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T J Hallam

Publications and source records attributed to T J Hallam.

At least 19 recordsLinked to original sources
PubMed1997

Inhibition of bovine nasal cartilage degradation by selective matrix metalloproteinase inhibitors.

N-terminal analysis of aggrecan fragments lost from bovine nasal cartilage cultured in the presence of recombinant human interleukin 1alpha revealed a predominant ARGSVIL sequence with an additional ADLEX sequence. Production of the ARGSVIL-containing fragments has been attributed to the action of a putative proteinase, aggrecanase. The minor sequence (ADLEX) corresponds to a new reported cleavage product; comparison of this sequence with the available partial sequence of bovine aggrecan indicates that this is the product of a cleavage occurring towards the C-terminus of the protein. Matrix metalloproteinase (MMP) inhibitors inhibited aggrecan loss from bovine nasal explants incubated in the presence of recombinant human interleukin 1alpha. A strong correlation between inhibition of aggrecan metabolism and inhibition of stromelysin 1 (MMP 3) (r=0.93) suggests a role for stromelysin or a stromelysin-like enzyme in cartilage aggrecan metabolism. However, the compounds were approx. 1/1000 as potent in inhibiting aggrecan loss from the cartilage explants as they were in inhibiting stromelysin. There was little or no correlation between inhibition of aggrecan metabolism and inhibition of gelatinase B (MMP 9) or inhibition of collagenase 1 (MMP 1). Studies with collagenase inhibitors with a range of potencies showed a correlation between inhibition of collagenase activity and inhibition of collagen degradation in the cartilage explant assay. This indicates that in interleukin 1alpha-driven bovine nasal cartilage destruction, stromelysin (or a closely related enzyme) is involved in aggrecan metabolism, whereas collagenase is principally responsible for collagen degradation.

Aggrecans↗
PubMed1995

Intracellular signaling events during positive and negative selection of CD4+CD8+ thymocytes in vitro.

We have used in vitro models of thymocyte positive and negative selection in conjunction with selective inhibitors of the TCR-mediated signaling cascade to investigate the intracellular signaling events that mediate these processes. We report that Ro 31.8425, a potent and selective inhibitor of protein kinase C, which blocks the activation of mature T cells in a dose-dependent fashion, has no effect on either positive or negative selection of CD4+8+ thymocytes. In contrast, cyclosporin A fails to prevent negative selection, but inhibits positive selection through a direct effect on developing thymocytes, rather than through the perturbation of stromal cell support. Thus, our data suggest that positive and negative selection may operate via distinct intracellular signaling pathways.

Animals↗
PubMed1994

Regulation of T-cell-receptor-stimulated bivalent-cation entry in Jurkat E6 cells: role of protein kinase C.

Stimulation of Jurkat E6 cells with anti-CD3 antibody results in a characteristic rise in [Ca2+]i which is due to both the release of Ca2+ from intracellular stores and the entry of external Ca2+. Individual components of the [Ca2+]i increase were investigated by measuring intracellular Ca2+ release in the absence of external Ca2+ and determining influx of bivalent cations by following the entry of Mn2+. The increase in [Ca2+]i induced by anti-CD3 antibody in the presence or absence of extracellular Ca2+ could be inhibited by the non-selective kinase inhibitor staurosporine, which also inhibits anti-CD3-stimulated phospholipase C activity. Staurosporine also inhibits the influx of bivalent cations induced by anti-CD3 antibody, but not that induced by depletion of intracellular Ca2+ stores using thapsigargin. The effect of staurosporine was compared with that of Ro 31-8425, a potent and selective inhibitor of protein kinase C (PKC). Ro 31-8425, at concentrations up to 10 microM, has no inhibitory effect on the anti-CD3 antibody-induced [Ca2+]i increase or phospholipase C activity. These studies are consistent with the concept that augmentation of [Ca2+]i by stimulated T-cell receptors requires activation of a kinase, probably a tyrosine kinase such as p56lck, ZAP-70 or p59fyn, and is independent of PKC. Phorbol esters inhibit the anti-CD3-stimulated [Ca2+]i increase and phospholipase C activity, showing that this can be negatively regulated by PKC. A small potentiation of the anti-CD3 antibody-induced [Ca2+]i rise in the presence of extracellular Ca2+ was detected in the presence of Ro 31-8425; this suggests that T-cell-receptor ligation can also limit the increase in [Ca2+]i via PKC activation.

Alkaloids↗
PubMed1994

Effect of a selective protein kinase C inhibitor, Ro 31-8425, on Mac-1 expression and adhesion of human neutrophils.

The role of protein kinase C (PKC) in mediating up-regulation of macrophage 1 adhesion protein (Mac-1) and adhesion of neutrophils in response to physiological agonists is not clear. Previous studies have relied on use of phorbol esters to activate PKC directly or on results obtained with non-selective inhibitors of protein kinases. 3-[8-(Aminomethyl)-6,7,8,9-tetrahydropyridol[1,2-a]-indol-10-yl]-4 -(1- methyl-3-indolyl)-1H-pyrrole-2,5-dione hydrochloride (Ro 31-8425) is a potent and highly selective inhibitor of PKC (Bit et al. J. Med. Chem. 1993. 36: 21). In these studies Ro 31-8425 has been used to define, more definitively, the role of PKC in mediating complement fragment C5a (C5a)-stimulated up-regulation of Mac-1 and adhesion of neutrophils to endothelial cells and to bovine serum albumin (BSA)-coated plastic. Phorbol 12, 13 dibutyrate (PBu2) increased surface expression of Mac-1 and stimulated adhesion of neutrophils to endothelial cells and to BSA-coated plastic. This confirms previous reports that activation of PKC can stimulate these responses. The PKC inhibitor, Ro 31-8425, inhibited the PBu2-stimulated responses, which confirms that Ro 31-8425 was effective in inhibiting PKC in these neutrophils. A more physiological agonist, C5a, also increased surface expression of Mac-1 and adhesion of neutrophils to endothelial cells and BSA-coated plastic. However, the responses to C5a were unaffected by Ro 31-8425. These results suggest that, although activation of PKC can promote up-regulation of Mac-1 and adhesion of neutrophils, this does not appear to be the physiological pathway. A non-selective protein kinase inhibitor, staurosporine, inhibited both PBu2 and C5a-stimulated adhesion. This suggests that a protein kinase other than PKC, possibly a tyrosine protein kinase, is likely to be involved in mediating C5a-stimulated Mac-1 up-regulation and adhesion. These results emphasise the need for caution in interpreting experiments and assuming a role for PKC. Use of a potent and selective inhibitor of PKC, Ro 31-8425, provides more definitive information.

Alkaloids↗
PubMed1994

Protein kinase C: is its pivotal role in cellular activation over-stated?

Evidence has emerged over the past decade to suggest that protein kinase C (PKC) is a widespread family of kinases responsible for many diverse and critical cellular functions. With the development of selective agents to activate or inhibit the individual PKC isoenzymes, it is now apparent that much of the literature that implicated PKC in many cellular functions needs to be appraised. In this article, Sandra Wilkinson and Trevor Hallam discuss the problems of the existing methods and the recent evidence that suggests that PKC isotypes are necessary for some, but not all, of those cellular responses where PKC had been thought to play an important role. Selective inhibitors of PKC isoenzymes may have potential for therapeutic use in auto-immune diseases, transplant rejection and oncology.

Animals↗
PubMed1994

Changes in protein kinase C subspecies protein expression and activity in a series of multidrug-resistant human KB carcinoma cell lines.

We have investigated the relationship between protein kinase C (PKC), levels of resistance and drug used for selection in a series of human KB carcinoma cell lines by comparing protein kinase C activity and PKC alpha, beta I, beta II, gamma, delta, epsilon, and zeta subspecies protein expression. PKC alpha protein expression was increased by 600% and 375% in KB-A1 and KB-C1 lines respectively over the parent KB-3-1 line; only KB-A1 cells showed increased PKC delta expression. Expression of other PKC subspecies was equal to that of KB-3-1 cells. There was considerable variation between the different KB cell lines in total cytosolic PKC activity, the KB-A1 and KB-C1 lines showing 400% and 350% increases respectively, KB-V1 and KB-8-5-11 about 180%, and KB-8-5 no increase relative to the parent KB-3-1 line. For calcium-independent PKC activity, the KB-C1 and KB-A1 lines only were increased over the KB-3-1 line. Immunoprecipitation with antisera to PKC subspecies confirmed that the increase in KB-A1 cytosolic total PKC activity was due largely to PKC alpha and partially to PKC delta. Membrane-associated PKC activity was increased by 500% and 350% in KB-A1 and KB-C1 lines respectively, by 250% and 270% in KB-V1 and KB-8-5-11, and not increased in KB-8-5 cells relative to the KB-3-1 cells. For KB-C1, KB-8-5-11, and KB-8-5 lines, which show decreasing resistance to colchicine, our results suggest a correlation between PKC and multidrug resistance in cells selected for resistance to this drug. There is no correlation between PKC and multidrug resistance for cells selected in different drugs. Our study therefore suggests that specific PKC subspecies are associated with the MDR phenotype of some KB cell lines, but that the extent of PKC involvement depends on the type of drug used for selection and its concentration.

Amino Acid Sequence↗
PubMed1994

Ro 32-0432, a selective and orally active inhibitor of protein kinase C prevents T-cell activation.

Several lines of circumstantial evidence support the assumption that protein kinase C (PKC) activation together with elevated levels of cytosolic Ca++ are necessary for T-cell activation and proliferation in response to a physiological stimulus, i.e., MHC class II restricted antigen presentation. By using a potent, cell-permeable and selective inhibitor of PKC, Ro 32-0432, we have tested this hypothesis. Ro 32-0432 inhibits interleukin-2 (IL-2) secretion, IL-2 receptor expression in, and proliferation of, peripheral human T-cells stimulated with phorbol ester together with phytohemagglutin or anti-CD3, but does not inhibit IL-2 induced proliferation in cells already stimulated to express IL-2 receptors. Proliferation of the influenza peptide antigen HA 307-319-specific human T-cell clone (HA27) after exposure to antigen-pulsed autologous presenting cells was also inhibited by Ro 32-0432. Oral administration of Ro 32-0432 inhibited subsequent phorbol ester-induced edema in rats demonstrating the systemic efficacy of the compound to inhibit PKC-driven responses. Induction of more physiologically T-cell driven responses such as host vs. graft responses and the secondary paw swelling in adjuvant-induced arthritis were also inhibited by Ro 32-0432. These data demonstrate the crucial role for PKC in T-cell activation and that selective p.o. bioavailable PKC inhibitors are efficacious in preventing T-cell driven chronic inflammatory responses in vivo. Inhibition of PKC represents an important mechanistic approach to prevent T-cell activation and compounds of this class may have important therapeutic applicability to chronic inflammatory and autoimmune diseases.

Administration, Oral↗
PubMed1991

The involvement of calcium and protein kinase C in modulating agonist-stimulated chemotaxis of human neutrophils.

Chemotaxis of human neutrophils in response to a gradient of the chemotactic peptide, fmet-leu-phe (FMLP), was measured by the under-agarose technique. The dose-response curve for FMLP was biphasic; low concentrations were stimulatory, and the response was reduced at higher concentrations. The response to FMLP was partially inhibited (about 50%) in the absence of extracellular Ca2 (EGTA added). NiCl2 dose-dependently inhibited FMLP-stimulated chemotaxis in the presence of extracellular Ca2+; the maximum inhibition obtainable with NiCl2 was similar to that with the absence of extracellular Ca2+. These results suggest that FMLP-stimulated chemotaxis is, at least partially, dependent on stimulation of Ca2+ influx. The phorbol ester, PMA, dose-dependently inhibited chemotaxis; the response was almost completely inhibited by 10 nM PMA. This result indicates that activation of protein kinase C inhibits chemotaxis. These results are discussed in relation to the physiological responses of neutrophils.

Calcium↗
PubMed1991

Octimibate, a potent non-prostanoid inhibitor of platelet aggregation, acts via the prostacyclin receptor.

1. Octimibate, 8-[(1,4,5-triphenyl-1H-imidazol-2-yl)oxy]octanoic acid, is reported to have antithrombotic properties. This is in addition to its antihyperlipidaemic effects which are due to inhibition of acylCoA:cholesterol acyltransferase (ACAT). The aim of this study was to investigate the mechanism of the antithrombotic effect of octimibate, and to determine whether the effects of octimibate are mediated through prostacyclin receptors. 2. In suspensions of washed (plasma-free) human platelets, octimibate is a potent inhibitor of aggregation; its IC50 is approx. 10 nM for inhibition of aggregation stimulated by several different agonists, including U46619 and ADP. The inhibitory effects of octimibate on aggregation are not competitive with the stimulatory agonist; the maximal response is suppressed but there is no obvious shift in potency of the agonist. In platelet-rich plasma, octimibate inhibits agonist-stimulated aggregation with an IC50 of approx. 200 nM. 3. Octimibate also inhibits agonist-stimulated rises in the cytosolic free calcium concentration, [Ca2+]i, in platelets. Both Ca2+ influx and release from intracellular stores are inhibited. The effects of octimibate on aggregation and [Ca2+]i are typical of agents that act via elevation of adenosine 3':5'-cyclic monophosphate (cyclic AMP). Similar effects are seen with forskolin, prostacyclin (PGl2) and iloprost (a stable PGl2 mimetic). 4. Octimibate increases cyclic AMP concentrations in platelets and increases the cyclic AMP-dependent protein kinase activity ratio. Octimibate stimulates adenylyl cyclase activity in human platelet membranes, with an EC50 of 200 nM. The maximal achievable activation of adenylyl cyclase by octimibate is 60% of that obtainable with iloprost. Octimibate has no effect on the cyclic GMP-inhibited phosphodiesterase (phosphodiesterase-ITI), which is the major cyclic AMP-degrading enzyme in human platelets.5. Octimibate inhibits, apparently competitively, the binding of [3H]-iloprost (a stable PGl2 mimetic) to platelet membranes; the estimated Ki is 150 nm. 6. The platelets of different species show considerable differences in the apparent potency of their inhibition of aggregation by octimibate; platelets from cynomolgus monkeys are 3 fold more sensitive than those from humans, while rat, cat and cow platelets are 50, 100, and 250 fold less sensitive than human platelets. The sensitivity of these different species to iloprost, however, varies over a range of only 10 fold with no obvious difference between primates and non-primates. 7. Octimibate appears to be a potent agonist (aggregation), or partial agonist (adenylyl cyclase), at prostacyclin receptors and is the first non-prostanoid agent of this type to be identified. The species differences in relative potency of octimibate and iloprost may reflect the existence of receptor subtypes.

Adenylyl Cyclases↗
PubMed1991

Primate vascular responses to octimibate, a non-prostanoid agonist at the prostacyclin receptor.

1. Octimibate is a potent inhibitor of human platelet aggregation, and appears to act (at least in part) through the prostacyclin receptor, as described in the preceding paper. Here, the vascular effects, both in vitro and in vivo, of octimibate have been compared to those of the stable prostacyclin (PGI2) mimetic, iloprost. Since octimibate shows extensive species variation and is potent at inhibiting platelet aggregation in primates, all of the experiments reported here have been carried out with primate tissue or in vivo in cynomolgus monkeys. 2. Activation of adenylyl cyclase in human lung membranes appears to involve stimulation of the vascular PGI2 receptor. Octimibate, as well as iloprost, stimulates adenylyl cyclase in this preparation. The EC50 values for iloprost and octimibate are 50 nM and 340 nM respectively. These values are similar to those seen with human platelet membranes. As with platelets, the maximal activation achievable with octimibate is 60% of that seen with iloprost. This result suggests that octimibate is a partial agonist for stimulation of adenylyl cyclase. 3. Iloprost (10-100 nM) relaxes human coronary and mesenteric artery precontracted with KCl, and also relaxes cynomolgus monkey aorta precontracted with phenylephrine. Octimibate appears to be a partial agonist for relaxation of human coronary artery precontracted with KCl; the intrinsic activity of octimibate (10 microM) is 0.15 compared to iloprost, and octimibate surmountably antagonizes the relaxant effects of iloprost with a Kp of 200 nM. Octimibate (up to 10 microM) evokes only weak relaxation of human mesenteric artery (precontracted with KCl) and cynomolgus monkey aorta (precontracted with phenylephrine). 4. The effects of iloprost and octimibate were compared in vivo in cynomolgus monkeys. In addition to inhibiting ex vivo platelet aggregation, both compounds cause hypotension with little effect on heart rate. The dose-response curves for inhibition of ex vivo platelet aggregation and a fall in mean arterial blood pressure were compared. The dose-separation (i.e., the relative differences in effective concentrations) for the two responses is similar with both iloprost and octimibate. 5. Since the pern; beral resistance vessels are intimately involved in regulation of systemic arterial blood pressure, the effects of both agents were tested on human peripheral resistance vessels (150-400pm diameter) in vitro. These vessels are relaxed by both iloprost and octimibate following precontraction with KCI. The IC50 value for iloprost is 44nM, and 1.7 microM octimibate evokes 50% of the maximal relaxation obtained with iloprost. Thus, the relative potencies of the two compounds in relaxing human subcutaneous resistance vessels are similar to their relative potencies in inhibiting platelet responses. This result correlates with the lack of platelet versus vascular selectivity seen with the in vivo monkey studies. 6. These results suggest that octimibate, a partial agonist at the prostacyclin receptor, is unable to discriminate between platelet and vascular prostacyclin receptors in primates.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1990

SK&F 96365, a novel inhibitor of receptor-mediated calcium entry.

A novel inhibitor of receptor-mediated calcium entry (RMCE) is described. SK&F 96365 (1-(beta-[3-(4-methoxy-phenyl)propoxy]-4-methoxyphenethyl)-1H- imidazole hydrochloride) is structurally distinct from the known 'calcium antagonists' and shows selectivity in blocking RMCE compared with receptor-mediated internal Ca2+ release. Human platelets, neutrophils and endothelial cells were loaded with the fluorescent Ca2(+)-indicator dyes quin2 or fura-2, in order to measure Ca2+ or Mn2+ entry through RMCE as well as Ca2+ release from internal stores. The IC50 (concn. producing 50% inhibition) for inhibition of RMCE by SK&F 96365 in platelets stimulated with ADP or thrombin was 8.5 microM or 11.7 microM respectively; these concentrations of SK&F 96365 did not affect internal Ca2+ release. Similar effects of SK&F 96365 were observed in suspensions of neutrophils and in single endothelial cells. SK&F 96365 also inhibited agonist-stimulated Mn2+ entry in platelets and neutrophils. The effects of SK&F 96365 were independent of cell type and of agonist, as would be expected for a compound that modulates post-receptor events. Voltage-gated Ca2+ entry in fura-2-loaded GH3 (pituitary) cells and rabbit ear-artery smooth-muscle cells held under voltage-clamp was also inhibited by SK&F 96365; however, the ATP-gated Ca2(+)-permeable channel of rabbit ear-artery smooth-muscle cells was unaffected by SK&F 96365. Thus SK&F 96365 (unlike the 'organic Ca2+ antagonists') shows no selectivity between voltage-gated Ca2+ entry and RMCE, although the lack of effect on ATP-gated channels indicates that it discriminates between different types of RMCE. The effects of SK&F 96365 on functional responses of cells thought to be dependent on Ca2+ entry via RMCE were also studied. Under conditions where platelet aggregation is dependent on stimulated Ca2+ entry via RMCE, the response was blocked by SK&F 96365 with an IC50 of 15.9 microM, which is similar to the IC50 of 8-12 microM observed for inhibition of RMCE. Adhesion and chemotaxis of neutrophils were also inhibited by SK&F 96365. SK&F 96365 is a useful tool to distinguish RMCE from internal Ca2+ release, and to probe the role of RMCE in mediating functional responses of cells. However, SK&F 96365 is not as potent (IC50 around 10 microM) or selective (also inhibits voltage-gated Ca2+ entry) as would be desirable, so caution must be exercised when using this compound.

Adenosine Diphosphate↗
PubMed1990

A role for calcium and protein kinase C in agonist-stimulated adhesion of human neutrophils.

Stimulated adherence of human neutrophils to plastic and changes in cytosolic free Ca2+ concn. [( Ca2+]i) were measured in the same cell preparations. [Ca2+]i-activation curves were constructed to compare the relation between [Ca2+]i and adhesion in response to ionomycin and formylmethionyl-leucyl-phenylalanine (FMLP). This showed that FMLP-stimulated adhesion required less increase in [Ca2+]i than did ionomycin's effect, a result suggesting that an additional stimulatory component might be involved in the response to FMLP. Protein kinase C activation was a possibility, and activation of protein kinase C with a phorbol ester (PMA) was found to stimulate adhesion with no change in [Ca2+]i. A low concentration of PMA was found to synergize with ionomycin to stimulate a greater adhesion response than with each alone, and the [Ca2+]i-activation curve for ionomycin in the presence of PMA was shifted towards that for FMLP. Thus, synergy between [Ca2+]i and protein kinase C (each of which is sufficient alone) probably explains the stimulatory effects of FMLP on adhesion of neutrophils.

Benzofurans↗
PubMed1990

The effects of siguazodan, a selective phosphodiesterase inhibitor, on human platelet function.

1. The effects of siguazodan (SK&F 94836) a selective phosphodiesterase (PDE) inhibitor with inotropic and vasodilator activity, were studied on human platelets. 2. Siguazodan selectively inhibited the major cyclic AMP-hydrolysing PDE in human platelet supernatants. The inhibited enzyme has been variously termed cyclic GMP-inhibited PDE or PDE-III. 3. In platelet-rich plasma (PRP), siguazodan inhibited U46619-induced aggregation more potently than that induced by ADP and collagen. Treatment of the PRP with aspirin had no effect on the potency of siguazodan. 4. In washed platelets, siguazodan increased cyclic AMP levels and reduced cytoplasmic free calcium [( Ca2+]i). ADP decreased the ability of siguazodan to raise cyclic AMP and this may explain its lower potency in inhibiting responses to ADP. 5. Siguazodan has anti-platelet actions over the same concentration range that it is an inotrope and vasodilator.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1989

Activation of protein kinase C in human neutrophils attenuates agonist-stimulated rises in cytosolic free Ca2+ concentration by inhibiting bivalent-cation influx and intracellular Ca2+ release in addition to stimulating Ca2+ efflux.

Stimulation of fura-2-loaded human neutrophils with formylmethionyl-leucyl-phenylalanine (FMLP) or ionomycin elevated the cytosolic free Ca2+ concentration, [Ca2+], to a maintained elevated level. Activation of protein kinase C (C-kinase) with phorbol 12-myristate 13-acetate, 4 beta-phorbol 12,13-didecanoate or dioctanoylglycerol caused decreases in [Ca2+]i from this level. 4 alpha-Phorbol didecanoate, which does not activate C-kinase, had no effect. These results confirm previous reports that C-kinase activation decreases neutrophil [Ca2+]i by stimulating removal of Ca2+ from the cytosol. Further experiments showed that activation of C-kinase attenuated the component of the FMLP-stimulated [Ca2+]i rise that was dependent on external Ca2+. C-kinase activation also inhibited FMLP-stimulated entry of the quenching cation, Mn2+, used as an indicator of bivalent-cation entry. In contrast, C-kinase activation caused only a partial inhibition of FMLP-stimulated release of Ca2+ from intracellular stores. 4 alpha-Phorbol didecanoate was ineffective in inhibiting Ca2+ entry, Mn2+ entry and intracellular Ca2+ release. Addition of FMLP also stimulated a decrease in the ionomycin-elevated [Ca2+]i, and this effect was blocked by staurosporine, a protein kinase inhibitor. These results show that, in addition to stimulating Ca2+ efflux, C-kinase activation in neutrophils inhibits FMLP-stimulated entry of bivalent cations, and partially inhibits intracellular release of Ca2+. Further, FMLP itself can modulate [Ca2+]i by activation of C-kinase.

Benzofurans↗
PubMed1989

Protein kinase C activation alters the sensitivity of agonist-stimulated endothelial-cell prostacyclin production to intracellular Ca2+.

Agonist-stimulated release of prostacyclin (PGI2) from endothelial cells requires elevation of the concentration of intracellular ionized calcium ([Ca2+]i) above a threshold value, and raised [Ca2+]i provides a sufficient transduction signal to account for the extent of PGI2 production. However, chronic activation of protein kinase C has been reported separately to potentiate PGI2 release, but to depress agonist-induced elevations of [Ca2+]i. We show here that pretreatment with phorbol 12-myristate 13-acetate (PMA) dose-dependently induces PGI2 release over many minutes after a significant lag period without any change in [Ca2+]i. In addition, PMA potentiates the transient release of PGI2 in response to agonists in a complex manner depending on the time of pre-incubation and the concentrations of both PMA and agonist. Concomitant measurement of [Ca2+]i and PGI2 release demonstrates that PMA pretreatment dose-dependently inhibits both the peak [Ca2+]i transient and the subsequent steady-state elevation of [Ca2+]i in response to agonists. Determination of the quantitative [Ca2+]i/PGI2 dose/response relationship, when PGI2 release is driven purely by elevating [Ca2+]i with ionomycin, demonstrates that PMA also enhances the Ca2+-sensitivity of PGI2 release. The observed effects of PMA on PGI2 release can be explained quantitatively by its abilities to lower the threshold [Ca2+]i required for PGI2 synthesis and to depress the peak [Ca2+]i evoked by agonist. We propose that these effects are due respectively to actions of PMA on phospholipase A2 and on a G-protein (Gp) that couples activated receptors to phospholipase C.

Calcium↗