Inositol Phosphate Accumulation In Cells Research Articles

A Miniaturized Column Chromatography Method for Measuring Receptor-Mediated Inositol Phosphate Accumulation

Inositol phosphates (IPs), such as 1,4,5-inositol-trisphosphate (IP(3)), comprise a ubiquitous intracellular signaling cascade initiated in response to G protein-coupled receptor-mediated activation of phospholipase C. Classical methods for measuring intracellular accumulation of these molecules include time-consuming high-performance liquid chromatography (HPLC) separation or large-volume, gravity-fed anion-exchange column chromatography. More recent approaches, such as radio-receptor and AlphaScreen assays, offer higher throughput. However, these techniques rely on measurement of IP(3) itself, rather than its accumulation with other downstream IPs, and often suffer from poor signal-to-noise ratios due to the transient nature of IP(3). The authors have developed a miniaturized, anion-exchange chromatography method for measuring inositol phosphate accumulation in cells that takes advantage of signal amplification achieved through measuring IP(3) and downstream IPs. This assay uses centrifugation of 96-well-formatted anion-exchange mini-columns for the isolation of radiolabeled inositol phosphates from cell extracts, followed by low-background dry-scintillation counting. This improved assay method measures receptor-mediated IP accumulation with signal-to-noise and pharmacological values comparable to the classical large-volume, column-based methods. Assay validation data for recombinant muscarinic receptor 1, galanin receptor 2, and rat astrocyte metabotropic glutamate receptor 5 are presented. This miniaturized protocol reduces reagent usage and assay time as compared to large-column methods and is compatible with standard 96-well scintillation counters.

Agonist-independent internalization of metabotropic glutamate receptor 1a is arrestin- and clathrin-dependent and is suppressed by receptor inverse agonists.

Three group I mGluR antagonists CPCCOEt, LY367385 and BAY36-7620, were analyzed for their effect on cell surface expression of metabotropic glutamate receptor 1a and 1b. All three antagonists inhibited glutamate-induced internalization of mGluR1a and mGluR1b. However, when added alone, either LY367385 or BAY36-7620 increased the cell surface expression of mGluR1a but not mGluR1b. Both LY367385 and BAY36-7620 displayed inverse agonist activity as judged by their ability to inhibit basal inositol phosphate accumulation in cells expressing the constitutively active mGluR1a. Interestingly, mGluR1a but not mGluR1b was constitutively internalized in HEK293 cells and both LY367385 and BAY36-7620 inhibited the constitutive internalization of this splice variant. Furthermore, coexpression of dominant negative mutant constructs of arrestin-2 [arrestin-2-(319-418)] or Eps15 [Eps15(E Delta 95-295)] increased cell surface expression of mGluR1a and blocked constitutive receptor internalization. In the presence of these dominant negative mutants, incubation of cells with LY367385 and BAY36-7620 produced no further increase in cell surface expression of mGluR1a. Taken together, these results suggest that the constitutive activity of mGluR1a triggers the internalization of the receptor through an arrestin- and clathrin-dependent pathway, and that inverse agonists increase the cell surface expression of mGluR1a by promoting an inactive form of mGluR1a, which does not undergo constitutive internalization.

Peptide and nonpeptide antagonist interaction with constitutively active human AT 1 receptors

Wild type human AT 1 receptors (WT-AT 1) and mutant receptors, in which Asn 111 was replaced by glycine (N111G), alanine (N111A) and serine (N111S), or in which Asp 281 was replaced by alanine (D281A) or in which N111G and D281A replacements were combined, were transiently expressed in CHO-K1 cells. While the biphenyltetrazole compound candesartan dissociated slowly and behaved as an insurmountable antagonist for WT-AT 1, it dissociated swiftly and only produced a rightward shift of the angiotensin Ang II- and -IV dose–response curves for inositol phosphate (IP) accumulation in cells expressing N111G. [ 3 H ]candesartan competition binding yielded the same potency order of the related biphenyltetrazoles for WT-AT 1 and mutated receptors, i.e. candesartan>EXP3174>irbesartan>losartan. Affinities were equal for WT-AT 1 and D281A and 40- to 400-fold lower for all Asn 111 mutants. Mutations did not affect the affinity of the peptide antagonist [Sar 1Ile 8]Ang II (SARILE). Basal IP accumulation in cells with WT-AT 1 was not affected by any biphenyltetrazole antagonists and was increased by SARILE to 19% of the maximal Ang II stimulation. Basal IP accumulation was higher for cells expressing the Asn 111-mutated receptors. For N111G, this accumulation was partially inhibited by all the biphenyltetrazoles upon long-term (18 hr) exposure. In these cells SARILE produced the same maximal stimulation as Ang II. Asn 111-mutated AT 1 receptors are thought to mimic the pre-activated state of the wild type receptor and comparing the efficacy and affinity of ligands for such mutated receptors facilitate the distinction of partial (SARILE) and inverse (biphenyltetrazoles) agonists from true antagonists.

A fusion protein of the human P2Y(1) receptor and NTPDase1 exhibits functional activities of the native receptor and ectoenzyme and reduced signaling responses to endogenously released nucleotides.

To begin to address the functional interactions between constitutively released nucleotides, ectonucleotidase activity, and P2Y receptor-promoted signaling responses, we engineered the human P2Y(1) receptor in a fusion protein with a member of the ectonucleoside triphosphate diphosphohydrolase family, NTPDase1. Membranes prepared from Chinese hamster ovary (CHO)-K1 cells stably expressing either wild-type NTPDase1 or the P2Y(1) receptor-NTPDase1 fusion protein exhibited nucleotide-hydrolytic activities that were over 300-fold greater than activity measured in membranes from empty vector-transfected cells. The molecular ratio for nucleoside triphosphate versus diphosphate hydrolysis was approximately 1:0.4 for both the wild-type NTPDase1 and P2Y(1)-NTPDase1 fusion protein. Stable expression of the P2Y(1)-NTPDase1 fusion protein conferred an ADP and 2MeSADP-promoted Ca(2+) response to CHO-K1 cells. Moreover, the maximal capacity of the nonhydrolyzable agonist ADPbetaS to stimulate inositol phosphate accumulation was similar, and the EC(50) of ADPbetaS was lower in the fusion protein than the wild-type receptor. In contrast, the substantial nucleotide-hydrolyzing activity of the fusion protein resulted in a greater than 50-fold shift to the right of the concentration-effect curve of ADP for activation of phospholipase C compared with the wild-type receptor. Heterologous expression of the P2Y(1) and other P2Y receptors results in marked increases in basal inositol phosphate levels. Given the high nucleotidase activity and apparently normal receptor signaling activity of the P2Y(1) receptor-NTPDase1 fusion protein, we quantitated basal inositol phosphate accumulation in cells stably expressing either the wild-type P2Y(1) receptor or the fusion protein. Although marked elevation of inositol phosphate levels occurred with wild-type P2Y(1) receptor expression, levels in cells expressing the fusion protein were not different from those in wild-type CHO-K1 cells.

Effects of age and gender on the AII-induced stimulation of prolactin release and inositol phosphate accumulation in rat anterior pituitary cells in vitro

The stimulatory effects of Angiotensin II (AII) on prolactin secretion and inositol phosphate accumulation were examined in dispersed anterior pituitary cells collected from young (3–4 month), mature (7–8 month) and old (18–20 month) male and female rats. Physiological doses of AII (0.01–10 nM) stimulated prolactin release from cells collected from mature female rats only. This effect was antagonized by pretreatment with Saralasin, an AII receptor antagonist. Significant accumulation of the inositol phosphates was observed in cells obtained from the mature, female donors and this increase preceded the prolactin response. Although there was a small increase in total inositol phosphate accumulation in cells obtained from the old female rats, this was transient and did not coincide with a similar increase in prolactin release. These results indicate that pituitary sensitivity to AII stimulation is related to the age and the gender of the donor animal. The physiological role of pituitary AII needs to be examined in sexually mature female animals.

Synergy between the inositol phosphate responses to transfected human adenosine A1-receptors and constitutive P2-purinoceptors in CHO-K1 cells.

1. The effect of adenosine A1-receptor and P2-purinoceptor agonists on [3H]-inositol phosphate accumulation has been investigated in CHO-K1 cells transfected with the human adenosine A1-receptor. 2. Adenosine receptor agonists stimulated [3H]-inositol phosphate accumulation in CHO-K1 cells with a rank potency order of N6-cyclopentyladenosine (CPA) > 5'-N-ethylcarboxamidoadenosine (NECA) > 2-chloroadenosine > N6-2-(4-aminophenyl) ethyladenosine (APNEA). The responses to both CPA and APNEA were antagonized by the A1 selective antagonist, 1,3-dipropylcyclopentylxanthine (DPCPX) yielding KD values of 1.2 nM and 4.3 nM respectively. 3. ATP, UTP and ATP gamma S were also able to stimulate [3H]-inositol phosphate accumulation in these cells with EC50 values of 1.9 microM, 1.3 microM and 5.0 microM respectively. 2-Methyl-thio-ATP was a weak agonist of this response (EC50 > 100 microM). 4. The [3H]-inositol phosphate response to CPA was completely attenuated by pertussis toxin treatment (24 h; 100 ng ml-1). In contrast, the responses to ATP, UTP and ATP gamma S were only reduced by circa 30% in pertussis toxin-treated cells. 5. The simultaneous addition of CPA and either ATP, UTP or ATP gamma S produced a large augmentation of [3H]-inositol phospholipid hydrolysis. This was due to an increase in the maximal response and was significantly greater than the predicted additive response for activation of these two receptor systems. The synergy was not observed in pertussis toxin-treated cells. 6. No synergy was observed between the [3H]-inositol phosphate responses to histamine and ATP in CHO-K1 cells transfected with the bovine histamine H1-receptor. In these cells the response to histamine was completely resistant to inhibition by pertussis toxin treatment. 7. This study provides a clear demonstration of a synergy between pertussis toxin-sensitive and insensitive receptor systems in a model cell system which is an ideal host for transfected cDNA sequences. This model system should provide a unique opportunity to unravel the mechanisms underlying this example of receptor cross-talk involving phospholipase C.

Acute effects of prostaglandin F2 alpha, luteinizing hormone, and estradiol on second messenger systems and on the secretion of oxytocin and progesterone from granulosa and early luteal cells of the ewe.

Previous reports have suggested that gonadotropins, estradiol, and prostaglandin F2 alpha (PGF2 alpha) have varying effects on progesterone and oxytocin synthesis or secretion in cultured granulosa and luteal cells collected at different stages of the estrous cycle. The experiments reported here were designed to investigate whether effects of these agonists on secretion of hormones and their coupling to second messenger systems changed around the time of ovulation. Granulosa cells and Day 2 luteal cells of the ewe were cultured for three days and then treated for 30 min with varying doses of PGF2 alpha, LH, or estradiol. LH increased intracellular cAMP at both stages, but granulosa cells were more responsive in terms of both minimum effective dose (10 compared with 100 ng/ml) and degree of stimulation. LH caused no change in intracellular inositol phosphate levels. Both granulosa and early luteal cells responded to LH treatment by an increase in progesterone output in a dose-responsive fashion. PGF2 alpha increased inositol phosphate accumulation in cells collected at both stages of the cycle. All doses tested (10(-6)-10(-8) M) stimulated the release of oxytocin into the culture medium from both granulosa and luteal cells. Progesterone secretion was also increased, but only at the highest dose (10(-6) M). Estradiol treatment (10(-6) M) did not affect either the inositol phosphate or cAMP second messenger systems, but it did inhibit the secretion of oxytocin from granulosa cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of substitution of threonine 654 of the epidermal growth factor receptor on epidermal growth factor-mediated activation of phospholipase C.

Addition of epidermal growth factor (EGF) to many cell types activates phospholipase C resulting in increased levels of diacylglycerol and intracellular Ca2+ which may lead to activation of protein kinase C. EGF treatment of cells can also lead to phosphorylation of the EGF receptor at threonine 654 (a protein kinase C phosphorylation site) which appears to attenuate some aspects of receptor signaling. Thus, a feedback loop involving the EGF receptor, phospholipase C, and protein kinase C may regulate EGF receptor function. In this report, the role of phosphorylation of threonine 654 of the EGF receptor in regulation of EGF-stimulated activation of phospholipase C was investigated. NIH-3T3 cells expressing the normal human EGF receptor or expressing EGF receptor in which an alanine residue had been substituted at residue 654 of the receptor were used. Addition of EGF to cells expressing wild-type receptor induced a rapid, but transient, increase in phosphorylation of threonine 654. EGF addition also caused the rapid accumulation of inositol phosphates in these cells. EGF-stimulated accumulation of inositol phosphates was significantly higher in cells expressing Ala-654 receptors compared to control cells. Treatment of cells with 12-O-tetradecanoylphorbol 13-acetate (TPA), which stimulated phosphorylation of threonine 654 to a greater degree than EGF, completely inhibited EGF-dependent inositol phosphate accumulation in cells expressing wild-type receptor, but caused only a 20-30% inhibition in Ala-654 expressing cells. EGF stimulated phosphorylation of phospholipase C-gamma on serine and tyrosine residues in cells expressing wild-type of Ala-654 receptors. However, TPA treatment of cells inhibited EGF-induced tyrosine phosphorylation of phospholipase C-gamma only in cells expressing wild-type receptors. Similarly, TPA inhibited tyrosine-specific autophosphorylation of the EGF receptor and tyrosine phosphorylation of several other proteins in wild-type receptor cells, but not in Ala-654 cells. TPA treatment abolished high affinity binding of EGF to cells expressing wild-type receptors, while decreasing the number of high affinity binding sites 20-30% in Ala-654 cells. These data suggest that phosphorylation of threonine 654 can regulate early events in EGF receptor signal transduction such as phosphoinositide turnover, probably through a feedback mechanism involving protein kinase C. Subsequent dephosphorylation of threonine 654 could reactivate the EGF receptor for participation in later signaling events.