gbeta(1)W332A mutant into Chinese hamster ovary cells blocked peptide-mediated activation of extracellular signal-regulated kinase (ERK), but it did not affect receptor-mediated Gbetagamma subunit-dependent ERK activation, indicating that Gbetagamma subunits are in fact the direct target in cells responsible for ERK activation. To determine whether free Galpha subunits were released from G protein heterotrimers upon peptide treatment, cells were transfected with Ric-8A, a guanine nucleotide exchange factor for free GalphaGDP, but not heterotrimeric G proteins. Ric-8A-transfected cells displayed enhanced myristoyl-SIRKALNILGYPDYD (mSIRK)-dependent inositol phosphate (IP) release and ERK activation. Ric-8A also enhanced ERK activation by the G(i)-linked G protein coupled receptor agonist lysophosphatidic acid. Inhibitors of Gbetagamma subunit function blocked Ric-8-enhanced activation of ERK and IP release. These results suggest that one potential function of Ric-8 in cells is to enhance G protein Gbetagamma subunit signaling. Overall, these experiments provide further support for the hypothesis that mSIRK promotes G protein subunit dissociation to release free betagamma subunits in intact cells.

Characterization of the GRK2 Binding Site of Gαq

Heterotrimeric guanine nucleotide-binding proteins (G proteins) transmit signals from membrane bound G protein-coupled receptors (GPCRs) to intracellular effector proteins. The G(q) subfamily of Galpha subunits couples GPCR activation to the enzymatic activity of phospholipase C-beta (PLC-beta). Regulators of G protein signaling (RGS) proteins bind to activated Galpha subunits, including Galpha(q), and regulate Galpha signaling by acting as GTPase activating proteins (GAPs), increasing the rate of the intrinsic GTPase activity, or by acting as effector antagonists for Galpha subunits. GPCR kinases (GRKs) phosphorylate agonist-bound receptors in the first step of receptor desensitization. The amino termini of all GRKs contain an RGS homology (RH) domain, and binding of the GRK2 RH domain to Galpha(q) attenuates PLC-beta activity. The RH domain of GRK2 interacts with Galpha(q/11) through a novel Galpha binding surface termed the "C" site. Here, molecular modeling of the Galpha(q).GRK2 complex and site-directed mutagenesis of Galpha(q) were used to identify residues in Galpha(q) that interact with GRK2. The model identifies Pro(185) in Switch I of Galpha(q) as being at the crux of the interface, and mutation of this residue to lysine disrupts Galpha(q) binding to the GRK2-RH domain. Switch III also appears to play a role in GRK2 binding because the mutations Galpha(q)-V240A, Galpha(q)-D243A, both residues within Switch III, and Galpha(q)-Q152A, a residue that structurally supports Switch III, are defective in binding GRK2. Furthermore, GRK2-mediated inhibition of Galpha(q)-Q152A-R183C-stimulated inositol phosphate release is reduced in comparison to Galpha(q)-R183C. Interestingly, the model also predicts that residues in the helical domain of Galpha(q) interact with GRK2. In fact, the mutants Galpha(q)-K77A, Galpha(q)-L78D, Galpha(q)-Q81A, and Galpha(q)-R92A have reduced binding to the GRK2-RH domain. Finally, although the mutant Galpha(q)-T187K has greatly reduced binding to RGS2 and RGS4, it has little to no effect on binding to GRK2. Thus the RH domain A and C sites for Galpha(q) interaction rely on contacts with distinct regions and different Switch I residues in Galpha(q).

Prostaglandin (PG) F2αReceptor Expression and Signaling in Human Endometrium: Role of PGF2αin Epithelial Cell Proliferation

Prostaglandin (PG) F(2alpha), a member of the prostanoid bioactive lipid family, is secreted by human endometrium throughout the menstrual cycle and is present in both menstrual fluid and medium of endometrial explants in culture. PGF(2alpha) mediates its effects through a seven-transmembrane G-protein-coupled receptor (FP). The aim of this study was to examine the temporal expression, signaling, and role of FP receptor in the human endometrium. Quantitative RT-PCR analysis demonstrated highest expression of FP receptor in the mid- to late-proliferative phase, compared with early-proliferative and secretory phase endometrium. In situ hybridization studies localized FP receptor mRNA expression to the epithelial cell compartment during the mid- to late-proliferative phase. Moreover, treatment of endometrial tissue with 1-100 nM PGF(2alpha) induced a concentration-dependent increase in inositol phosphate mobilization, indicating functional FP receptor expression. The Ishikawa human endometrial epithelial cell line was used to investigate further the signaling and role of PGF(2alpha) in endometrial epithelial cells. Ishikawa cells endogenously express the FP receptor, and treatment with 1-100 nM PGF(2alpha) elicits a concentration-dependent increase in inositol phosphate release. Moreover, treatment of Ishikawa cells with 100 nM PGF(2alpha) induced phosphorylation of ERK1/2 that was abolished when cells were cotreated with 50 micro M PD98059 (MAPK kinase inhibitor) or 10 micro M U73122 [phospholipase C (PLC) inhibitor]. Treatment of Ishikawa cells with PGF(2alpha) for 24 h induced a significant concentration-dependent increase in Ishikawa cell proliferation. Coincubation of the cells with 50 micro M PD98059 or 2 micro M U73122 demonstrated that PLC inhibition significantly reduced PGF(2alpha)-induced proliferation, whereas MAPK kinase inhibition had no effect. In summary, these studies demonstrate increased FP receptor expression in endometrial epithelial cells during the proliferative phase of the menstrual cycle and identify a role for PGF(2alpha) in epithelial cell proliferation via a PLC-dependent pathway.

Rapid desensitization of the serotonin(2C) receptor system: effector pathway and agonist dependence.

The serotonin(2C) (5-HT(2C)) receptor couples to multiple effector mechanisms, including phospholipase A(2)-mediated arachidonic acid (AA) release and phospholipase C-mediated production of inositol phosphates (IP). Agonist relative efficacy differs depending upon which response (AA release or IP accumulation) is measured. In this study, we investigated the characteristics and agonist dependence of rapid desensitization of 5-HT(2C) receptor-mediated AA release and IP accumulation measured simultaneously from the same cell population. Pretreatment with 5-HT reduced the ability of a maximal concentration of 5-HT to elicit AA release and IP accumulation by about 60%; however, the AA response desensitized more rapidly (t(1/2) = 1.3 min) than the IP response (t(1/2) = 6.9 min). In addition, desensitization of the IP response was more sensitive (occurred at lower receptor occupancy levels) than the AA response. Moreover, in response to submaximal 5-HT concentrations, after an initial transient desensitization, the AA response was enhanced by up to approximately 250%. After maximal desensitization, both responses recovered, but recovery of the AA response was complete and faster than that for IP. Desensitization of both responses was also agonist-dependent, and the capacity of agonists to elicit desensitization was not related to their efficacy to activate signaling. These data suggest that desensitization of the 5-HT(2C) receptor system is both agonist- and effector pathway-dependent and underscore the need to study multiple cellular responses to multiple agonists to understand receptor-mediated signaling systems.

Receptor-selective Effects of Endogenous RGS3 and RGS5 to Regulate Mitogen-activated Protein Kinase Activation in Rat Vascular Smooth Muscle Cells

Regulators of G protein signaling (RGS) proteins compose a highly diverse protein family best known for inhibition of G protein signaling by enhancing GTP hydrolysis by Galpha subunits. Little is known about the function of endogenous RGS proteins. In this study, we used synthetic ribozymes targeted to RGS2, RGS3, RGS5, and RGS7 to assess their function. After demonstrating the specificity of in vitro cleavage by the RGS ribozymes, rat aorta smooth muscle cells were used for transient transfection with the RGS-specific ribozymes. RGS3 and RGS5 ribozymes differentially enhanced carbachol- and angiotensin II-induced MAP kinase activity, respectively, whereas RGS2 and RGS7 ribozymes had no effect. This enhancement was pertussis toxin-insensitive. Thus RGS3 is a negative modulator of muscarinic m3 receptor signaling, and RGS5 is a negative modulator of angiotensin AT1a receptor signaling through G(q/11). Also, RGS5 ribozyme enhanced angiotensin-stimulated inositol phosphate release. These results indicate the feasibility of using the ribozyme technology to determine the functional role of endogenous RGS proteins in signaling pathways and to define novel receptor-selective roles of endogenous RGS3 and RGS5 in modulating MAP kinase responses to either carbachol or angiotensin.

CD28 co-stimulates TCR/CD3-induced phosphoinositide turnover in human T lymphocytes.

Upon engagement of TCR with peptide-MHC complexes displayed on the surface of antigen-presenting cells, T lymphocytes undergo a sustained elevation of intracellular Ca(2+) concentration([Ca(2+)](i)), which is required for cytokine production. In the present work, we investigate how inositol lipid metabolism can be activated for a prolonged time to ensure a sustained link between receptor triggering and downstream signaling effectors. Four lines of evidence indicate that an extensive phosphoinositide turnover induced by TCR and CD28 engagement allows this task to be accomplished: (i) continuous phosphoinositide breakdown is required for a sustained [Ca(2+)](i )increase in antigen-stimulated T cells; (ii) TCR triggering results in a continuous release of inositol phosphates from the cell membrane paralleled by a massive and sustained phosphoinositide re-synthesis due to free inositol re-incorporation; (iii) TCR-induced phosphoinositide turnover is strongly increased by CD28 ligation; and (iv) CD28 engagement augments and sustains the TCR-induced [Ca(2+)](i )increase. Our results show that the T cell pool of phosphoinositides is continuously re-formed during T cell-APC cognate interaction, thereby explaining how sustained receptor triggering can transduce an equally sustained [Ca(2+)](i) increase. Importantly, our data identify a novel step in the signaling cascade where co-stimulation converges with TCR-generated signals to sustain and amplify the activation process.

Identification and characterization of a second melanin-concentrating hormone receptor, MCH-2R.

Melanin-concentrating hormone (MCH) is a 19-aa cyclic neuropeptide originally isolated from chum salmon pituitaries. Besides its effects on the aggregation of melanophores in fish several lines of evidence suggest that in mammals MCH functions as a regulator of energy homeostasis. Recently, several groups reported the identification of an orphan G protein-coupled receptor as a receptor for MCH (MCH-1R). We hereby report the identification of a second human MCH receptor termed MCH-2R, which shares about 38% amino acid identity with MCH-1R. MCH-2R displayed high-affinity MCH binding, resulting in inositol phosphate turnover and release of intracellular calcium in mammalian cells. In contrast to MCH-1R, MCH-2R signaling is not sensitive to pertussis toxin and MCH-2R cannot reduce forskolin-stimulated cAMP production, suggesting an exclusive G(alpha)q coupling of the MCH-2R in cell-based systems. Northern blot and in situ hybridization analysis of human and monkey tissue shows that expression of MCH-2R mRNA is restricted to several regions of the brain, including the arcuate nucleus and the ventral medial hypothalamus, areas implicated in regulation of body weight. In addition, the human MCH-2R gene was mapped to the long arm of chromosome 6 at band 6q16.2-16.3, a region reported to be associated with cytogenetic abnormalities of obese patients. The characterization of a second mammalian G protein-coupled receptor for MCH potentially indicates that the control of energy homeostasis in mammals by the MCH neuropeptide system may be more complex than initially anticipated.

A23187 causes release of inositol phosphates from cultured rat Kupffer cells

The Ca 2+ ionophore A23187 is routinely used to illustrate the extracellular Ca 2+-dependence of a variety of cellular reactions. We found that A23187-induced hydrolysis of phosphoinositides to various inositol phosphates in rat Kupffer cells was accompanied by their release from the cells. The synthesis and release of inositol phosphates was A23187 concentration-dependent (0.5–10 μM), and was apparent at the lowest concentration tested. A23187-induced release of inositol phosphates increased time-dependently, was apparent at 5 s of stimulation and maximal at 20 min. The effects of A23187 were reversed by EGTA. The integrity of the cells was not affected by A23187 treatment as indicated by their exclusion of trypan blue and the lack of release of lactate dehydrogenase. We propose that such effects should be considered while evaluating the Ca 2+-dependence of biological processes based on the actions of A23187.

Single Amino Acid of Gα16 (Ala228) Is Responsible for the Ability of Chemoattractant C5a Receptor to Induce Gα16-Mediated Inositol Phosphate Release

Our previous study suggested that the region encompassing residues 220–240 on Gα16 is important in coupling with C5a receptor (Lee et al. (1995) Mol. Pharmacol. 47, 218–223). When aligned sequences are compared in the residue 220–240 segment of Gα16, there is a block of eight amino acids extending from residue 227 to residue 234 (227-Ile-Ala-Leu-Ile-Tyr-Leu-Ala-Ser-234) in Gα16 that is replaced by a heterologous block extending from amino acid residue 224 to residue 231 (224-Thr-Ser-Ile-Met-Phe-Leu-Val-Ala-231) in Gα11. In order to identify the specific amino acid residue necessary for coupling to C5a receptor within the extension of eight amino acids in Gα16, a series of chimeric Gα11/Gα16 cDNA constructs and mutant Gα16 cDNAs were expressed. Then the ability of chimeras and mutant proteins to mediate C5a-induced release of inositol phosphate in transfected Cos-7 cells was tested. The results show that single amino acid Ala228 is responsible for conferring about 40–50% of the activity of Gα16 induced by C5a receptor stimulation.

Suppression of inositol phosphate release by cardiac myocytes isolated from fish oil-fed pigs.

Fatty acid composition of cardiac myocytes and release of inositol phosphates in pigs fed a fish oil supplemented diet was examined. Two groups of female pigs were fed diets supplemented with either 50 g/kg diet beef tallow (as control) or 50 g/kg diet fish oil (MaxEPA) rich in n-3 fatty acids. After 6 weeks of supplementation, the pigs were anesthetized and hearts were removed. Cardiac myocytes were isolated, lipid extracted and separated into non-polar and polar lipids by thin-layer chromatography. Fatty acid composition of individual neutral and polar lipid classes were examined by gas chromatography. To study the effect of membrane phospholipid modification on the phospholipase C (PLC) mediated release of inositol phosphates, cardiac myocytes were labelled with 4 microCi/mL myo-[2-(3)H]inositol for 48 h. After stimulation with epinephrine and phenylephrine, the water soluble [3H]inositol products were extracted, separated from [3H]inositol and [3H]glycerophosphoinositol by chromatography on Dowex AG 1-X8 and quantitated by scintillation counting. Cardiac myocytes isolated from fish oil-fed pigs had higher levels of n-3 polyunsaturated fatty acid in the non-esterified fatty acid and phospholipid fraction. Similarly, these cardiac myocytes had increased level of n-3 fatty and decreased n-6 fatty acids in all the phospholipid fractions, PE, PC, P1 and PS (p < 0.05). After stimulation, the levels of [3H]inositol trisphosphate (IP3) and [3H]inositol tetrakisphosphate (IP4) in cardiac myocytes isolated from fish oil-fed pigs were significantly reduced (p < 0.05) compared to myocytes isolated from beef tallow fed-pigs. This study for the first time has utilised adult cardiac myocytes to demonstrate the effect of n-3 PUFA supplementation on cardiac myocyte phospholipid fatty acid composition and release of second messengers.

Cholesterol-induced Growth Stimulation, Macromolecule Synthesis, and Increased Phosphoinositide Metabolism of Ascites Tumour Cells in Culture

Ascites tumour cells have previously been shown by us to require exogenous cholesterol for growth. To investigate further this phenomenon, we have used, in addition to free cholesterol, cholesterol complexed to digitonine, to elaborate the specificity of this growth-controlling process using a chemically defined medium. Our data show that only free cholesterol stimulates cell growth and macromolecule synthesis in a dose-dependent manner, suggesting that the proper embedding of the sterol into the membrane is a prerequisite for its function. Furthermore, studies have been performed on the influence of cholesterol on the phosphoinositide metabolism of our cells, as phosphoinositides furnish important second messenger molecules in the cascade of signal transduction. We could show that cholesterol stimulates a transient release of inositol trisphosphate and other inositol phosphates by inducing the activation of phospholipase C (PLC). PLC activation by a factor of about 3 with phosphatidylinositol 4-phosphate and phosphatidyl inositol 4,5-bisphosphate as substrates could be measured directly by using a partially purified membrane preparation. This enzyme activity was found to be strongly dependent on free Ca 2+ ions with optimal concentrations of 100 nM for cholesterol- and 50 nM for cholesterol-digitonide-treated cells. Ca 2+ concentration for half-maximum activation, however, was identical under both conditions. Phospholipase C activity could be synergistically increased about 2-fold with 25 μg GTP γS in cholesterol-digitonide-treated cells as well, suggesting that the coupling between phospholipase C and the G-protein was not disturbed by the complex. These data demonstrate a functional role of cholesterol on cell growth, macromolecule synthesis, and phosphoinositide metabolism mediating the release of important second messenger molecules.

Action of the opioid agonist FK 33-824 on porcine small and large luteal cells from the mid-luteal phase: effect on progesterone, cAMP, cGMP and inositol phosphate release.

The present study was designed to investigate the effect of the opioid agonist FK 33-824 on basal and hCG-induced progesterone (P4), cAMP and cGMP secretion and on the phosphoinositide-specific phospholipase C signalling system in separated porcine small (SLCs) and large luteal cells (LLCs). Unit gravity sedimentation was used to produce cultures of small and large luteal cells from corpora lutea (CL) on days 8-10 of the oestrous cycle. In order to examine the effect of FK 33-824 on P4 and cyclic nucleotide release, SLCs and LLCs were incubated in M199 medium at 37 degrees C in 5% CO2:95% air, for 12 h. Small and large luteal cells were treated with hCG (100 ng/ml) alone, FK 33-824 (10(-9) M) alone or were co-treated with FK 33-824 and hCG and with the opioid antagonist, naloxone (NAL, 10(-5) M). FK 33-824 alone did not influence P4 secretion by LLCs and SLCs. However, FK 33-824 completely abolished the stimulatory effect of hCG on P4 secretion by SLCs. The addition of FK 33-824 was followed by a significant increase in cAMP release (p<0.01) by LLCs and a decrease in cGMP secretion by SLCs (p<0.05). The effect of FK 33-824 was blocked by NAL, which strongly suggests that the observed influence of this opioid agonist was achieved through its binding to opioid receptors in luteal membranes. In the presence of hCG, cAMP secretion by both SLCs and LLCs was many-fold higher than in the control group. As regards cGMP output, only LLCs showed elevated secretion of this cyclic nucleotide under the influence of hCG. With the aim of examining the influence of FK 33-824 on phosphatidylinositol hydrolysis, LLCs, SLCs and mixed small and large cells were labelled with [3H]-myo-inositol (100 microCi/ml) for 3 h at 37 degrees C. The cells were then incubated in M199 medium supplemented with 10 mM LiCl, 1% BSA, and antibiotics in the presence and absence of FK 33-824 (10(-9) M) at 37 degrees C for 30 min. Liberated labelled inositol mono-, bis-, and trisphosphates (IPs) were isolated and quantified by affinity chromatography on columns of AG 1-X8 resin, followed by liquid scintillation spectroscopy. Inositol phosphate accumulation in LLCs, SLCs, and mixed small and large cells was not altered by treatment with FK 33-824 at the dose used. In view of these findings we suggest that opioid peptides affect pig corpus luteum steroid secretion, and the response is probably mediated through cyclic nucleotides, but not IPs.