Transport of molybdate by Clostridium pasteurianum.

The transport of 99MoO42- into dinitrogen-fixing cells of Clostridium pasteurianum was investigated. Transport of molybdate in this organism is energy dependent; sucrose is required in the minimal media, and the system is inhibited by the glycolysis inhibitors, NaF, iodoacetic acid, and arsenate. The cells accumulate molybdate against a concentration gradient, and the uptake shows a marked dependence on temperature (optimum 37 C) and pH (optimum 6.0). The rate of molybdate uptake with increasing molybdate concentrations shows saturation kinetics with an apparent Km and Vmax of 4.8 X 10(-5) M and 55 nmol/g of dry cells per min, respectively. Inhibition studies with the anions SO42-, S2O32-, WO42-, and VO32- show that SO42- and WO42- competitively inhibit MoO42- uptake (apparent Ki [SO42-] is 3.0 X 10(-5) M; apparent Ki [WO42-] is 2.4 X 10(-5), whereas S2O32- and VO32- have no inhibitory effect. Exchange experiments with MoO42- show that only a small percentage of the 99MoO42- taken up by the cells is exchangeable. Exchange experiments with WO42- and SO42- indicate that once inside the cells WO42- and SO42- cannot substitute for MoO42-.     

Secretion of chondroitin SO4 by monolayer cultures of chick embryo chondrocytes

Monolayer cultures of chick embryo tibial chondrocytes incorporate 35SO42- into chondroitin SO4 which is rapidly secreted from the cells into two extracellular pools. Part of the extracellular chondroitin SO4 is recovered in a soluble form in the culture medium, and the remainder is associated with the cell matrix from which it is released by isotonic trypsinization. At 38 degrees C labeled chondroitin SO4 appears in the cell matrix fraction within 5 min after addition of 35SO42- and in the culture medium fraction 15 min after 35SO42- is added. The intracellular pool of labeled chondroitin SO4 reaches a steady state level of 150 to 200 pmol of bound SO4 per 10(6) cells in 60 min, while the cell matrix and medium fractions increase at rates of 3 and 1 nmol of bound SO4 per h per 10(6) cells, respectively. After 4 h of labeling, less than 20% of the newly synthesized cell-associated chondroitin SO4 is in the intracellular fraction. By labeling cells for 15 min at 25 degrees C 80% of the cell-associated chondroitin 35SO4 is obtained in the intracellular fraction. This material is chased without lag into both the cell matrix fraction and the medium fraction. A mixture of NaF and NaCN, both at 30 mM, lowers the cellular ATP level to 15% of normal and blocks secretion of the intracellular chondroitin SO4 into both extracellular fractions. Colchicine at 10(-6) M gives a partial inhibition of both synthesis and secretion of chondroitinSO4. Sucrose density gradient sedimentation analysis of the intracellular chondroitin SO4 and the two extracellular fractions shows that all three fractions contain both a heavy and light proteoglycan fraction. The intracellular light proteoglycan fraction is secreted preferentially into the culture medium where it represents 30% of the total culture medium pool. The ratio of 6-sulfated GalNAc to 4-sulfated GalNAc in the heavy proteochondroitin SO4 fraction is approximately twice that found for the light fraction.     

Induction and inhibition of estradiol hydroxylase activities in MCF-7 human breast cancer cells in culture

The effects of estradiol, progesterone, and tamoxifen on the activity of estradiol 2- and 16 alpha-hydroxylases were studied in human breast cancer cell cultures using a radiometric assay. After 5 days' exposure to these compounds, incubations in the presence of either [2-3H]estradiol or [16 alpha-3H]estradiol as substrate were carried out. In MCF-7 cells, estradiol (10(-8) M), progesterone (10(-6) M) and tamoxifen (10(-6) M) significantly increased 16 alpha-hydroxylase activity (estradiol; 21% progesterone 10% to 32%; tamoxifen 21% to 31%; P less than 0.01). Synergistic effects were observed when the cells were successively exposed to tamoxifen and progesterone. Simultaneous treatment with tamoxifen plus estradiol or estradiol plus progesterone showed no change from estradiol alone. On the other hand, although estradiol had no direct effects on 2-hydroxylase activity, tamoxifen decreased this enzymatic activity significantly at 10(-6) M (23% to 37%). Progesterone acted synergistically to further decrease this reaction. Treatment with only progesterone caused an increase in 2-hydroxylation. In contrast, a subline of MCF-7 cells with low estrogen receptor levels showed only minimal enzyme-hormone responses. Likewise, treatment of the estrogen receptor-negative MDA-MB-231 human breast cancer cell line with these compounds showed no effects on either 2- or 16 alpha-hydroxylase activity. In the progesterone receptor-rich T47D cell line, estradiol decreased both activities while progesterone increased both.(ABSTRACT TRUNCATED AT 250 WORDS)     

Eosinophil-induced release of acetylcholine from differentiated cholinergic nerve cells

One immunological component of asthma is believed to be the interaction of eosinophils with parasympathetic cholinergic nerves and a consequent inhibition of acetylcholine muscarinic M2 receptor activity, leading to enhanced acetylcholine release and bronchoconstriction. Here we have used an in vitro model of cholinergic nerve function, the human IMR32 cell line, to study this interaction. IMR32 cells, differentiated in culture for 7 days, expressed M2 receptors. Cells were radiolabeled with [3H]choline and electrically stimulated. The stimulation-induced release of acetylcholine was prevented by the removal of Ca2+. The muscarinic M1/M2 receptor agonist arecaidine reduced the release of acetylcholine after stimulation (to 82 +/- 2% of control at 10(-7) M), and the M2 receptor antagonist AF-DX 116 increased it (to 175 +/- 23% of control at 10(-5) M), indicating the presence of a functional M2 receptor that modulated acetylcholine release. When human eosinophils were added to IMR32 cells, they enhanced acetylcholine release by 36 +/- 10%. This effect was prevented by inhibitors of adhesion of the eosinophils to the IMR32 cells. Pretreatment of IMR32 cells with 10 mM carbachol, to desensitize acetylcholine receptors, prevented the potentiation of acetylcholine release by eosinophils or AF-DX 116. Acetylcholine release was similarly potentiated (by up to 45 +/- 7%) by degranulation products from eosinophils that had been treated with N-formyl-methionyl-leucyl-phenylalanine or that had been in contact with IMR32 cells. Contact between eosinophils and IMR32 cells led to an initial increase in expression of M2 receptors, whereas prolonged exposure reduced M2 receptor expression.     

Real-time kinetics of ligand/cell surface receptor interactions in living cells: binding of epidermal growth factor to the epidermal growth factor receptor

We describe a system for extending stopped-flow analysis to the kinetics of ligand capture and release by cell surface receptors in living cells. While most mammalian cell lines cannot survive the shear forces associated with turbulent stopped-flow mixing, we determined that a murine hematopoietic precursor cell line, 32D, is capable of surviving rapid mixing using flow rates as great as 4.0 mL/s, allowing rapid processes to be quantitated with dead times as short as 10 ms. 32D cells do not express any endogenous epidermal growth factor (EGF) receptor or other ErbB family members and were used to establish monoclonal cell lines stably expressing the EGF receptor. Association of fluorescein-labeled H22Y-murine EGF (F-EGF) to receptor-expressing 32D cells was observed by measuring time-dependent changes in fluorescence anisotropy following rapid mixing. Dissociation of F-EGF from EGF-receptor-expressing 32D cells was measured both by chase experiments using unlabeled mEGF and by experiments in which equilibrium was perturbed by dilution. Comparison of these dissociation experiments showed that little, if any, ligand-induced dissociation occurs in the chase dissociation experiments. Data from a series of association and dissociation experiments, performed at various concentrations of F-EGF in the nanomolar range and at multiple cell densities, were simultaneously analyzed using global analysis techniques and fit to a two independent receptor-class model. Our analysis is consistent with the presence of two distinct receptor populations having association rate constants of k(on1) = 8.6 x 10(6) M(-1) s(-1) and k(on2) = 2.4 x 10(6) M(-1) s(-1) and dissociation rate constants of k(off1) = 0.17 x 10(-2) s(-1) and k(off2) = 0.21 x 10(-2) s(-1). The magnitudes of these parameters suggest that under physiological conditions, in which cells are transiently exposed to nanomolar concentrations of ligand, ligand capture and release may function as the first line of regulation of the EGF receptor-induced signal transduction cascade.     

Retinoic acid inhibits phospholipid turnover and protein kinase C activity in RA-sensitive but not in RA-resistant cells

Treatment with 10(-5) M retinoic acid causes loss of anchorage-independent growth in src-transformed RR1022 cells but not in ras-transformed KNRK cells. In an effort to elucidate the mechanisms underlying this difference, we investigated the effect of RA on phospholipid turnover and PKC activity in these two cell lines. 10(-5) M RA treatment caused a drastic inhibition of 32P incorporation into PI and PA and a large increase in 32P incorporation into PC in RR1022 cells. Similar treatment of KNRK cells yielded no change in PC or PA labelling and a much smaller decrease in PI labelling. Furthermore, 10(-5) M RA treatment causes a large decrease in PKC activity in RR1022 cells (35% of control) but only a small decrease in KNRK cells (78% of control). We suggest that these effects are part of an altered signal transduction pathway which mediates the differential effects of RA on anchorage-independent growth in these two cell lines.     

Evidence for a platelet-activating factor receptor on human lymphoblastoid B cells: activation of the phosphatidylinositol cycle and induction of calcium mobilization

In this report we demonstrate evidence which strongly suggests that a receptor for platelet-activating factor (PAF) exists on a lymphoblastoid B cell line, LA350. PAF ranging in concentration from 10(-6)-10(-9)M initiated the incorporation of 32P into phosphatidic acid (PA) and phosphatidylinositol (PI) with no change in phosphatidylethanolamine (PE) and phosphatidylcholine (PC) over baseline. Lyso-PAF, the inactive precursor, at 10(-7)M had no effect on membrane phospholipid metabolism. In addition, PAF from 10(-6)-10(-8)M when added to Fura-2 containing B cells induced a rapid and significant rise of calcium within the cell, with lyso-PAF having no effect. These data suggest that PAF binds to a receptor on B cells and induces the hydrolysis of PI and a subsequent increase of intracellular calcium.     

Action of atrial natriuretic peptide and angiotensin II on the myocardium: studies in isolated rat ventricular cardiomyocytes

Isolated calcium-tolerant rat ventricular cardiomyocytes were used to characterize the effects of atrial natriuretic peptide (ANP), Angiotensin II (AII) and their interaction on the myocardial contraction-/relaxation pattern free of interference from other types of cardiac cells. Binding of 125I-ANP showed a KD of 12 pM and approximately 600 binding sites per cell. At 37 degrees C (rate 140 bpm) ANP decreased the contraction maximum with an EC50 of about 70 pM, maximal decrease was 35%. ANP (10(-7) M) raised cellular cyclic-GMP from 0.76+/-0.12 to 1.32+/-0.13 pmole/10(6) cells (73%, p less than 0.05). Angiotensin II increased contractility by a maximum of 32% at 10(-7) M; the EC50 was 8 x 10(-10) M. AII markedly delayed relaxation (reduction of maximum relaxation velocity from 0.092 to 0.063 mm/s; p less than 0.05). ANP (10(-7) M) increased the effect of AII (10(-8) M) on contractility by 66% without changing relaxation parameters significantly. This unexpected interaction may be relevant in pathological conditions where both AII and ANP are stimulated, such as heart failure or secondary hypertension.     

Effect of dimethyl sulfoxide on post-thaw viability assessment of CD45+ and CD34+ cells of umbilical cord blood and mobilized peripheral blood

BACKGROUND: The effect of dimethyl sulfoxide (Me2SO) on enumeration of post-thaw CD45+ and CD34+ cells of umbilical cord blood (HPC-C) and mobilized peripheral blood (HPC-A) has not been systematically studied. METHODS: Cells from leukapheresis products from multiple myeloma patients and umbilical cord blood cells were suspended in 1, 2, 5, or 10% Me2SO for 20 min at 22 degrees C. Cells suspended in Me2SO were then immediately assessed or assessed following removal of Me2SO. In other samples, cells were suspended in 10% Me2SO, cooled slowly to -60 degrees C, stored at -150 degrees C for 48 h, then thawed. The thawed cells in 10% Me2SO were diluted to 1, 2, 5, or 10% Me2SO, held for 20 min at 22 degrees C and then immediately assessed or assessed after the removal of Me2SO. CD34+ cell viability was determined using a single platform flow cytometric absolute CD34+ cell count technique incorporating 7-AAD. RESULTS: The results indicate that after cryopreservation neither recovery of CD34+ cells nor viability of CD45+ and CD34+ cells from both post-thaw HPC-A and HPC-C were a function of the concentration of Me2SO. Without cryopreservation, when Me2SO is present recovery and viability of HPC-C CD34+ cells exposed to 10% Me2SO but not CD45+ cells were significantly decreased. Removing Me2SO by centrifugation significantly decreased the viability and recovery of CD34+ cells in both HPC-A and HPC-C before and after cryopreservation. DISCUSSION: To reflect the actual number of CD45+ cells and CD34+ cells infused into a patient, these results indicate that removal of Me2SO for assessment of CD34+ cell viability should only be performed if the HPC are infused after washing to remove Me2SO.     

Undersulfated proteoglycans are secreted by cultured chondrocytes in the presence of the ionophore monensin

The monovalent ionophore, monensin, inhibits secretion of many different proteins from a wide variety of cells. The site of blockage is at the golgi complex. We have exposed chick embryo chondrocytes in suspension culture to monensin, at concentrations ranging from 10(-8) to 10(-6) M. At the higher concentrations, between 10(-7) and 10(-6) M, monensin inhibited secretion of type II procollagen, which accumulated in the chondrocytes. At these concentrations of the ionophore, proteoglycan synthesis was inhibited, as measured by radioactive serine incorporation into core proteins and by radioactive glucosamine or SO4 incorporation into glycosaminoglycans. However, at a monensin concentration of 3 x 10(-8) M, the incorporations of serine and glucosamine were close to normal while SO4 incorporation was at 30% of control values. The ratio of glucosamine to serine in pronase-released glycosaminoglycans from culture media was unaffected by 3 x 10(-8) M monensin but the sulfate to serine ratio decreased to 29% of control values. Examination of the glycosaminoglycans by gel filtration showed a progressive increase in Kav values as sulfation decreased. Undersulfation was demonstrated by radiochromatographic analysis of the digestion products following incubation with chondroitinase ABC. The composite results show that monensin interferes with sulfation of newly synthesized proteoglycans.     

The 32-kilodalton envelope protein of vaccinia virus synthesized in Escherichia coli binds with specificity to cell surfaces.

The nature of interaction between vaccinia virus and the surface of host cells as the first step in virus infection is undefined. A 32-kDa virus envelope protein has been identified as a cell surface binding protein (J.-S. Maa, J. F. Rodriguez, and M. Esteban, J. Biol. Chem. 265:1569-1577, 1990). To carry out studies on the structure-function relationship of this protein, the 32-kDa protein was obtained from Escherichia coli cells harboring the expression plasmid pT7Ek32. The recombinant polypeptide was found to have structural properties similar to those of the native virus envelope protein. Binding studies of 125I-labeled 32-kDa protein to cultured cells of various origins revealed that the E. coli-produced 32-kDa protein exhibited selectivity, specificity, and saturability. Scatchard analysis indicated about 4.5 x 10(4) sites per cell with a high affinity (Kd = 1.8 x 10(-9) M), suggesting interaction of the 32-kDa protein with a specific receptor. The availability of large quantities of the 32-kDa virus protein in bacteria will permit further structural and functional studies of this virus envelope protein and facilitate identification of the specific cell surface receptor.     

Stable isotope fractionation by Clostridium pasteurianum. 2. Regulation of sulfite reductases by sulfur amino acids and their influence on sulfur isotope fractionation during SO32- and SO42- reduction

In addition to an assimilatory sulfite reductase, studies of cultures of Clostridium pasteurianum supplemented with methionine, cysteine, and 35SO42- provides evidence for another reductase which is induced by SO32-. This inducible reductase appears to be dissimaltory because of the copious sulfide production arising when the cells are grown on SO32-. Cysteine can repress the assimilatory sulfite reductase but does not affect the inducible reductase. During late logarithmic growth on 1 mM SO42- + 10mM cysteine, depression of the inducible reductase occurred along with increased sulfide production. The presence of 1 mM cysteine and (or) 1 mM cysteine and (or) 1 mM methionine does not affect the inverse sulfur isotope effect for evolved H2S. However, 5 and 10 mM cysteine reduce the maximum delta34S value for released H2S from +40 to 10%. A small conversion of cysteine to H2S by C. pasteurianum occurs, but only in the stationary phase.     

Sodium Selenite Acts as an Otoprotectant against Neomycin-Induced Hair Cell Damage in a Zebrafish Model

Sodium selenite is a trace element essential for many physiological functions in the body. It is involved in various biological processes; it acts as a cofactor for antioxidant enzymes that protect against free radicals and is reported to limit metal-mediated oxidative DNA damage. In the present study, we investigated the effect of sodium selenite on neomycin ototoxicity in wild-type and transgenic zebrafish (Brn3C: EGFP). Five or six days post-fertilization, zebrafish larvae were co-exposed to 125 μM neomycin and various concentrations (10 μM, 100 μM, 250 μM, and 500 μM) of sodium selenite for 1 h. Hair cells within neuromasts of the supraorbital (SO1 and SO2), otic (O1), and occipital (OC1) lateral lines were analyzed by fluorescence microscopy (n = 10 fish per treatment). Hair cell survival was estimated as the ratio of the hair cell numbers in each group compared to those of the control group that were not exposed to neomycin. Apoptosis and hair cell damage of neuromasts were evaluated using the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) assay and 2-[4-(dimethylamino) styryl]-N-ethylpyridinium iodide (DASPEI) assay, respectively. Ultrastructural changes were evaluated using scanning electron microscopy and transmission electron microscopy. Neuromast hair cells were preserved in zebrafish exposed to 125 μM neomycin and 500 μM sodium selenite for 1 h. Sodium selenite protected against neomycin-induced hair cell loss of neuromasts, reduced apoptosis, and prevented zebrafish ultrastructural changes. We propose that sodium selenite protects against neomycin-induced hair cell damage by inhibiting apoptosis, decreasing the disarray of stereocilia, and preventing ultrastructural changes in the neuromast hair cells of the zebrafish.     

Isolation, purification, and partial characterization of suppressin, a novel inhibitor of cell proliferation

Pituitary tissues were investigated for the presence of regulatory molecules that would alter the function of lymphoid cells. A novel endogenous polypeptide inhibitor of basal and mitogen-stimulated splenocyte DNA synthesis and proliferation, suppressin, was isolated from bovine pituitary glands. Suppressin is a potent inhibitor of basal and mitogen-stimulated splenocyte proliferation at picomole and nanomole concentrations with 50% inhibition occurring 2.8 x 10(-9) M. Suppressin was purified to apparent homogeneity using sequential (NH4)2SO4 precipitation, ion-exchange chromatography, and preparative native gel electrophoresis. Biochemical characterizations of suppressin showed that this inhibitory molecule was a monomeric polypeptide with (i) a Mr = 63,000 and (ii) a pI of 8.1. Finally, metabolic labeling studies using a rat pituitary tumor cell line, GH3, showed that suppressin was synthesized de novo and secreted by these cells.     

Synthesis and release of cell surface-derived and sulfated lactosaminoglycans by human ovarian carcinoma cells.

Ovarian carcinoma cell clusters were isolated from patient effusions. Cell-surface glycoconjugates were radiolabelled by a galactose oxidase-borotritide method. The surface-labelled glycoconjugates and metabolically labelled glycoconjugates released to culture medium were characterized. The surface-derived glycoconjugates were highly heterodisperse and had the same molecular weight distribution as the metabolically labelled components. Lectin precipitation assays showed that both classes of glycoconjugates contained N-linked oligosaccharides bearing N-acetyllactosamine moieties. A121 ovarian carcinoma cells also synthesized and released a heterodisperse array of glycoconjugates to culture medium. Ricinus communis agglutinin I (RCAI) precipitated glycoconjugates of MW greater than 100 kDa for both A121 cells and cells from effusions. Cells of different ovarian carcinoma histology yielded similar results. Metabolic labelling experiments with 35SO4 showed that the RACI-bound glycoconjugates released by A121 cells were sulfated. The RCAI-bound sulfated lactosaminoglycans may be associated with malignant transformation and/or metastasis since similar components were not produced by mesothelial cells isolated from effusions [Allen, H.J., M. Gamarra M.S. Piver and E.A.Z. Johnson (1989). Cancer Biochem. Biophys. 10, 219-226].     

Sulfur isotope fractionation by Salmonella heidelberg: inverse isotope effects during growth on high concentrations of Na2SO3

During growth on minimal salts - glucose media supplemented with high concentrations of Na2SO3 (10-3 and 10-2 M), Salmonella heidelberg exhibited cytological and growth responses which indicated increased cellular toxicity with increasing sulfite concentrations. The large quantities of sulfide evolved during growth at both SO32- concentrations were accompanied by large normal and inverse isotope effects. Consistent with earlier findings, this organism was found capable of rapidly metabolizing both the sulfane and sulfonate sulfur of thiosulfate. Therefore, the isotope effects do not appear to be caused by extracellular chemical thiosulfate formation.     

Synthesis and processing of bone sialoproteins during de novo bone formation in vitro.

Bone sialoprotein (BSP) and osteopontin (OPN) are sulphated and phosphorylated sialoglycoproteins that regulate the formation of hydroxyapatite crystals during de novo bone formation. To gain insights into the relationship between the synthesis and posttranslational modification of BSP and OPN and the mineralization of bone, pulse-chase studies were conducted on cultures of newly forming bone nodules produced by fetal rat calvarial cells in vitro. Cultures were pulse labelled with 35SO4, or with either 32PO4 or [gamma-32P]ATP to study intracellular and extracellular phosphorylation, respectively, and chased in isotope-free medium for various times up to 24 h. The presence of radiolabelled BSP and OPN was determined in the cells, in culture medium, and in various tissue compartments obtained by dissociative extraction with 4 M GuHCl (G1), 0.5 M EDTA (E), and again with 4 M GuHCl (G2) and a bacterial collagenase digestion of the demineralized collagenous tissue residue. With each isotope employed, radiolabelled BSP and OPN were detected in the E extract within the 1-h chase period and increased in amount with time. Similarly, 35SO4- and 32PO4-labelled BSP increased in the G2 extract, but OPN was not detected. In the G1 extract the 35SO4-labelled BSP decreased with chase time, whereas the 32PO4-labelled BSP increased. No differences were evident in the profiles of BSP labelled with 32PO4 or [gamma-32P]ATP. In the absence of beta-glycerophosphate, which is required for optimal mineralization of the bone nodules, 35SO4-labelled BSP was increased in the medium and G1 extract and decreased in the E extract and G2 extract after 3 h. In addition to differences in the tissue compartmentalization of BSP and OPN, these studies indicate that 35SO4 is lost from BSP during mineralization and that isoforms of BSP exist with a selective affinity for the organic and mineral phases. Moreover, the additional phosphorylation of BSP and OPN catalyzed by ectokinase activity does not appear to alter the distribution of these sialoproteins.     

Human mammary cancer cell mutants with altered hormone receptor activity

We have recently isolated retinoic acid-resistant clones U-2 and U-3 from human breast cancer cell line MCF-7 (Ueda et al. (1985) Cancer Res. 45, 3332-3338). Growth of MCF-7 cells was found to be stimulated by estradiol but that of U-2 or U-3 was not. Cytosol from U-2 or U-3 cells contained no detectable estradiol receptor activity, whereas that from the parental MCF-7 cells showed estradiol receptor activity of 32 fmol/mg cytosol protein with a Kd of 2.6 X 10(-10) M by Scatchard analysis. Sucrose gradient centrifugation analysis of the cytosol fraction confirmed the presence of estradiol receptor activity in MCF-7 but not in U-2. Cytosol from MCF-7 and U-2 cells showed progesterone receptor activities of 106 fmol/mg protein with a Kd of 7.4 X 10(-10) M and 13 fmol/mg protein with a Kd of 9.9 X 10(-10) M, respectively. Addition of estradiol to the culture medium of the cells increased the level of progesterone receptor about 2-fold in MCF-7, but not in U-2. U-2 or U-3 cells showed about 5-fold higher resistance to an antiestrogen, tamoxifen, than MCF-7, and they were also 300- to 1,000-fold more resistant to other antiestrogens, epitiostanol and medroxyprogesterone, than MCF-7. The altered cellular sensitivity of U-2 or U-3 to the hormone antagonists is discussed in relation to the absence or presence of hormone receptors.     

Protein phosphorylation in intact coronary endothelial cells by bradykinin

In cultured coronary endothelial cells obtained from guinea pig hearts, bradykinin (10(-6) M) stimulated the 32Pi-incorporation into 5 substrate proteins with molecular weights corresponding to 27, 32, 60, 86 and 100 kDa. The time course of phosphorylation of the 60, 86 and 100 kDa proteins was rapid (within 30 s), but transient (max. within 1-2 min.), while the 32Pi incorporation into the 27 and 32 kDa protein was delayed but increased within 10 minutes. Ca+(+)-ionophore A 23187 (10(-5) M) and 12-O-tetradecanoylphorbol-13-acetate (TPA) (10(-5) M) both mimicked the effects of the bradykinin induced phosphorylation pattern. While A 23187 enhanced the phosphorylation of the 27, 60 and 100 kDa substrates, TPA increased the 32Pi-incorporation into the 32 and 86 kDa proteins. Furthermore the time course of protein phosphorylation elicited by A 23187 and TPA showed marked similarities to those obtained with bradykinin. Our findings are consistent with the view, that stimulation of coronary endothelial bradykinin-receptors activates both Ca+(+)-dependent protein kinases and protein kinase C.