Differential modulation of PI3-kinase/Akt pathway during all-trans retinoic acid- and Am80-induced HL-60 cell differentiation revealed by DNA microarray analysis

All-trans retinoic acid (ATRA) and Am80 are natural and synthetic derivatives of Vitamin A and have been used in the fields of oncology and dermatology for years. Their action was considered to be achieved mainly through binding to nuclear hormone receptors, retinoic acid receptors (RARs), although they have been observed to have different biological effects. For example, the two compounds have similar effects on differentiation but different effects on proliferation in human promyelocytic leukemia cell line HL-60 cells. To elucidate the genes responsible for this and other differences, we attempted for the first time to determine the genes whose expressions were differentially modulated during the time course of HL-60 cell differentiation by ATRA and Am80 treatment up to 72h utilizing DNA microarray and clustering analyses. As a result, the expressions of 204 genes were found to be modulated differentially by ATRA and Am80. Among them, we focused on two components of the PI3-kinase/Akt signal transduction pathway, phosphoinositide-3-kinase, beta-catalytic subunit and ribosomal protein S6 kinase polypeptide 1, which are related to the regulation of cell proliferation and apoptosis. Their expressions were specifically suppressed by ATRA, which coincided with the suppressive effects of ATRA on the HL-60 cell proliferation. Moreover, PI3-kinase inhibitors suppressed the proliferation of Am80-treated cells to the same extent as ATRA did. These results indicated that these gene products play a role in HL-60 cell growth suppression during the late stage of differentiation. The complete data and a list of the genes are available at .     

Andrographolide inhibits growth of acute promyelocytic leukaemia cells by inducing retinoic acid receptor‐independent cell differentiation and apoptosis

Objectives The growth inhibiting potential of andrographolide was evaluated in three acute promyelocytic leukaemia cell line models (HL‐60, NB4 and all‐trans retinoic acid (ATRA)‐resistant NB4‐R2). Methods In elucidating the mechanisms of growth inhibition, a special emphasis was placed on assessing the induction of differentiation and apoptosis by andrographolide in the primary acute promyelocytic leukaemia NB4 cells. Key findings The compound was 2‐ and 3‐fold more active in inhibiting the growth of HL‐60 and NB4‐R2 cells compared with NB4 cells, respectively. At IC50 (concentration at which growth of 50% of the cells (compared with medium only treated control cells) is inhibited; 4.5 μM) the compound exhibited strong cell‐differentiating activity in NB4 cells, similar to ATRA (IC50 1.5 μM). In the presence of a pure retinoic acid receptor antagonist AGN193109, the growth inhibition of NB4 cells by ATRA was reversed, whereas the activity of andrographolide was not affected. This clearly suggested that andrographolide's cell differentiating activity to induce growth inhibition of NB4 cells most likely occurred via a retinoic acid receptor‐independent pathway. At higher concentration (2 × IC50), andrographolide was an efficient inducer of apoptosis in NB4 cells. Conclusions Taken together, these results suggest andrographolide and its derivatives, apparently with a novel cell differentiating mechanism and with ability to induce apoptosis, might be beneficial in the treatment of primary and ATRA‐resistant acute promyelocytic leukaemia.     

Proteomic analysis of changes in the protein composition of MCF-7 human breast cancer cells induced by all-trans retinoic acid, 9-cis retinoic acid,and their combination

Retinoic acid (all-trans and 9-cis) isomers represent important therapeutic agents for many types of cancers, including human breast cancer. Changes in protein composition of the MCF-7 human breast cancer cells were induced by all-trans retinoic acid, 9-cis retinoic acid, and their combination and subsequently proteomic strategies based on bottom-up method were applied. Proposed approach was used for the analysis of proteins extracted from MCF-7 human breast cancer cell line utilizing a commercially manufactured kit RIPA and separated on two dimensional (2D) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) after treatment with both retinoic acid isomers. We found significant differences in occurrence of proteins probably affecting the cell migration process in tumour cells. Heat shock protein 27, ribonucleoprotein SmD3, and cofilin-1 were significantly upregulated after treatment with combination of individual retinoic acid isomers. On the other hand, AP-5 complex subunit beta-1 shows the different response. Thus, the results might help to find the answer to important medical questions on (i) the identification of signaling pathways affected by retinoic acid isomers or (ii) how the observed proteomic pattern might reflect the effectiveness of retinoic acids treatment.     

Matrix and serine protease expression during leukemic cell differentiation induced by aclacinomycin and all-trans-retinoic acid

In myeloid leukemia, immature leukemic cells are able to egress into peripheral blood to infiltrate extra-medullary organs. We therefore analyzed the migrating and invasive potential of human HL-60 and NB4 cell lines, representative of acute myelogenous leukemia, their ability to express matrix metalloproteases (MMPs), tissue inhibitors of metalloproteases (TIMPs) and urokinase plasminogen activator (uPA) in response to differentiating agents. Granulocytic differentiation by all-trans-retinoic acid (ATRA) and aclacinomycin (ACLA) strongly increased HL-60 and NB4 cell migration and invasion. At mRNA and protein levels, these cell lines produced significant amounts of MMP-9 (HL-60相似文献   

Inhibition of human recombinant T-type calcium channels by the endocannabinoid N-arachidonoyl dopamine

Background and purpose:

N-arachidonoyl dopamine (NADA) has complex effects on nociception mediated via cannabinoid CB₁ receptors and the transient receptor potential vanilloid receptor 1 (TRPV1). Anandamide, the prototypic CB₁/TRPV1 agonist, also inhibits T-type voltage-gated calcium channel currents (I_Ca). These channels are expressed by many excitable cells, including neurons involved in pain detection and processing. We sought to determine whether NADA and the prototypic arachidonoyl amino acid, N-arachidonoyl glycine (NAGly) modulate T-type I_Ca

Experimental approach:

Human recombinant T-type I_Ca (Ca_V3 channels) expressed in HEK 293 cells and native mouse T-type I_Ca were examined using standard whole-cell voltage clamp electrophysiology techniques.

Key results:

N-arachidonoyl dopamine completely inhibited Ca_V3 channels with a rank order of potency (pEC₅₀) of Ca_V3.3 (6.45) ≥ Ca_V3.1 (6.29) > Ca_V3.2 (5.95). NAGly (10 µmol·L⁻¹) inhibited Ca_V3 I_Ca by approximately 50% or less. The effects of NADA and NAGly were voltage- but not use-dependent, and both compounds produced significant hyperpolarizing shifts in Ca_V3 channel steady-state inactivation relationships. By contrast with anandamide, NADA and NAGly had modest effects on Ca_V3 channel kinetics. Both NAGly and NADA inhibited native T-type I_Ca in mouse sensory neurons.

Conclusions and implications:

N-arachidonoyl dopamine and NAGly increase the steady-state inactivation of Ca_V3 channels, reducing the number of channels available to open during depolarization. These effects occur at NADA concentrations at or below to those affecting CB₁ and TRPV1 receptors. Together with anandamide, the arachidonoyl neurotransmitter amides, NADA and NAGly, represent a new family of endogenous T-type I_Ca modulators.     

Stimulation of intracellular Ca2+ elevation in neutrophils by thiol-oxidizing phenylarsine oxide

Phenylarsine oxide (PAO), a trivalent arsenical compound, stimulated [Ca2+]i elevation in rat neutrophils in a Ca2+-containing medium but caused no appreciable response in a Ca2+-free medium. PAO also induced external Mn2+ entry, which was inhibited by N-acetyl-L-cysteine (NAC), but failed to elicit any appreciable Ba2+ and Sr2+ entry. Pretreatment of neutrophils with thiol-reducing agents including dithiothreitol (DTT), NAC, 2,3-dimercapto-1-propanol (DMP), 2,3-dimercaptopropane-1-sulfonic acid (DMPS) and tris-(2-carboxyethyl)phosphine (TCEP), all greatly inhibited PAO-induced [Ca2+]i elevation. Addition of Ni2+ or La3+ followed by PAO stimulation also attenuated the Ca2+ signals in a concentration-dependent manner. PAO had no significant effect on the production of reactive oxygen intermediates (ROI) and nitric oxide (NO) nor did it decrease cellular low molecular weight thiols levels. PAO-induced [Ca2+]i elevation was significantly inhibited by 1-[6-[17beta-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U-73122), the inhibitor of phospholipase C-coupled processes, genistein, a general tyrosine kinase inhibitor, phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activator, calyculin A, a cortical actin stabilizer, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY 294002), a phosphoinositide 3-kinase inhibitor, 1-[beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole (SKF-96365), and cis-N-(2-phenylcyclopentyl)azacyclotridec-1-en-2-amine (MDL-12,330A), the blockers of receptor-gated and store-operated Ca2+ channels, whereas there was no appreciable effect exerted by aristolochic acid, a phospholipase A2 inhibitor, 7-nitroindazole and N-(3-aminomethyl)benzylacetamidine (1400W), the blockers of NO synthase, and by suspension in a Na+-deprived medium. In contrast, 2-aminoethoxydiphenyl borane (2-APB), the blocker of IP3 receptor and Ca2+ influx, enhanced the PAO-induced response. PAO had no effect on the plasma membrane Ca2+-ATPase (PMCA) activity in the pharmacological isolated neutrophil preparation and the neutrophil membrane fractions. These results indicate that PAO stimulates [Ca2+]i rise in rat neutrophils mainly through the oxidation of vicinal thiol groups on the cell surface membrane to activation of a non-store operated Ca2+ entry (non-SOCE) without affecting the activity of PMCA and the plasmalemmal Na+/Ca2+ exchanger.     

Induction of G2/M arrest and apoptosis by sesquiterpene lactones in human melanoma cell lines

Malignant melanoma is a highly aggressive tumor which frequently resists chemotherapy, therefore, the search for new agents for its treatment is of great importance. In this study, we purified the sesquiterpene lactones (SLs), Tomentosin and Inuviscolide from Inula viscosa (Compositae) leaves and studied their anti-cancer potency against human melanoma cell lines in order to develop new agents for melanoma treatment. SLs inhibited the proliferation of three human melanoma cell lines: SK-28, 624 mel and 1363 mel in a dose-dependent manner. We further investigated SLs mechanism of action using SK-28 as a representative cell line model. SLs caused cell-cycle arrest at G(2)/M, accompanied by the appearance of a sub-G0 fraction, indicative of apoptotic cell death. Induction of apoptosis was further confirmed by changes in membrane phospholipids, changes in mitochondrial membrane potential (DeltaPsi) and by detection of Caspase-3 activity. Rapid inhibitory phosphorylation of Cdc2 (Thr14 and Tyr15) was seen early after treatment, followed by a later decrease in the expression level of both Cyclin b1 and Cdc2. Induction of p53 and p21(waf1) proteins and phosphorylation of p53 at Ser15 were also detected early after treatment. The anti-apoptotic proteins, p65 subunit of nuclear factor kappaB (NF-kappaB), and Survivin were reduced in a dose-dependent manner. Taken together, these changes partially explain the ability of the SLs to induce G(2)/M arrest and apoptosis. Induction of apoptosis by Tomentosin and Inuviscolide in human aggressive melanoma cell lines has high pharmacological value and implies that SLs might be developed as new agents for melanoma treatment.     

Potentiation of 1,25-dihydroxyvitamin D(3)-induced differentiation of human promyelocytic leukemia cells into monocytes by costunolide,a germacranolide sesquiterpene lactone

Costunolide, a germacranolide sesquiterpene lactone that exists in several medicinal plants, is known to be a possible anti-cancer and chemopreventive agent for tumorigenesis. In this report, we investigated the effect of costunolide on cellular differentiation in the human promyelocytic leukemia HL-60 cell culture system. Costunolide markedly increased the degree of HL-60 leukemia cell differentiation when simultaneously combined with 5nM 1,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)). Costunolide by itself had very weak effects on the differentiation of HL-60 cells. Cytofluorometric analysis and cell morphologic studies indicated that costunolide potentiated 1,25-(OH)(2)D(3)-induced cell differentiation predominantly into monocytes. Inhibitors for PKC, PI3-K, and ERK markedly inhibited HL-60 cell differentiation induced by costunolide in combination with 1,25-(OH)(2)D(3). In addition, pretreatment of HL-60 cells with costunolide before the 1,25-(OH)(2)D(3) addition also potentiated cell differentiation in a concentration- and time-dependent manner, and the enhanced levels of cell differentiation closely correlated with the inhibitory levels of NF-kappaB-binding activity by costunolide. These results indicate that PKC, PI3-K, ERK and NF-kappaB may be involved in 1,25-(OH)(2)D(3)-mediated cell differentiation enhanced by costunolide.     

Regulation of CYP26A1 expression by selective RAR and RXR agonists in human NB4 promyelocytic leukemia cells

All-trans retinoic acid (ATRA) can induce complete remission in acute promyelocytic leukemia (APL), but resistance to this treatment develops rapidly partly due to increased ATRA metabolism. Among the cytochrome P450s (CYPs) involved in ATRA metabolism, the ATRA-inducible cytochrome P450 26A1 (CYP26A1) is particularly active although the molecular mechanisms involved in its regulation are not well defined in the target leukemia cells. To study CYP26A1 expression and regulation in APL cells, we used the NB4 promyelocytic leukemia cell line. CYP26A1 constitutive expression was barely detectable in NB4 cells, but ATRA could induce high levels of CYP26A1 expression, which reached a maximum at 72h. To further define CYP26A1 induction mechanisms in the NB4 leukemia cells, we used RARs and RXR selective agonists. The RARalpha agonist BMS753 could elicit maturation, as expected, but not CYP26A1 expression. Treatment with the RARbeta agonist BMS641, or the RARbeta/gamma agonist BMS961, could not elicit maturation, as expected, nor induce CYP26A1 expression. Because CYP26A1 expression could not be induced by RAR ligands alone, NB4 cells were then co-treated with the RXR agonist BMS649. The RXR agonist alone could not induce CYP26A1 expression, nor in combination with either the RARbeta agonist or the RARbeta/gamma agonist. However, the combination of the RXR agonist and the RARalpha agonist could elicit a marked induction of CYP26A1 expression. In conclusion, we have shown that CYP26A1 induction is not essential for the granulocytic maturation of NB4 leukemia cells, and that CYP26A1 induction requires the activation of both RARalpha and RXR in these cells.     

Induction of apoptosis without redox catastrophe by thioredoxin-inhibitory compounds

The dithiol-reducing thioredoxin/thioredoxin reductase system normally maintains the reduced state of key enzymes responsible for the cell's anti-oxidant defences. We therefore addressed the question of whether AW 464--a novel thioredoxin inhibitor--as well as broad spectrum dithiol ligands diamide and phenylarsine oxide are able to induce and execute a regular apoptotic sequence of events without overwhelming the cell's ability to detoxify reactive oxygen species. All three agents were found to target the thioredoxin system in a cell-free assay. In HL-60 leukaemia cells, they were also found to induce Bak activation, cytochrome c release from mitochondria, decreasing Delta Psi m, chromatin condensation, phosphatidyl serine exposure and Tdt-sensitive DNA nicks. At the onset of apoptosis there was no evidence of increases in oxygen free radicals or peroxide in cells treated with AW 464 or diamide. Phenylarsine oxide induced both free radicals and hydrogen peroxide, but this did not appear to interfere with apoptosis. We conclude that pharmacological targeting of thioredoxin can induce a well-orchestrated apoptotic programme.     

13-cis retinoic acid and isomerisation in paediatric oncology--is changing shape the key to success?

Retinoic acid isomers have been used with some success as chemotherapeutic agents, most recently with 13-cis retinoic acid showing impressive clinical efficacy in the paediatric malignancy neuroblastoma. The aim of this commentary is to review the evidence that 13-cis retinoic acid is a pro-drug, and consider the implications of retinoid metabolism and isomerisation for the further development of retinoic acid for cancer therapy. The low binding affinity of 13-cis retinoic acid for retinoic acid receptors, low activity in gene expression assays and the accumulation of the all-trans isomer in cells treated with 13-cis retinoic acid, coupled with the more-favourable pharmacokinetic profile of 13-cis retinoic acid compared to other isomers, suggest that intracellular isomerisation to all-trans retinoic acid is the key process underlying the biological activity of 13-cis retinoic acid. Intracellular metabolism of all-trans retinoic acid by a positive auto-regulatory loop may result in clinical resistance to retinoic acid. Agents that block or reduce the metabolism of all-trans retinoic acid are therefore attractive targets for drug development. Devising strategies to deliver 13-cis retinoic acid to tumour cells and facilitate the intracellular isomerisation of 13-cis retinoic acid, while limiting metabolism of all-trans retinoic acid, may have a major impact on the efficacy of 13-cis retinoic acid in paediatric oncology.     

Role of sodium in intracellular calcium elevation and leukotriene B4 formation by receptor-mediated activation of human neutrophils

The role of Na(+) and Na(+) exchangers in intracellular Ca(2+) elevation and leukotriene B(4) (LTBs) formation was investigated in granulocyte macrophage colony-stimulating factor (GM-CSF)-primed, fMLP-stimulated human neutrophils. Isotonic substitution of extracellular Na(+) with N-methyl-D-glucamine(+) (NMDG(+)) resulted in over 85% inhibition of the LTBs generation observed (from 14.1+/-0.9pmol/10(6) neutrophils to 1.7+/-1.0pmol/10(6) neutrophils at 0.3 microM fMLP). Isotonic substitution of Na(+) with NMDG(+) also induced a significant inhibition of fMLP-induced rise in cytosolic Ca(2+) concentration ([Ca(2+)](i)) (from 2.17- to 0.78-fold increase over basal levels). Pretreatment with an inhibitor of the Na(+)/Ca(2+) exchanger (benzamil) did not inhibit either [Ca(2+)](i) rise or LTBs production, indicating that the observed effects of extracellular Na(+)-deprivation were unrelated to the Na(+)/Ca(2+) exchanger in receptor-mediated Ca(2+) influx, as previously hypothesized. LTBs production by thapsigargin-activated neutrophils was not affected by Na(+) depletion, but was totally abolished in the presence of EGTA, suggesting that store depletion-driven extracellular Ca(2+) influx is required for leukotriene synthesis and that this process is independent of Na(+)-deprivation. Exposure to Na(+)-free medium for the time of GM-CSF priming led to a significant decrease of intracellular pH values, suggesting a role of the Na(+)/H(+) exchanger in intracellular Na(+) depletion. Reducing the time of Na(+)-deprivation totally reversed the observed effect on LTBs production, resulting in enhanced, rather than inhibited, formation of LTBs. These results indicate that LTBs generation and [Ca(2+)](i) rise in human neutrophils primed by GM-CSF and stimulated with fMLP is dependent on intracellular Na(+) concentration, and, at variance with previously published results, unrelated to the Ca(2+) influx through the Na(+)/Ca(2+) exchanger.     

Inhibition of [3H]-U69593 binding and the cardiac effects of U50,488H by calcium channel blockers in the rat heart

1. The calcium channel blockers (CCBs), nifedipine, nicardipine, diltiazem and verapamil, were used to displace the binding of [³H]-{"type":"entrez-nucleotide","attrs":{"text":"U69593","term_id":"4205069","term_text":"U69593"}}U69593 ((5a,7a,8b)-(+)-N-methyl-N-(7-[1-pyrrolidinyl]-1-oxaspiro[4,5]dec-8-yl)-benzeneacetamide), a specific κ-opioid agonist, in the rat cardiac sarcolemma. The CCBs competed with the binding of [³H]-{"type":"entrez-nucleotide","attrs":{"text":"U69593","term_id":"4205069","term_text":"U69593"}}U69593 (4 nM) in a dose-dependent manner. The displacing potency of verapamil was 55 times greater than that of nifedipine.
2. The effects of two CCBs, verapamil and nifedipine, on the arrhythmogenic action of κ-receptor stimulation by a specific κ-receptor agonist, U50,488H (trans-(±-3,4-dichloro-N-methyl-N-(2-[1-pyrrolidinyl] cyclohexyl) benzeacetamide methanesulphonate), were also studied in the rat isolated perfused heart. U50,488H 80–800 nmol dose-dependently induced arrhythmias, which were completely abolished by a selective κ-receptor antagonist, nor-BNI (nor-binaltorphimine,17,17′-(dicyclopropylmethyl)-6,6′,7,7′-6,6′-imino-7,7′-binorphinan-3,4′,14, 14′-tetrol), at 100 nmol. The arrhythmogenic effect was also attenuated by both verapamil and nifedipine in a dose-dependent manner. The ED₅₀ values for verapamil and nifedipine were 2.75 and 63.7 nmol, respectively. The antiarrhythmic potencies of these two CCBs were correlated to their displacing potencies and inversely related to their well known potencies in inhibiting transmembrane Ca²⁺ influx in the cardiac muscle.
3. Measurement of [Ca²⁺]_i in the absence of free extracellular Ca²⁺ by a spectrofluorometric method, with fura-2 as Ca²⁺ indicator, showed that U50,488H 5×10⁻⁵ M slowly increased [Ca²⁺]_i in single ventricular myocytes and this effect was abolished by pretreatment with nor-BNI (5 μM), or ryanodine (5 μM). Verapamil 1 and 10 μM abolished the effect of U50,488H in 37.5% (3 out of 8) and 100% (12 out of 12) of the cells studied, respectively. On the other hand, nifedipine 10 and 100 μM had no effect at all. Neither verapamil nor nifedipine exerted any significant effect on the caffeine-induced Ca²⁺ transient.
4. The observations suggest that CCBs may inhibit the actions of κ-receptor stimulation at the level of the κ-receptor.

     

Antipsoriatic effects of avarol-3'-thiosalicylate are mediated by inhibition of TNF-alpha generation and NF-kappaB activation in mouse skin

BACKGROUND AND PURPOSE: Avarol is a marine sesquiterpenoid hydroquinone with anti-inflammatory and antipsoriatic properties. The aim of this study was to evaluate the in vitro and in vivo pharmacological behaviour of the derivative avarol-3'-thiosalicylate (TA) on some inflammatory parameters related to the pathogenesis of psoriasis. EXPERIMENTAL APPROACH: Human neutrophils and monocytes as well as the human keratinocyte cell line HaCaT were used to study the effect of TA on oxidative stress, the arachidonic acid pathway, tumour necrosis factor-alpha (TNF-alpha) release and nuclear factor-kappaB (NF-kappaB) activation. All these parameters were also determined in vivo using the zymosan induced mouse air pouch model and the 12-O-tetradecanoylphorbol-13-acetate (TPA) induced mouse epidermal hyperplasia model. KEY RESULTS: TA showed antioxidant properties in human neutrophils and in the hypoxanthine/xanthine oxidase assay. This compound reduced, in a concentration-dependent manner, leukotriene B(4), prostaglandin E(2) and TNF-alpha production in activated leukocytes. Oral and intrapouch administration of TA in the mouse air pouch model produced a dose-dependent reduction of all these inflammatory mediators. TA also inhibited secretory phospholipase A(2) activity and NF-kappaB DNA-binding in HaCaT keratinocytes. In TPA-induced mouse epidermal hyperplasia, topical administration of TA reduced oedema, leukocyte infiltration, eicosanoid levels and TNF-alpha in skin. In addition, interleukin (IL)-1beta and IL-2 production were also inhibited. Finally, TA was also capable of suppressing NF-kappaB nuclear translocation in vivo. CONCLUSIONS AND IMPLICATIONS: TA inhibited several key biomarkers up-regulated in the inflammatory response of psoriatic skin and this compound could be a promising antipsoriatic agent.     

Modification of intracellular free calcium in cultured A10 vascular smooth muscle cells by exogenous phosphatidic acid

Exogenous phosphatidic acid (PA) was observed to produce a concentration-dependent increase in [Ca(2+)](i) in cultured A10 vascular smooth muscle cells. Preincubation of cells with sarcoplasmic reticulum Ca(2+)-ATPase inhibitors (cyclopiazonic acid and thapsigargin), a phospholipase C inhibitor (2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate), inositol 1,4,5-trisphosphate receptor antagonists (2-aminoethoxydiphenyl borate and xestospongin), and an activator of protein kinase C (PKC) (phorbol 12-myristate 13-acetate) depressed the PA-evoked increase in [Ca(2+)](i). Although EGTA, an extracellular Ca(2+) chelator, decreased the PA-induced increase in [Ca(2+)](i), sarcolemmal Ca(2+)-channel blockers (verapamil or diltiazem) did not alter the action of PA. On the other hand, inhibitors of PKC (bisindolylmaleimide I) and G(i)-protein (pertussis toxin) potentiated the increase in [Ca(2+)](i) evoked by PA significantly. These results suggest that the PA-induced increase in [Ca(2+)](i) in vascular smooth muscle cells may occur upon the activation of phospholipase C and the subsequent release of Ca(2+) from the inositol 1,4,5-trisphosphate-sensitive Ca(2+) pool in the sarcoplasmic reticulum. This action of PA may be mediated through the involvement of PKC.