Involvement of cholinoceptors in cadmium-induced endothelial dysfunction.

Cadmium (Cd) toxicity was produced in male rats to study the role of cholinoceptors in Cd-induced endothelial dysfunction. The changes in the tension of the aortic rings to constrictor and dilator agonists were compared with those of controls. A Cd-induced significant increase in phenylephrine response was associated with a decrease in basal dilator prostanoid release. In Cd-exposed rings, despite an obvious depression in the acetylcholine (ACh) response, the receptor-independent dilation to the calcium ionophore A23187, which elicits a receptor-independent endothelial relaxation, was slightly elevated (p<0.01), but the smooth muscle cell response to the NO donor, sodium nitroprusside (SNP) remained unaltered. Cadmium decreased both the maximal response to ACh (10(-5) M) and its pirenzepine (Prz) sensitive component. The M1 type cholinoceptor-mediated response to ACh decreased in Cd-exposed rings to 10.30 +/- 5.00% from 38.40 +/- 6.90% (p<0.001). Cadmium also reduced the share of indomethacin 1.64% to 13.92 +/- 2.89% (p<0.01), which correlated well with the changes in the M1-mediated response (r=0.991, p<0.0001). Most of the deleterious effect of Cd appears to be restricted to the M1-dependent ACh response. These findings suggest that Cd produces an endothelial dysfunction by impairing the M1 type cholinoceptor mediated response, which seems to be involved in prostanoid release.

Evaluation of 5-lipoxygenase inhibitors, zileuton, A-78773 and ICI-D-2138 in an ionophore (A-23187)-induced pleural inflammation model in the rat.

Intrapleural injection of A-23187 (10 micrograms), a calcium ionophore, elicited rapid increase in biosynthesis of prostaglandins and leukotrienes in a time-dependent manner. 6-Keto-prostaglandin-F1 alpha (6-KPA) was the principal cyclooxygenase product with modest increases in levels of thromboxane B2 and prostaglandin-E2. Orally administered indomethacin, a selective cyclooxygenase inhibitor, and three selective 5-lipoxygenase inhibitors, zileuton, A-78773 and ICI-D-2138 markedly attenuated respective arachidonate pathways with projected ED50 values of < 1-2 mg/kg. Furthermore, a single oral administration of either ICI-D-2138 or A-78773 (each 20 mg/kg, po) resulted in persistent inhibition of 5-lipoxygenase pathway for up to 24 hr. These results indicate zileuton, A-78773 and ICI-D-2138 to be potent and selective inhibitors of 5-LO and document the utility of A-23187-induced pleural inflammation in evaluating efficacy of inhibitors of arachidonic acid metabolism in vivo.

Duck lymphocytes--V. Transformation responses to phorbol ester and calcium ionophore.

1. Lymphocytes purified from duck blood and spleen were cultured in the presence of phorbol 12-myristate 13-acetate (PMA) and the calcium ionophore A23187. Stimulation was assessed by the incorporation of [3H]thymidine after 3 days' culture. 2. PMA stimulated over a wide range of concentrations, with maximum stimulation at final concentrations of 5 x 10(-7)-5 x 10(-8) M/litre. A23187 was effective in the range 5 x 10(-6)-5 x 10(-7) M/litre and also, in some experiments using spleen lymphocytes, at 5 x 10(-11)-5 x 10(-12) M/litre. 3. Synergism was observed between PMA and A23187, the pattern depending on the concentrations of these reagents employed. Synergism was also observed between PMA and suboptimum concentrations of phytohaemagglutinin (PHA), wheat germ agglutinin (WGA), pokeweed mitogen (PWM) and Bandeiraea simplicifolia seed extract (BSS), but not with concanavalin A (Con A), lentil lectin (LL) or Helix pomatia lectin (HP). Similarly, synergism occurred between A23187 and WGA or PWM, but not with PHA, BSS, Con A, LL or HP. 4. Mitomycin C and cycloheximide inhibited the response of duck lymphocytes to PMA, A23187 and lectins. Cyclosporin A inhibited responses to lectins but not to PMA or A23187. Neither hydrocortisone nor indomethacin inhibited responses to lectins, PMA or A23187. 5. These results indicate that activation of duck lymphocytes occurs by virtue of similar intracellular messenger pathways to those operating in mammalian lymphocytes.

A radioautographic study of [3H]-A 23187 (calcimycin) in components of the brain. Distribution in different cell types of the diencephalo-mesencephalic roof.

The distribution of the ionophore [3H]-A 23187 was examined by means of light and electron microscopy in elements of the central nervous system located in the diencephalo-mesencephalic roof. A 23187 is not evenly distributed in the components studied (ependyma, secretory ependyma of the subcommissural organ and neurons of the mesencephalon). At the cellular level, A 23187 appears preferentially associated with the cytoplasmic membrane as well as with the internal membranous system.

How to evaluate the distribution of an "invisible" amphiphile between biological membranes and water.

To evaluate the distribution of an amphiphile or its binding to membranes whose properties are affected by such binding, it is only necessary to establish to what extent the dose-response to the amphiphile depends on the membrane concentration. The measured response only needs to reflect local events. This method of evaluation does not depend on the precise shape of the dose-response curve and is particularly useful for amphiphiles devoid of properties like fluorescence or radioactivity which would allow their direct assay. In this work, we establish the validity of this approach by comparing it with direct conventional determinations. Two parameters are especially suitable for such evaluation: the perturbation of an enzyme's activity, produced by many amphiphiles, and the fluorescence quenching of membrane-embedded proteins by chromophoric amphiphiles through long-range Förster transfer. We illustrate this approach in sarcoplasmic reticulum membranes containing Ca2(+)-ATPase as the main protein constituent. The equilibrium distribution of the antioxidant 4-nonylphenol was deduced from its inhibition of ATPase activity, whereas the equilibrium distribution of the calcium ionophore calcimycin (A23187) and of its brominated analog 4-bromo-A23187 were determined from their quenching of ATPase fluorescence. Apparent partition coefficients K* in the range of 10(5) (expressed as (moles of lipid/liter)-1) were obtained for these highly hydrophobic molecules.

Receptor-mediated stimulation of taurocholate efflux from the rat hepatocyte and the ex vivo perfused rat liver.

The peptide hormone, arginine-vasopressin[( Arg8]vasopressin, AVP), stimulates efflux of the bile salts taurocholate and glycocholate from the rat hepatocyte in suspension via its association with the V1 receptor on the hepatic cell membrane. At a concentration ratio of 5:1 (antagonist to hormone), the V1 vasopressin antagonist, (dCH2)5Tyr(Me)AVP, inhibits the vasopressin induced efflux of taurocholate by approximately 82%, and of glycocholate, by approximately 85%. In contrast, the V2 antagonist (d(CH2)5[D-Ile2,Ala4]AVP, does not interfere with the stimulation of taurocholate and glycocholate efflux by vasopressin. In the isolated perfused rat liver, vasopressin (5 X 10(-10) M) causes an immediate increase of 55 +/- 12% over baseline in [14C]taurocholate secretion and a corresponding increase in bile flow. A more gradual and prolonged increase in [14C]taurocholate secretion, reflecting an increased biliary concentration of [14C]taurocholate, is observed beginning 6 min after vasopressin, reaching a plateau of 23 +/- 12% over baseline by 14 min and returning to baseline by 30 min. The mean rate of 14C secretion during the 30 min following administration of vasopressin (non-steady state) is increased by 14.3 +/- 6.4% over pre-infusion steady-state baseline (P less than 0.05). Prior administration of the V1 receptor antagonist d(CH2)5Tyr(Me)AVP attenuates these effects of vasopressin. The combination of these in vitro and in vivo findings suggest that vasopressin may play a role in regulating bile salt efflux. Furthermore, these studies in the isolated hepatocyte and the intact liver may provide a unique approach for defining biochemical changes associated with bile salt transport from the hepatic cell.

Tumor necrosis factor-enhanced leukotriene B4 generation and chemotaxis in human neutrophils.

In an in vivo study of five normal volunteers infused with endotoxin (20 U/kg of US reference endotoxin lot EC-5), increased neutrophil (PMN) generation of leukotriene B4 and chemotaxis to leukotriene B4 were found concomitantly with elevated plasma tumor necrosis factor (TNF) levels. To clarify the role of TNF in PMN activation, neutrophil responsiveness after in vitro treatment with TNF was examined. Neutrophils from seven normal subjects were incubated with TNF for 30 minutes and tested for chemotaxis to leukotriene B4, formyl-methionyl-leucyl-phenylalanine and zymosan-activated serum, or the calcium ionophore A23187 to assess leukotriene B4 generation. A range of 10(-13) to 10(-9) mol/L of TNF was used for these assays. When 10(-9) mol/L of TNF was used, the amount of leukotriene B4 that was produced was significantly greater than in control cells. The effect of TNF on PMN chemotaxis was uniformly inhibitory for the three stimuli at 10(-10) mol/L compared with untreated cells. At a picomolar range, PMN migration to leukotriene B4, but not to zymosan-activated serum or formyl-methionyl-leucyl-phenylalanine, was significantly increased over that of PMNs not exposed to TNF. This suggests that TNF has a specific facilitatory effect on PMN responsiveness for both leukotriene B4 production and chemotaxis to leukotriene B4 and may be the same signal for this phenomenon in endotoxemic patients.

Comparison of signals delivered through CD3 and CD2 for T-cell activation: the role of calcium influx and interleukin 1.

In the absence of monocytes, resting T lymphocytes extensively purified from human peripheral blood failed to proliferate when stimulated with a mixture of calcium ionophore, which elevates intracellular calcium levels, and TPA, which activated protein kinase C. A third signal, i.e., the triggering via CD3 or CD2 molecules, was necessary in order to observe proliferation. These highly purified T cells required the presence of monocytes in both CD3 and CD2 systems for their proliferation. Exogenous interleukin 1 clearly substituted for monocytes in CD2- but not in CD3- triggered T-cell proliferation. In contrast, the effect of CD2 and CD3 antibodies on Ca++ influx was apparently not dependent on the presence of monocytes. In the presence or absence of the monocytes, CD3, as well as certain combinations of CD2 monoclonal antibodies including the D66 monoclonal antibody, were able to increase the intracellular calcium concentration as measured by Quin 2 fluorescence. EGTA, a Ca++ chelator, completely inhibited CD2- and CD3- mediated T-cell proliferation, indicating that calcium uptake is necessary during the T-cell proliferation. The addition of TPA abrogated the inhibitory effect of EGTA and completely restored the response of the T cells stimulated by CD3, but not by CD2, monoclonal antibodies. In the CD2 pathway, EGTA-inhibited proliferation of T cells could be completely restored by addition of exogenous interleukin 2 as well as exogenous recombinant interleukin 1. Our results indicate that EGTA inhibits the production of interleukin 1 but has no direct effect on either interleukin 2 production or on Tac antigen expression. In this system, recombinant interleukin 1 alpha demonstrated a more potent ability for restoring the T-cell response than did recombinant interleukin 1 beta. These results suggest that interleukin 1 could act as a potent costimulatory factor in the non-antigen-specific T-cell activation.