Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Pepstatins”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 325 records · Page 18Linked to original sources
PubMed2002

Alterations in membrane-associated CD14 expression and the simultaneous liberation of soluble CD14 fragment in adherent macrophages mediated by a leukocyte carboxyl/aspartate protease.

Investigations sought to discover the biochemical mechanisms in macrophages that mediate the 'shedding' of soluble CD14 fragment. Stimulated macrophages display both increased liberation of soluble CD14 fragment and decreases in residual membrane-associated CD14 complexes following exposure to activating agents (fMLP/A23187). Application of 'class-specific' protease inhibitors revealed that a thiol/cysteine was involved in the biochemical production of soluble CD14 fractions and that a metalloprotease enzymatically degraded soluble CD14 fragment. Exposure of macrophages to individual proteases revealed that both cathepsin-D and elastase promoted variable depletion of membrane-associated CD14 complexes. Additionally, cathepsin-D, and to a lesser extent elastase, generated soluble CD14 fragment. Related studies isolated a carboxyl/aspartate protease from activated macrophages using pepstatin-A affinity chromatography. The physical and functional properties of macrophage pepstatin-A binding protein fractions closely corresponded with the known characteristics of cathepsin-D with respect to: (i) cellular origin; (ii) binding-avidity of carboxyl/aspartate proteases for pepstatin-A; (iii) non-specific proteolysis of haemoglobin detected by Hb-PAGE zymography; and (iv) hydrolysis of a synthetic cathepsin-D-specific peptide substrate. Interpretation of these findings collectively implies that activated leukocytes can biochemically alter membrane-associated CD14 complex expression and promote the liberation of soluble CD14 fragment in both activated and non-activated cell populations.

Animals↗
PubMed1982

Biochemical and immunological characterization of ectopic tumoral renin.

Biochemical and immunological characteristics of renin secreted by two malignant renin-secreting tumors [pulmonary (PT) and paraovarian (POT)] were studied. They both contain inactive renin (IR), as renin activity of tumoral extracts was able to be increased after acid activation or trypsin treatment (10.1 to 20.8 Goldblatt units/g tissue for PT and 1.4 to 3.71 for POT). Renin activity after activation reached the value obtained by direct RIA of human renin (23 and 3.4, respectively), as both forms are recognized by renin antiserum. Both enzymatic activities could be completely inhibited by renin antiserum. Displacement curves for the two tumoral renins paralleled the MRC renin in the direct RIA. After chromatography on affigel blue, active renin was not bound to the gel, and inactive renin eluted only with 1 M NaCl. On pepstatin A Sepharose and CBL-pepstatin Sepharose (an N-modified-pepstatin), a separation of the two forms of pulmonary renin was obtained; inactive renin eluted with breakthrough proteins, whereas active renin was strongly bound to the gel. After this affinity chromatography, the molecular weights of inactive and active renin, determined on Ultrogel, were very close (46,000 and 42,500). We conclude that 1) ectopic renin in these cases in similar to the renal enzyme; 2) renin can be secreted in an inactive form, supporting the hypothesis of an inactive initial state of renin; and 3) molecular weight differences between the two forms are very slight.

Adenocarcinoma↗
PubMed2001

Proteolytic activity and recombinant protein production in virus-infected Sf-9 insect cell cultures supplemented with carboxyl and cysteine protease inhibitors.

In insect cell-baculovirus expression systems for recombinant protein production, it is sometimes necessary to supplement cultures with protease inhibitors to protect recombinant proteins against proteolysis. To date, however, there is no information available concerning protease activities in inhibitor-supplemented cultures. The aim of the present study was to investigate intracellular and extracellular protease activities in cultures of virus-infected Sf-9 insect cells which were supplemented with inhibitors against carboxyl and cysteine proteases produced during culture. Prior to the supplementation culture, the cell toxicity of several protease inhibitors was determined. As a result, pepstatin A (carboxyl protease inhibitor) and E64, cystatin, leupeptin, and antipain (cysteine protease inhibitors) tested in this study showed no apparent negative effects on the growth and viability of noninfected Sf-9 insect cells at low concentrations. In addition, E64 and pepstatin A could rapidly permeate virus-infected Sf-9 cells and inhibit the respective intracellular protease activities. A virus-infected culture with a multiplicity of infection of 1 was carried out with E64 and pepstatin A which were added to the culture medium at 2 d post-infection. As a result of inhibitor supplementation, the cellular activity for recombinant protein biosynthesis was reduced by 5-30%. However, a significant reduction in carboxyl and cysteine protease activities was observed not only in the medium but also intracellularly. This is the first study that directly demonstrates a reduction in extracellular and intracellular protease activities in protease inhibitor-supplemented cultures of virus-infected insect cells.

Journal Article↗
PubMed2003

Characterization of a serine protease activity in Sarcocystis neurona merozoites.

Sarcocystis neurona merozoites were examined for their ability to invade and divide in bovine turbinate (BT) cell cultures after treatment with cysteine (iodoacetamide), aspartic (pepstatin A), metallo-(1,10-phenanthroline and ethylene glycol-bis(aminoethylether)-tetraacetic acid [EGTA]), or serine (4-[2-aminoethyl]-benzenesulfonyl fluoride hydrochloride [AEBSF], phenylmethane sulphonyl fluoride [PMSF], and tosyl lysyl chloramethyl ketone [TLCK]) protease inhibitors. Significant (P < 0.01) inhibition of serine protease activity by PMSF and TLCK led to a reduction of 86 and 78% in merozoites produced in BT cell cultures, respectively, whereas AEBSF (1 mM) led to a 68% reduction in merozoites produced in BT cell cultures and a reduction of 84 and 92% at higher AEBSF concentrations (2 and 3 mM, respectively). Pepstatin A and iodoacetamide failed to cause any inhibition in merozoite production, whereas 1,10-phenanthroline and EGTA caused slight, but not significant, inhibition at 6 and 17%, respectively. In zymograms, 2 bands of protease activity between 65- and 70-kDa molecular weight were seen. The protease activity was inhibited by AEBSF but not by E-64 (cysteine protease inhibitor), EGTA, iodoacetamide, or pepstatin A. In native zymograms, the protease activity was highest between a pH range of 8 and 10. These data suggest that merozoites of S. neurona have serine protease activity with a relative molecular weight range between 65 and 70 kDa and optimal pH range between 8 and 10, which is essential for host cell entry at least in vitro. The protease activity described here could be a potential target for chemotherapy development.

Animals↗
PubMedDate not supplied

[Isolation of aspartic proteinases at different stages of development of Boophilus microplus tick].

The magnitud of tisular expression of aspartic proteinases at juvenile stages was analyzed in adult young Boophilus microplus ticks as well as the effect of one of the most widely used aspartic proteinase inhibitors (pepstatin A) on their development. The isolation and quantification of acid proteolytic activity was performed in samples of parasites with different stages of the digestive process where aspartic-proteinase activity was confirmed and a tendency to increased values of enzymatic activity per miligram of total weight was observed during the development of adults female ticks, with a lowering of this ratio just when they are full up. Adding pepstatin A to the ingesta at 14 and 70 mmol concentrations had no effect on the viability of young female ticks while they were kept in vitro for 24 h, so this rules out the immediate toxic effects of this compound. However, in those experiments where adult female ticks after receiving the various ingestas, are put together with sexually competent male ticks in isolators for 6-7 days, the viability showed different results (p< 0,01) at a 70 mmol concentration. The effect on the development of young adult female ticks was slight, but it indicates hat there might be a selective inhibitory action of pepstatin A on aspartic-dependent enzymes.

Animals↗
PubMed1993

Comparison of postmortem autolysis in cardiac and skeletal muscle.

To understand the mechanism in postmortem autolysis better, processes in the postmortem degradation of myofibril proteins in the presence of protease inhibitors were studied. Male Wistar rats were given injections of the carboxyl-, thiol-, and serine-protease inhibitors, pepstatin, Ep-475[L-transepoxysuccinyl-leucylamide(3-methyl) butane; E-64-C], and chymostatin, via the femoral vein. Control rats were similarly treated with saline. Then, myofibril proteins were isolated from their cardiac and femoral muscles and from those of control animals at various times after death, and degradation of these myofibril proteins with time was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In cardiac muscle, alpha-actinin was degraded rapidly, followed by the heavy chain of myosin and light chain of myosin (L2). Actin and the light chain of myosin (L1) were degraded slowly. the degradations of the heavy chain of myosin, alpha-actinin, tropomyosin and L2 after 14 days were not inhibited by pepstatin, but were inhibited by Ep-475 and chymostatin. In skeletal muscle, L1 and L2 were degraded rapidly, followed by the heavy chain of myosin and alpha-actinin. Actin was degraded slowly and was still unchanged 2 weeks after death. The degradations of protein components were inhibited by pepstatin, Ep-475 and chymostatin. These results indicated that after death the components of myofibrils are degraded by various proteases at various rates depending on their properties or structures. This degradation is fundamentally the same in cardiac and skeletal muscles, but inhibitors have somewhat different effects on the postmortem degradation processes after death in the two types of muscle.

Animals↗
PubMed1994

Western blotting and enzymatic activity analysis of cathepsin D in breast tissue and sera of patients with breast cancer and benign breast disease and of normal controls.

Increased total antigen amounts of cathepsin D in breast tissue have been reported to be associated with increased disease recurrence, more frequent metastasis, and increased mortality in breast cancer patients. In the present study, Western blotting analysis has been used for the first time to determine the relative amounts of precursor and processed forms of cathepsin D in sera and breast tissue of patients with breast cancer, benign breast disease, and normal controls. Sera gave similar blots for breast cancer patients and controls with two major forms of cathepsin D (M(r) 52,000 and 27,000). Malignant breast tissue contained the two forms of cathepsin D found in sera and an additional M(r) 31,000 form which was found in significantly increased (P < 0.001) relative amounts in breast tissue from 43 breast cancer patients [24 +/- 12% (SD)] when compared to 51 benign breast disease patients (13 +/- 8.9%) and 23 normal controls (1.8 +/- 4.4%). Preliminary analysis of subgroups of benign breast disease patients suggested no significant difference (P = 0.41) in relative amounts of the M(r) 31,000 form of cathepsin D between proliferative-type and non-proliferative-type fibrocystic breast disease. A cathepsin D assay has been optimized for human breast tissue and used to demonstrate for the first time significantly increased (P < 0.001) amounts of pepstatin-inhibitable, cathepsin D-specific activity in breast tissue from 36 breast cancer patients (2.2 +/- 1.4 units/mg of protein) when compared to 47 benign breast disease patients (0.63 +/- 0.43) and 23 normal controls (0.24 +/- 0.21). Preliminary analysis of subgroups of benign breast disease patients suggested no significant difference (P = 0.21) in pepstatin-inhibitable, cathepsin D-specific activity between proliferative-type and nonproliferative-type fibrocystic breast disease. The positive correlation (r = 0.82) of increased amounts of the M(r) 31,000 form of cathepsin D and increased pepstatin-inhibitable, cathepsin D enzymatic activity in malignant breast tissue suggests that the M(r) 31,000 form is the proteolytically active form of the enzyme which may be involved in the development and/or metastatic spread of breast cancer.

Adult↗
PubMed1979

Cathepsin D: the lysosomal aspartic proteinase.

Cathepsin D was originally known simply as 'cathepsin' and was first purified in the late 1930s. Nowadays the enzyme is purified by conventional column chromatography, and by isoelectric focusing (which resolves isoforms), but affinity chromatography with pepstatin--Sepharose is also important. Cathepsin D is a glycoprotein of about 42,000 molecular weight; sometimes it comprises a single polypeptide chain but often this is found to have been 'nicked' about two-thirds of the way from one end. Cathepsin D is an 'aspartic proteinase' and may be one of the more primitive members of the family. The activity of cathepsin D is expressed exclusively at acidic pH values and the specificity shows a strong preference for cleavage near hydrophobic amino acids. Specific inhibition of cathepsin D with antibodies and pepstatin has provided strong evidence that the enzyme plays a part in intralysosomal proteolysis but there is as yet little evidence for extracellular activity.

Animals↗
PubMed2006

Aspartic proteinases in the digestive tract of marine decapod crustaceans.

Decapod crustaceans synthesize highly active proteolytic enzymes in the midgut gland and release at least a part of them into the stomach where they facilitate the first step in peptide hydrolysis. The most common proteinases in the gastric fluid characterized so far are serine proteinases, that is, trypsin and chymotrypsin. These enzymes show highest activities at neutral or slightly alkaline conditions. The presence of acid proteinases, as they prevail in vertebrates, has been discussed contradictorily yet in invertebrates. In this study, we show that acid aspartic proteinases appear in the gastric fluid of several decapods. Lobsters Homarus gammarus showed the highest activity with a maximum at pH 3. These activities were almost entirely inhibited by pepstatin A, which indicates a high share of aspartic proteinases. In other species (Panulirus interruptus, Cancer pagurus, Callinectes arcuatus and Callinectes bellicosus), proteolytic activities were present at acid conditions but were distinctly lower than in H. gammarus. Zymograms at pH 3 showed in each of the studied species at least one, but mostly two-four bands of activity. The apparent molecular weight of the enzymes ranged from 17.8 to 38.6 kDa. Two distinct bands were identified which were inhibited by pepstatin A. Acid aspartic proteinases may play an important role in the process of extracellular digestion in decapod crustaceans. Activities were significantly higher in clawed lobster than in spiny lobster and three species of brachyurans. Therefore, it may be suggested that the expression of acid proteinases is favored in certain groups and reduced in others.

Animals↗
PubMed1993

Identification and localization of a novel cathepsin S-like proteinase in guinea pig spermatozoa.

Guinea pig spermatozoa were found to contain a novel cysteine proteinase that most closely resembled lysosomal cathepsin S. The responsible enzyme was shown to have an acrosomal localization. Like the cathepsin S of lymphocyte-rich tissues, e.g., lymph nodes and spleen, the spermatozoal enzyme digested protein substrates such as hemoglobin and Azocoll optimally at about pH 3.5 in the presence of pepstatin. No appreciable action was manifested in the range of pH 5-6 on protein substrates nor on the 7-(4-methyl)coumarylamide (NMec) derivatives of peptides commonly employed for the fluorometric assay of cathepsins B (benzyloxycarbonyl (Z)-Arg-Arg-NMec), H (Arg-NMec), and L (Z-Phe-Arg-NMec). Like spleen cathepsin S, the sperm enzyme selectively hydrolyzed Z-Phe-Arg-NMec, but its activity was uncharacteristically limited to a pH range of 3.0-3.5, where it displayed a typical high sensitivity to sulfhydryl reagents (HgCl2, mersalyl acid, iodoacetate), leupeptin, Z-Phe-Phe-CHN2, and L-trans-epoxysuccinylleucylamido(3-methyl)butane. Gossypol (a male antifertility agent) was also inhibitory. Inhibition by the mercurial sulfhydryl reagents was completely reversible with dithiothreitol. Pepstatin, a potent inhibitor of aspartic proteinases, and serine proteinase inhibitors (phenylmethylsulfonyl fluoride, benzamidine) were ineffective. Other properties displayed by the sperm extract that were uniquely characteristic of cathepsin S included stability under alkaline conditions, and a greater rate of hydrolysis when the P2-phenylalanine of the assay substrate was replaced by two aliphatic residues, as in Z-Leu-Leu-Arg-NMec.

Acrosome↗
PubMed2000

Inhibition of cathepsin D prevents free-radical-induced apoptosis in rat cardiomyocytes.

Apoptosis was inhibited in rat cardiomyocytes pretreated with the aspartic protease inhibitor pepstatin A and subsequently exposed to naphthazarin (5,8-dihydroxy-1,4-naphthoquinone). Cathepsin D was released from lysosomes to the cytosol upon exposure to naphthazarin, and the enzyme activity decreased simultaneously. Later, cathepsin D reappeared in granules of increased size, and enzyme activity was restored. Activation of caspase-3-like proteases was detected, and the number of cells showing apoptotic morphology increased with time. Pepstatin A pretreatment did not prevent release of cathepsin D from lysosomes but did significantly inhibit subsequent naphthazarin-induced caspase activation and apoptotic morphology. This suggests that cathepsin D exerts its apoptosis-stimulating effect upstream of caspase-3-like activation.

Animals↗
PubMed1996

Promotion of heat-induced apoptosis in FM3A cells by protease inhibitors.

Although it has been shown that proteases may play a positive role in in causing apoptosis of some cells, we report here that, on the contrary, protease inhibitors can promote heat-induced apoptosis in FM3A cells. Cysteine protease inhibitor, trans-Epoxy-succinyl-L-leucylamido-(4-guanidino)butane (E-64, 100 micrograms/ml) and aspartate protease inhibitor, pepstatin-A (100 micrograms/ml) were used to test hyperthermic effect on FM3A cells and showed remarkable cytotoxicity when they were present in cell suspension during heating at 44 degrees C. The cytotoxicity was due to promotion of heat-induced apoptosis as judged by DNA agarose electrophoresis. Furthermore, using flow cytometric analysis, we observed a decrease in the G0/G1 phase cell and an increase in the S phase cell as well as increased apoptosis after heat shock. E-64 and pepstatin-A exhibited a promotive effect on the changes of cell cycle induced by heat. The data presented suggest that the enhancement of hyperthermic cell killing by protease inhibitors may be related to promotion of heat-induced apoptosis and changes of cell cycle.

Animals↗
PubMed1994

Kinetic studies on phagocytosis and lysosomal digestion of rod outer segments by human retinal pigment epithelial cells in vitro.

Using novel methodology, this study describes the kinetics of rod outer segment (ROS) phagocytosis and digestion by human retinal pigment epithelial (RPE) cells in vitro and examines the effect of certain lysosomal enzyme inhibitors on ROS digestion in these cells. Human RPE cells displayed saturation of phagocytosis with respect to both ROS concentration and time. While surface-binding and ingestion phases of ROS phagocytosis saturated after 24-36 h, the rate of ROS digestion reached a maximal level within 24 h. Increasing the concentration of zinc in the culture medium from 1.9 to 100 microM had no significant effect on ROS digestion. The effects of swainsonine (an alpha-mannosidase inhibitor), pepstatin (an aspartic protease inhibitor), and leupeptin (a cysteine protease inhibitor) were also examined. At 6 h, ROS digestion was reduced 27.3 +/- 15.3% by swainsonine, 69.4 +/- 20.9% by pepstatin, and 77.0 +/- 14.4% by leupeptin. The effect of these inhibitors declined with increasing time. This study is the first to demonstrate the functional importance of cysteine and aspartic proteases in the digestion of ROS by RPE cells in vitro.

Animals↗
PubMed1993

Crystallization of inhibited aspartic proteinase from Candida albicans.

A major secreted aspartic proteinase from Candida albicans has been crystallized in the presence of inhibitors to prevent autodegradation. With pepstatin a cleaved form of the enzyme was nevertheless found in the crystals whereas with the inhibitor A70450 the enzyme remained intact. The crystals containing pepstatin were not suitable for X-ray data collection while the crystals containing A70450 grew by vapour diffusion as tetragonal bipyramids, space group P4(3)2(1)2 (or P4(1)2(1)2), a = b = 76.2 A, c = 126.1 A, with one molecule in the asymmetric unit and they diffract to beyond 2.2 A.

Aspartic Acid Endopeptidases↗
PubMed1994

Purification of crystallizable recombinant SIVmac251-32H proteinase.

We have cloned a simian immunodeficiency virus (SIV) proteinase gene directly from proviral DNA of the infectious viral stock SIVmac251-32H (11/88 pool). The deduced amino acid sequence from this proteinase gene is similar to that for the published SIVmac239 molecular clone. SIVmac251-32H proteinase (SIV PR) and its flanking pol sequences were expressed in Escherichia coli as a fusion protein with most of the T7 bacteriophage gene 10 protein. The expressed protein formed cytoplasmic inclusion bodies which were solubilized in 8 M urea, and the recombinant SIV PR was refolded, yielding active, self-processed enzyme. The SIV PR was purified to homogeneity using a single pepstatin A affinity chromatography step, and had a specific peptidolytic activity of 20 mumol/min/mg. Enzymatic characteristics similar to those previously documented for other immunodeficiency virus proteinases (EC 3.4.23) were observed. These include an acidic pH optimum (pH 5.3), sensitivity to sodium chloride concentration, and complete inhibition by pepstatin A. In addition to these properties we have observed quantitative crystallization from low protein concentrations. We describe the first crystal habit for the proteinase from the HIV-2/SIV class of immunodeficiency virus, which is distinctly different from that for HIV-1 proteinase crystals.

Amino Acid Sequence↗
PubMed1988

Probable involvement of vascular angiotensin II formation in the beta 2-adrenoceptor-mediated facilitation of the neurogenic vasopressor response in the pithed rat.

Rats were pithed, vagotomized and adrenalectomized and the effect of procaterol on the pressor response to electrical stimulation of the thoracolumbar preganglionic sympathetic outflow from the spinal cord or to exogenous noradrenaline was studied in the absence and presence of beta-adrenoceptor antagonists and drugs interfering with the renin-angiotensin system. 1. Basal diastolic blood pressure was decreased by captopril, ramiprilate (angiotensin converting enzyme inhibitors), saralasin (an angiotensin II receptor antagonist), pepstatin A (a protease inhibitor with renin antagonistic properties) and by functional nephrectomy (ligation of both renal hili), but was not affected by procaterol (a beta 2-adrenoceptor agonist), nebivolol (a beta 1-adrenoceptor antagonist) and ICI 118,551 (erythro-dl-1-(7-methylindan-4-yloxy)-3-isopropylaminobut an-2-ol; a beta 2-adrenoceptor antagonist). 2. The vasopressor response induced by electrical stimulation of the preganglionic sympathetic nerve fibres was increased by procaterol, whereas the increase in blood pressure evoked by exogenous noradrenaline was not affected. The pressor response to both electrical stimulation and exogenous noradrenaline was decreased by captopril, ramiprilate, saralasin and nephrectomy but was not affected by nebivolol and ICI 118,551. 3. The facilitatory effect of procaterol on the neurogenic, electrically induced pressor response, which was also obtained when basal blood pressure was decreased by nephrectomy and increased by Lys8-vasopressin, was abolished by ICI 118,551 but not affected by nebivolol. Under none of these experimental conditions did procaterol alter the vasopressor response to exogenous noradrenaline. 4. The facilitatory effect of procaterol on the neurogenic, electrically induced rise in blood pressure was abolished by captopril, ramiprilate, saralasin and pepstatin A.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenergic beta-Agonists↗
PubMed1979

Macromolecular, anionic pulmonary permeability factor.

Aqueous extracts of fresh or acetone-powdered calf lung have been found to contain a factor which increases the permeability of the microcirculation when injected into the skin of rats. This permeability factor, which is not found in similar extracts of muscle or kidney, was concentrated by Amicon ultrafiltration at a molecular weight range of between 50,000 and 100,000 daltons and via isoelectric focusing at an IEP of pH 4.2. After preparative acrylamide gel electrophoresis, this permeability factor was homogeneous by electrophoretic criteria and in SDS acrylamide gel electrophoresis had a molecular weight of approximately 82,000 daltons. This apparently homogeneous permeability factor from lung was inhibited by pepstatin, and yet possessed no acid proteolytic activity against any substrate. Its activity was completely inhibited by pretreatment of the animals with antihistamines. This pepstatin-inhibitable permeability factor was found largely in the lysosomal fraction of fresh lung and could also be obtained by extraction of large amounts of alveolar "washout" macrophages. Since macrophages, during phagocytosis, are known to leak significant quantities of their lysosomal contents, the fact that one of these components is a material which could release histamine from mast cells and thereby increase locally the permeability of the microcirculation may be of importance in the defense system of the lung.

Animals↗
PubMed1988

Function and stimulus-specific effects of phorbol 12-myristate 13-acetate on human polymorphonuclear neutrophils: autoregulatory role for protein kinase C in signal transduction.

Exposure of polymorphonuclear neutrophils (PMNs) to phorbol 12-myristate 13-acetate (PMA) resulted in a concentration-dependent (1-10 ng/ml) inhibition of granule exocytosis induced with the receptor-specific ligands, N-formyl-methionyl-leucyl-phenylalanine (FMLP), pepstatin A, 5(S),12(R)-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid (LTB4), and acetyl-sn-glyceryl-3-phosphorylcholine (AGEPC). PMA exerted a marginal inhibitory effect on calcium ionophore A23187-induced PMN degranulation, and the PMA analog, 4 alpha-phorbol 12,13-didecanoate (4 alpha-PDD), was inactive. However, PMA potentiated AGEPC, pepstatin A, FMLP, LTB4, and A23187-stimulated superoxide anion (O2-) production. The mobilization of intracellular sequestered calcium (Ca2+) by the receptor-specific ligands, as reflected by a rise in the cytosolic-free Ca2+ concentration ([Ca2+]i) in PMNs loaded with the Ca2+-sensitive dye, Fura-2, was suppressed by PMA. A protein kinase C (PKC) inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) reversed the PMA-mediated inhibition of PMN degranulation and intracellular CA2+ mobilization. However, another, but less potent PKC inhibitor, N-(2-guanidino-ethyl)-5-isoquinolinesulfonamide (HA1004), had no effect on the inhibition of PMN activation by PMA.

Exocytosis↗