Inositol Polyphosphate Accumulation Research Articles

Enhanced calcium signaling to bradykinin in airway smooth muscle from hyperresponsive inbred rats.

Inbred Fischer 344 rats display airway hyperresponsiveness (AHR) in vivo compared with the normoresponsive Lewis strain. Fischer AHR has been linked with increased airway smooth muscle (ASM) contraction ex vivo and enhanced ASM cell intracellular Ca(2+) mobilization in response to serotonin compared with Lewis. To determine the generality of this association, we tested whether bradykinin (BK) also stimulates greater contraction of Fischer airways and greater Ca(2+) mobilization in Fischer ASM cells. Explants of Fischer intraparenchymal airways constricted faster and to a greater degree in response to BK than Lewis airways. BK also evoked higher Ca(2+) transients in Fischer than in Lewis ASM cells. ASM cell B(2) receptor expression was similar between the two strains. BK activated both phosphatidylinositide-specific phospholipase C (PI-PLC) and phosphatidylcholine-specific PLC to mobilize Ca(2+) in Fischer and Lewis ASM cells. PI-PLC activity, as measured by inositol polyphosphate accumulation, was similar in the two strains. PKC inhibition with GF109203X, Go6973, or Go6983 attenuated BK-mediated Ca(2+) transients in Fischer cells, whereas GF109203X potentiated while Go6976 and Go6983 did not affect Ca(2+) transients in Lewis cells. Enhanced Ca(2+) mobilization in ASM cells can arise from variations in PKC and may be an important component of nonspecific, innate AHR.

Signaling properties of VEGF receptor-1 and -2 homo- and heterodimers

Vascular endothelial growth factor (VEGF-A) exerts its effects through receptor tyrosine kinases VEGF receptor-1 (VEGFR-1) and VEGFR-2, which are expressed on most endothelial cell types in vitro and in vivo. We have examined VEGF-A-induced signal transduction in porcine aortic endothelial (PAE) cells individually expressing VEGFR-1 or VEGFR-2, and cells co-expressing both receptor types. We show that VEGF-A-stimulated PAE cells co-expressing VEGFR-1 and -2 contain receptor heterodimers. VEGF-A-stimulation of all three cell lines (expressing VEGFR-1, -2 and -1/2) resulted in signal transduction with different efficiencies. Thus, tyrosine phosphorylation of phospholipase Cγ, and accumulation of inositol polyphosphates were efficiently transduced in the VEGFR-1/2 cells whereas cells expressing VEGFR-1 responded poorly in these assays. In contrast, VEGF-A-induced activation of phosphoinositide 3-kinase and induction of Ca 2+ fluxes were transduced well by VEGFR-1 and VEGFR-2 homo- and heterodimers. The pattern of Ca 2+ fluxes was unique for each type of VEGF receptor dimer. Our data show that signal transduction induced by VEGF-A is transduced in distinct manners by homo- and heterodimers of VEGF receptors.

Inositol (1,4,5)Trisphosphate Metabolism and Enhanced Calcium Mobilization in Airway Smooth Muscle of Hyperresponsive Rats

Airway hyperresponsiveness (AHR) is a phenotype of asthma and can be modeled by the inbred Fisher strain of rat, which is hyperresponsive in vivo relative to the Lewis strain. Enhanced airway smooth muscle (ASM) contractility and Ca(2+) mobilization are associated with the AHR observed in Fisher rats. In this study, we investigated whether the interstrain differences in Ca(2+) mobilization to serotonin (5HT) result from differences in inositol (1,4,5)trisphosphate (IP(3)) metabolism and/or IP(3) receptor (IP(3)R) sensitivity. Ca(2+) mobilization by 5HT in cultured ASM cells from both rat strains was phospholipase C (PLC) dependent. Inositol polyphosphate accumulation, and hence PLC activity, was similar in both rat strains, but a specific IP(3) transient was detectable only in Fisher myocytes in response to 5HT. These findings suggested that IP(3) degradation rather than production differed between the two strains. The Vmax and Michaelis constant (K(m)) of IP(3)-specific 5-phosphatase activity were higher in the particulate fraction of Lewis than in Fisher ASM cell homogenates and appeared to be related to a greater expression of two isoforms of 5-phosphatase (type I and type II) in Lewis cells as shown by Western blot analysis. The sensitivity of the IP(3)R to IP(3) was similar between Fisher and Lewis ASM cells, indicating that the interstrain intracellular Ca(2+) differences were unrelated to IP(3)R function. We propose that interstrain variations in 5-phosphatase activity and expression may give rise to the interstrain differences in IP(3)-mediated Ca(2+) release in ASM and may be a determinant of AHR.

The phosphatidylinositol polyphosphate 5-phosphatase SHIP and the protein tyrosine phosphatase SHP-2 form a complex in hematopoietic cells which can be regulated by BCR/ABL and growth factors.

We report here that interleukin-3 (IL-3) and erythropoietin (EPO) induce formation of a complex composed of two SH2-containing phosphatases, the tyrosine phosphatase SHP-2 and the SH2 containing inositol 5-phosphatase (SHIP). Both SHP-2 and SHIP are known to be involved in growth factor signal transduction, but their potential interaction in the same pathway is novel. SHIP has previously been shown to associate with SHC, and potentially to be involved in regulating apoptosis. In contrast, in some model systems, SHP-2 has been demonstrated to positively regulate cell growth. Both phosphatases in the complex were tyrosine phosphorylated, and the amount of SHIP coprecipitating with SHP-2 was inversely related to the amount of SHIP coprecipitating with SHC. In hematopoietic cells transformed by the BCR/ABL oncogene, this phosphatase complex was found to be constitutively present with both components heavily tyrosine phosphorylated. Also, other proteins were detected in the complex, including BCR/ABL itself and c-CBL. However, transformation by BCR/ABL was associated with a reduced SHIP protein expression, which could further affect the accumulation of various inositol polyphosphates in these leukemic cells. These data suggest that the function of SHIP and SHP-2 in normal cells are linked and that BCR/ABL alters the function of this signaling complex.

Inhibition by NMDA of carbachol-stimulated inositol tetrakisphosphate accumulation in rat brain cortical slices

The present studies examined the effect of NMDA on carbachol-stimulated accumulation of inositol polyphosphates, with emphasis on the accumulation of inositol 1,3,4,5-tetrakisphosphate (Ins 1,3,4,5-P 4), at short time periods in rat brain cortical slices. There was a stimulatory effect of NMDA on accumulation of labeled inositol mono-, bis- and trisphosphates but not on labeled inositol tetrakisphosphates. In the presence of carbachol Ins 1,3,4,5-P 4 accumulation was preferentially inhibited by NMDA at early time periods (within 30 seconds after NMDA addition). Subsequently, total phosphoinositide breakdown was inhibited by NMDA. NMDA did not stimulate accumulation of total Ins 1,3,4,5-P 4 but immediately inhibited carbachol stimulated accumulation of Ins 1,3,4,5-P 4. The inhibitory effect of NMDA (1 mM) was not mimicked by increasing K + in the medium from 10 to 30 mM. However 30 mM K + reversed the inhibitory effect of 1 mM NMDA on carbachol-stimulated Ins 1,3,4,5-P 4. Parallel experiments with veratridine (a sodium channel activator) suggest that the early inhibitory effects of NMDA on Ins 1,3,4,5-P 4 accumulation are not due to decreases in ATP availability or elevations in intracellular Na +. These data indicate that NMDA increases inositol mono-, bis- and trisphosphate accumulation while blocking muscarinic cholinergic stimulated accumulation of Ins 1,3,4,5-P 4.

Involvement of metabotropic and ionotropic glutamate receptors in inositol polyphosphate formation in carp retinal slices.

The contribution of ionotropic and metabotropic glutamate receptors to inositol polyphosphate accumulation in carp retinal slices was investigated using myo-[2-3H]inositol prelabelling. In the presence of the glutamate agonists quisqualate, (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and trans-(+/-)-1-amino-1,3-cyclopentane-dicarboxylic acid (t-ACPD), formation of [3H]inositol phosphate was significantly increased in a dose-dependent manner, with EC50 values of 350 nM, 1.5 microM and 10 microM respectively. The complete AMPA-induced response and a large component of the quisqualate-induced response were inhibited in a competitive manner when the ionotropic antagonist 6-cyano-7-nitroquinoxalin- 2,3-dione (CNQX) was present. Furthermore, the remaining level of quisqualate-induced [3H]inositol phosphate formation closely matched that produced by ACPD alone, and coincubation of AMPA and ACPD showed additive effects, suggesting that the quisqualate-induced response resulted from coactivation of metabotropic and ionotropic glutamate receptors. The ionotropic component was partially reduced in the presence of cobalt, suggesting indirect effects resulting from synaptic interactions. We could exclude indirect effects through depolarization-induced release of other neurotransmitters. Only serotonin (EC50 1 microM) and carbachol (at a concentration of 1 mM) stimulated [3H]inositol phosphate formation, but their antagonists did not affect the quisqualate response and coactivation with quisqualate and serotonin or carbachol resulted in additive effects. The ionotropic component was completely suppressed when Ca2+ was omitted from the medium and cobalt was present. This makes it likely that the ionotropic component resulted from Ca2+ entry through AMPA-gated channels and subsequent Ca(2+)-dependent activation of phospholipase C.

Peptides containing the RERMS sequence of amyloid beta/A4 protein precursor bind cell surface and promote neurite extension

Amyloid beta/A4 protein precursor (APP) is secreted into medium by most cultured cells and can function as an autocrine factor. To study the biological function of secreted forms of APP (sAPP) on neurons, we used a clonal CNS neuronal line, B103, which does not synthesize detectable levels of APP. B103 cells transfected with APP construct developed neurites faster than the parent B103 cells when plated in a serum-free defined medium. Neurite outgrowth of B103 cells was promoted by the conditioned medium of APP-695-over-producing cells or by the bacteria-produced sAPP-695 (named KB75). A series of peptides having sequences between Ala-319 and Met-335 of APP-695 also stimulated neurite outgrowth of B103 cells. The sequence of five amino acids, RERMS (APP 328-332), within this stretch of sequence, was the shortest active peptide, although the concentration required for the neuritotropic activity was higher than that of KB75. Binding assay using 125I-labeled APP 17-mer peptide corresponding to Ala-319 to Met-335 of APP-695 as a ligand demonstrated specific and saturable cell-surface binding sites. The predicted KD value was 20 +/- 5 nM and the Bmax value was 80 +/- 8 fmol/10(6) cells. The binding could be displaced with KB75. A 17-mer peptide with reverse sequence neither induced neurite outgrowth nor competed for the binding. A bacteria-produced sAPP fragment lacking the active 17-mer sequence (named KB75 delta) did not compete with 125I-labeled 17-mer for binding or stimulate neurite extension. A peptide of sequence RMSQ (APP 330-333), which partially overlaps the active sequence RERMS, could block the neuritotropic effects of both KB75 and the 17-mer at higher concentrations. APP 17-mer was also found to induce the accumulation of inositol polyphosphates, suggesting that the APP 17-mer effects involve activation of inositol phospholipid signal transduction systems. These data indicate that sAPP induces neurite extension through cell-surface binding and that the domain containing the RERMS sequence (APP 328-332) represents the active site responsible for this function.

Effects of membrane depolarization and changes in intra- and extracellular calcium concentration on phosphoinositide hydrolysis in bovine tracheal smooth muscle

Agonist-stimulated phosphoinositide metabolism plays a central role in pharmacomechanical coupling in airways smooth muscle (ASM). In many other tissues and cells, most noteably excitable cells, membrane depolarization or an increase in intracellular Ca 2+ ([Ca 2+] i) generated by inositol 1,4,5-trisphosphate (Ins(1,4,5)P 3)-induced Ca 2+ release or agonist-mediated Ca 2+influx is able to trigger or augment phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2) hydrolysis and/or initiate PtdIns4P/ PtdIns hydrolysis by direct stimulation of PIC. To assess the importance of these mechanisms in ASM the effects of KCl-induced membrane depolarization, extracellular Ca 2+([Ca 2+] e) chelation, and addition of ionomycin to elevate [Ca 2+] i on basal and agonist-stimulated Ins(1,4,5)P 3 concentration and [ 3H]-InsP x accumulation have been examined. Reducing [Ca 2+] e from 1.8 mM to 6 or 0.8μM caused a progressive inhibition of agonist-stimulated [ 3H]inositol polyphosphate accumulation over 30 min with the histamine-stimulated response being significantly more sensitive to [Ca 2+] e chelation than the response to carbachol. In contrast, the initial accumulation of Ins(1,4,5)P 3 was completely unaffected by such reductions in [Ca 2+] e. Incubation of [ 3H]inositol-prelabelled BTSM slices with buffer containing 80 mM KC1 failed to stimulate [ 3H]InsP x accumulation, causing instead a small inhibition of carbacholstimulated [ 3H]InsP x accumulation with a similar effect seen with respect to Ins(1,4,5)P 3 accumulation. Addition of 5 μM ionomycin to BTSM slices similarily did not stimulate Ins(1,4,5)P 3 generation and only increased [ 3H]InsP x accumulation after prolonged stimulation in the presence of high (mM) [Ca 2+] e. These data indicated that in ASM, membrane depolarization or physiological increases in [Ca 2+] i did not result in either independent activation of PIC or augmentation of initial agoniststimulated PtdIns(4,5)P 2 hydrolysis. However, while the initial agonist-stimulated generation of Ins(1,4,5)P 3 was not dependent on [Ca 2+] e, a normal plasmalemmal Ca 2+ gradient was required to sustain maximal rates of agonist-stimulated PtdIns(4,5)P 2 hydrolysis.

Characteristics of inositol polyphosphate metabolism in cultured adrenal chromaffin cells

Sasakawa, Nobuyuki, Toshio Nakaki and Ryuichi Kato: Characteristics of Inositol polyphosphate Metabolism in cultured adrenal chromaffin cells. Prog. NeuroPsychopharmacol. & Biol. Psychiat. 1993, : 17(5): 825–834. 1. 1. Nicotine, high K + and maitotoxin caused the inositol polyphosphate accumulation concomitant with 45Ca 2+ uptake. 2. 2. Angiotensin II (Ang II) and ATP induced the inositol polyphosphate accumulation without 45Ca 2+ uptake. 3. 3. Nifedipine-treatment and Ca 2+-deprivation inhibited the high K +-induced inositol polyphosphate accumulation but failed to inhibit the Ang II-induced inositol polyphosphate accumulation. 4. 4. 12-O-tetradecanoylphorbol-13-acetate inhibited the Ang II-induced inositol polyphosphate accumulation but failed to inhibit the high K +-induced one 5. 5. These results suggest that the formation of inositol polyphosphates may be regulated by two mechanisms i.e. Ca 2+ uptake-dependent mechanisms represented by high K +, and Ca 2+ uptake-independent mechanisms represented by Ang II.

Increase in inositol tris-, pentakis- and hexakisphosphates by high K + stimulation in cultured rat cerebellar granule cells

Effects of high K + stimulation on inositol polyphosphate accumulations and intracellular free calcium concentration ([Ca 2+] i) were investigated in cultured rat cerebellar granule cells. When the [ 3H]inositol-labelled cells were stimulated with KCl, concentration-dependent accumulations of [ 3H]Ins(1,4,5)P 3, [ 3H]InsP 5 and [ 3H]InsP 6 were observed. Nifedipine (3 μM), a calcium channel antagonist, inhibited the high (KCl, 90 mM) K +-induced accumulations of these inositol polyphosphates. In Ca 2+-depleted and EGTA-containing (0.1 mM) medium, the high K +-induced inositol polyphosphate accumulation were completely inhibited. Similar results were also observed in the case of [Ca 2+] i. These results suggest that the rise in [Ca 2+] i caused by activation of voltage-dependent calcium channels plays an important roles in the high K +-induced accumulation of [ 3H]Ins(1,4,5)P 3, [ 3H]InsP 5 and [ 3H]InsP 6 in cultured rat cerebellar granule cells.

Bradykinin Receptors and Signal Transduction Pathways in Human Fibroblasts: Integral Role for Extracellular Calcium

Bradykinin receptors have been identified in human gingival fibroblasts; the primary signal transduction pathways and their dependence on calcium have been characterized. Binding data revealed a calcium-independent binding of bradykinin to the cell membrane with a receptor density of 25,000 receptors per cell and a Kd of 1.6 nM. The bradykinin receptor-mediated activation of phospholipase C (PLC) resulted in an extensive and rapid stimulation of phosphoinositide metabolism. Using radioreceptor assay techniques, in the absence of LiCl, the inositol 1,4,5-trisphosphate (Ins 1,4,5 P3) generation was found to be transient, with maximal levels attained within 15 s. An EC50 of 12 nM was observed for the accumulation of total inositol polyphosphates. The activation of phospholipase A2 (PLA2), and the subsequent release of arachidonic acid and the primary metabolite prostaglandin E2, also was found to be time- and concentration-dependent. Stimulation of tyrosine kinase activity by bradykinin was concentration-dependent and resulted in the phosphorylation of three substrates of unknown identity. Bradykinin stimulation did not activate adenylate cyclase as there occurred no increase in the generation of cyclic AMP. The mobilization of intracellular calcium stores followed closely the Ins 1,4,5 P3 kinetics and had an EC50 of 11 nM. Chelation of extracellular calcium reduced significantly the duration of the calcium response, while only minimally lowering the rapid, maximal increase in intracellular free calcium concentration ([Ca2+]i). A sustained elevation of [Ca2+]i was found to be essential in PLC and PLA2 signaling, as well as in tyrosine kinase activation, suggesting a major role for membrane calcium channels in bradykinin stimulation of cellular responses in these cells. Bradykinin was found to inhibit dramatically epidermal growth factor-induced DNA synthesis in confluent cells, although to a much lesser degree in subconfluent cells. This pattern was similar to the observed maximal specific increase in bradykinin binding with confluency. Together these results demonstrate the presence of bradykinin receptors in human gingival fibroblasts; these receptors are coupled to signal transduction mechanisms involving the PLC, PLA2, and tyrosine kinase effector systems, all of which require extracellular calcium to achieve maximal activation.

Inhibitors of the arachidonic acid metabolism attenuate the thyroliberin (TRH) stimulated prolactin production without modifying the production of inositolphosphates in GH4C1 pituitary cells.

Some arachidonic acid metabolites might be among the intracellular signalling substances that regulate hormone release. We report that the phospholipase A2 and diacylglycerol lipase inhibitor quinacrine (1-10 mumol l-1) inhibited the thyroliberin stimulated prolactin (rPRL) production in a dose-dependent way in a rat pituitary tumour cell line (GH4Cl cells). The lipoxygenase inhibitor nafazatrom (5-50 mumol-1) also dose-dependently inhibited the thyroliberin stimulated rPRL production, while the cyclo-oxygenase inhibitor indomethacin had no such effect on rPRL production. The inhibitors of the arachidonic acid metabolism (quinacrine, ETYA and nafazatrom) had no effect on the accumulation of inositolpolyphosphates indicating that the arachidonic acid metabolites are not involved in the regulation of the phospholipase C activity.

Role of protein kinase C in the regulation of histamine and bradykinin stimulated inositol polyphosphate turnover in adrenal chromaffin cells.

1. The possibility that bradykinin- or histamine-stimulated inositol polyphosphate accumulation may be regulated by protein kinase C (PKC) in bovine adrenal chromaffin cells has been addressed. 2. Initial experiments confirmed that the phorbol ester 12-O-tetradecanoyl-phorbol 13-acetate (TPA) dramatically inhibited agonist-stimulated [3H]-inositol phosphate accumulations in [3H]-inositol prelabelled cells. In contrast, the PKC inhibitor, Ro 31-8220, did not affect this response. 3. Histamine (100 microM) or bradykinin (100 nM) evoked rapid increases in inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4) mass accumulations (maximal accumulations within 10 s and 30 s, respectively) which declined towards basal values over a 10 min incubation period. TPA (1 microM) significantly attenuated the peak Ins(1,4,5)P3 response to bradykinin and histamine by 30% and 70% respectively. In contrast, TPA did not significantly affect agonist-stimulated Ins(1,3,4,5)P4 responses. 4. Ro 31-8220 (10 microM) significantly enhanced the maximal Ins(1,4,5)P3 accumulations elicited by both bradykinin and histamine. 5. The results indicate that the initial Ins(1,4,5)P3 response to either bradykinin or histamine in bovine adrenal chromaffin cells can be attenuated by PKC activation by phorbol ester and enhanced by PKC inhibition by Ro 31-8220. In contrast, agonist-stimulated Ins(1,3,4,5)P4 accumulation does not appear to be affected by these manipulations of PKC activity. Possible bases for differential modulation of Ins(1,4,5)P3 and Ins(1,3,4,5)P4 are discussed.

Endothelin stimulates multiple responses in isolated adult ventricular cardiac myocytes.

We have examined the responses to endothelin (ET) in isolated adult cardiac myocytes from both rodent and feline species to assess whether endothelin may have a role in the induction or mediation of cardiac hypertrophy in the adult animal. We have evidence that ET acts by more than one mechanism to promote cell-signaling events believed important in growth regulation. In isolated adult cardiac ventricular myocytes labeled overnight with [3H]inositol, endothelin (ET) promoted a two- to fourfold increase in the accumulation of inositol polyphosphates in a dose-dependent manner with an half-maximal effective concentration (EC50) of approximately 5 nM. In contrast, picomolar concentrations of ET promoted an increase in both the extent and velocity of sarcomere shortening in electrically stimulated myocytes. Pretreatment of cells with pertussis toxin had no effect on the ET-stimulated phosphoinositide hydrolysis, but blocked the ET-stimulated positive inotropic effect. In addition to these early responses to ET, results obtained by Northern blot analysis demonstrate that exposure of isolated cardiac myocytes to 100 nM ET promoted the expression of c-fos and c-zif in both mammalian species. These data demonstrate that ET stimulates multiple cell-signaling pathways in adult mammalian cardiac myocytes. A paracrine mechanism of regulation of adult myocardium is suggested.

Agonist-stimulated inositol polyphosphate formation in cerebellum.

The accumulation of inositol polyphosphates in the cerebellum in response to agonists has not been demonstrated. Guinea pig cerebellar slices prelabeled with [3H]inositol showed the following increases in response to 1 mM serotonin: At 15 s, there was a peak in 3H label in the second messenger inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], decreasing to a lower level in about 1 min. The level of 3H label in the putative second-messenger inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] increased rapidly up to 60 s and increased slowly thereafter. The accumulation of 3H label in various inositol phosphate isomers at 10 min, when steady state was obtained, showed the following increases due to serotonin: inositol 1,3,4-trisphosphate [Ins(1,3,4)P3], eight-fold; Ins(1,3,4,5)P4, 6.4-fold; Ins(1,4,5)P3, 75%; inositol 1,4-bisphosphate [Ins(1,4)P2], 0%; inositol 3,4-bisphosphate, 100%; inositol 1-phosphate/inositol 3-phosphate, 30%; and inositol 4-phosphate, 40%. [3H]Inositol 1,3-bisphosphate was not detected in controls, but it accounted for 7.2% of the total inositol bisphosphates formed in the serotonin-stimulated samples. The fact that serotonin did not increase the formation of Ins(1,4)P2 could be due to the fact that Ins(1,4)P2 is rapidly degraded or that Ins(1,4,5)P3 is metabolized primarily by Ins(1,4,5)P3-3'kinase to form Ins(1,3,4,5)P4. In the presence of pargyline (10 microM), [3H]Ins(1,3,4,5)P4 and [3H]Ins(1,3,4)P3 levels were increased, even at 1 microM serotonin. Ketanserin (7 microM) completely inhibited the serotonin effect, indicating stimulation of serotonin2 receptors. Quisqualic acid (100 microM) also increased the levels of [3H]Ins(1,4,5)P3, [3H]Ins(1,3,4,5)P4, and [3H]Ins(1,3,4)P3, but the profile of these increases was different.(ABSTRACT TRUNCATED AT 250 WORDS)

Depolarization and Agonist‐Stimulated Changes in Inositol 1,4,5‐Trisphosphate and Inositol 1,3,4,5‐Tetrakisphosphate Mass Accumulation in Rat Cerebral Cortex

Muscarinic receptor stimulation or depolarization with elevated extracellular K+ induced rapid and sustained increases in mass accumulations of myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and myo-inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] in cerebral cortex slices. Synergistic but transient responses of both inositol polyphosphate second messengers were observed when slices were stimulated with carbachol under depolarizing conditions; this synergy was observed as an increase in the maximal responsiveness, with no significant change in EC50 values for carbachol. Omission of buffer Ca2+ ([Ca2+]e 10-20 microM) reduced basal Ins(1,4,5)P3 and Ins(1,3,4,5)P4 concentrations; the relative stimulatory effects of muscarinic receptor stimulation were maintained, but the effects of depolarization were markedly attenuated under these conditions. A component of the response to depolarization appeared to be indirectly mediated by the release of acetylcholine, because the K(+)-evoked increase in Ins(1,3,4,5)P4 was enhanced by the cholinesterase inhibitor physostigmine, and was partially attenuated by atropine. An additive suppression by nitrendipine suggests that entry of Ca2+ through L-type Ca2+ channels may serve to accelerate phosphorylation of Ins(1,4,5)P3 by 3-kinase. Norepinephrine did not significantly increase Ins(1,4,5)P3 or Ins(1,3,4,5)P4 accumulation; however, in the presence of depolarizing K+, norepinephrine caused a dramatic increase in Ins(1,3,4,5)P4 mass accumulation. In contrast, the excitatory amino acid quisqualate caused significant increases in the mass accumulations of both inositol polyphosphates measured, with no further increase being observed under depolarizing conditions. The results are discussed with respect to the interactive effects of agonist and depolarization stimuli on inositol polyphosphate accumulation which might more accurately reflect the conditions pertaining in vivo.

Stimulatory and Inhibitory Effects of N‐Methyl‐D‐Aspartate on 3H‐Inositol Polyphosphate Accumulation in Rat Cortical Slices

The actions of the excitatory amino acid N-methyl-D-aspartate (NMDA) on the accumulation of 3H-inositol polyphosphate isomers in rat cerebral cortex slices have been examined over short (less than 5 min) incubation periods. NMDA caused the dose-dependent accumulation of only [3H]inositol monophosphate and [3H]inositol bisphosphate (maximal effect between 0.3 and 1 mM), with no increase in [3H]inositol trisphosphate ([3H]InsP3) and [3H]inositol tetrakisphosphate ([3H]InsP4). HPLC analysis confirmed this, showing no increases in the breakdown products of [3H]Ins(1,3,4,5)P4. When present with the muscarinic agonist carbachol (1 mM), high concentrations of NMDA (1 mM) could almost totally inhibit carbachol-induced accumulation of 3H-inositol polyphosphates. In contrast, at lower concentrations of NMDA (10 microM), the inhibitory effect was replaced with a synergistic accumulation of inositol polyphosphates, especially [3H]InsP4 and [3H]InsP3. The inhibitory effects of NMDA were only apparent when extracellular Ca2+ was present, although incubation in media with no added Ca2+ resulted in somewhat reduced stimulatory responses to NMDA alone, but suppressed totally the inhibitory effects of 1 mM NMDA and reduced the synergistic effects of 10 microM NMDA on carbachol responses. These studies, therefore, reveal Ca(2+)-dependent effects of NMDA indicative of indirect mechanisms of action and show that care must be made in interpreting the effects of NMDA on phosphoinositide metabolism unless the inositol polyphosphate composition has been fully characterised.

Ca 2+ channel agonists enhance thyrotropin-releasing hormone-induced inositol phosphates and prolactin secretion

The dihydropyridine Ca 2+ channel activator BAY K 8644 (1 μM) stimulated basal prolactin secretion from perifused primary cultures of anterior pituitary cells and potentiated the stimulation of prolactin secretion by 1 μM thyrotropin-releasing hormone (TRH) 5-fold over 30 min. This potentiation was mimicked by other dihydropyridine agonists CGP 28392 and (+)-SDZ 202–791 and by (−)-BAY K. 8644 (1 μM), but not by (+)-BAY K 8644. The Ca 2+ channel antagonist nimodipine, at a concentration sufficient to block BAY K 8644-stimulated 45Ca 2+ uptake in GH 4C 1 anterior pituitary tumor cells, decreased basal prolactin secretion and blocked the enhancement of basal and TRH-stimulated secretion by BAY K 8644. These results suggest that dihydropyridine agonists potentiate TRH-induced secretion through interaction with known stereospecific sites on Ca 2+ channels. In gh 4c 1 cells, BAY K 8644 alone did not affect inositol polyphosphate accumulation, but potentiated TRH-stimulated accumulation of inositol 1,3,4-trisphosphate and inositol 1,3,4,5-tetrakisphosphate. Accumulation of the Ca 2+ -mobilizing isomer inositol 1,4,5-trisphosphate was not potentiated, suggesting that potentiation of TRH-stimulated hormone secretion by BAY K 8644 does not result from synergistic stimulation of phospholipase C, but may correlate with enhanced inositol trisphosphate-3-kinase activity.

Pertussis toxin produces differential inhibitory effects on basal, P2-purinergic, and chemotactic peptide-stimulated inositol phospholipid breakdown in HL-60 cells and HL-60 cell membranes.

P2-purinergic receptor agonists (UTP) and formylated peptide receptor agonists (FMLP) were found to be equally efficacious in eliciting rapid 6-7-fold increases in inositol polyphosphate accumulation in differentiated HL-60 granulocytes. The activation of this response by either agonist was substantially but incompletely inhibited in cells treated with pertussis toxin. Thus, in cells containing only 1-10% of the control level of non-ADP-ribosylated Gi-2/3, UTP induced rapid 2-fold increases in inositol polyphosphate accumulation whereas smaller 50% increases were observed in FMLP-stimulated cells. Washed membranes prepared from control and toxin-treated HL-60 cells were used to characterize this toxin-insensitive activation of phospholipase C further. The agonist-independent stimulation of phospholipase C by either millimolar Ca2+ or the nonhydrolyzable GTP analog guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) was only modestly attenuated by toxin treatment. There was a 70-80% decrease in the rate and extent of phospholipase C activity stimulated by GTP per se in the absence of receptor agonists. The rate and extent of FMLP-induced potentiation of GTP-dependent phospholipase C activity were also inhibited by greater than 80% in toxin-treated membranes. Conversely, the potency and efficacy characterizing UTP-induced potentiation of GTP-dependent phospholipase C activity were only modestly attenuated (less than 20% inhibition). The results indicate that P2-purinergic receptors (and perhaps other Ca2(+)-mobilizing receptors) activate both pertussis toxin-sensitive and toxin-insensitive pathways for phospholipase C regulation in phagocytic leukocytes.

P 2-Purinergic receptors activate a guanine nucleotide-dependent phospholipase C in membranes from HL-60 cells

We have previously determined that human neutrophils and monocytes, as well as neutrophil/monocyte progenitor cells, express a subtype of P 2-purinergic receptors (for ATP) which activate the inositol phospholipid signalling system. In the present study, membranes prepared from HL-60 promyelocytic leukemia cells were used to examine the mechanism by which these ATP receptors activate phosphatidylinositol-specific phospholipase C (PI-PLC) under defined in vitro conditions. Micromolar concentrations of the receptor agonists ATP, UTP, and ATPγS stimulated the GTP-dependent formation of inositol bisphosphate (IP 2) and inositol trisphosphate (IP 3) in washed membranes prepared from undifferentiated HL-60 cells prelabeled with [ 3H]inositol. The stimulatory effects of these nucleotides on PI-PLC appeared to be mediated through a GTP binding protein since minimal inositol polyphosphate accumulation was observed in the absence of guanine nucleotides. The increased inositol polyphosphate formation triggered by these nucleotide receptor agonists did not result from inhibition of GTP breakdown. Neither was it a consequence of increased [ 3H]polyphosphatidylinositol levels resulting from enhanced activity of membrane-associated PI- or PIP-kinases. Instead, the stimulated phospholipase activity was apparently receptor-mediated. The rank order of potency observed in these in vitro membrane assays (ATP = UTP > ATP γS ⪢ TTP > CTP ⪢ β, γ-CH-ATP) was similar to that observed with intact HL-60 cells. This order of potency appears to distinguish the P 2-purinergic receptors expressed by human phagocytic leukocytes from the P 2y-purinergic receptors which activate PI-PLC in turkey erythrocyte membranes.