Comparison of immunoglobulin formation in vitro by leukocytes of normal donors and steroid- and nonsteroid-treated asthmatic patients

To characterize further our observed differences between asthmatic patients and normal adults with regard to lymphocyte immunoglobulin (Ig) formation in vitro, we studied the response of lymphocytes from steroid-treated asthmatic patients to hydrocortisone, isoproterenol, and aminophylline. Cells from these donors showed no response to either hydrocortisone or aminophylline, but significantly enhanced Ig formation with isoproterenol (10^?9 to 10^?6 M). Experiments with lymphocytes from nonsteroid-treated asthmatic patients showed no response to isoproterenol, but significantly enhanced Ig formation with aminophylline (10^?7 to 10^?4 M). Corresponding experiments with cells from normal adults showed significantly enhanced Ig formation with both isoproterenol (10^?9 to 10^?6 M) and aminophylline (10^?7 to 10^?5 M). To explore the possible mediation of Ig formation by cyclic 3′,5′-adenosine monophosphate (cAMP), we studied: (1) combinations of aminophylline and isoproterenol, (2) cAMP, and (3) dibutyryl cAMP. Cells from nonsteroid-treated asthmatic patients showed significantly enhanced Ig formation with combinations of aminophylline and isoproterenol, and this enhanced Ig formation was greater than that observed with aminophylline alone. In similar experiments with cells from normal subjects, the enhanced Ig formation with the combinations was of the same magnitude as that with aminophylline alone. Cells from nonsteroid-treated asthmatic patients showed no response to cAMP. Cells from normal subjects had significantly enhanced Ig formation with two concentrations of cAMP (10^?6 and 10^?5 M) and no response to dibutyryl cAMP. Although our data do not add substantial support to the possible mediation of Ig formation by cAMP, they do demonstrate striking differences in response of lymphocytes from asthmatic patients and normal subjects.     

Effect of hydrocortisone and isoproterenol on immunoglobulin synthesis by peripheral blood lymphocytes of patients with asthma

Suspensions of peripheral lymphocytes from asthmatic patients (not receiving steroid therapy) were briefly exposed to various concentrations of hydrocortisone, isoproterenol, or 10⁻⁷M hydrocortisone plus isoproterenol and incubated for 72 hours at 37 °C. After this incubation, the amount of immunoglobulin (Ig) synthesized and secreted into the culture supernatants was quantitated by radioimmunoassay. In contrast to our data with normal human peripheral lymphocytes, the lymphocytes from asthmatic patients were markedly hyporesponsive to hydrocortisone, isoproterenol, and 10⁻⁷M hydrocortisone plus isoproterenol. Significantly increased Ig formation compared to control levels was observed only with 10⁻⁶M hydrocortisone. The amount of Ig formation observed with all the other concentrations of the compounds was not significantly different from the control values.     

Modulation by 310-1310-1310-1stimulation of IgE-mediated14C-serotonin release from rat mast cells

The formaldehyde method was used to examine the effects of clonidine and methoxamine on IgE-mediated¹⁴C-serotonin release from rat mast cellsin vitro. Clonidine (10^?11?10^?8 M) caused dose-related enhancement of the mediator release 7min after the antigen challenge yohimbine (10^?6 M) blocked this enhancement by clonidine (10^?6 M), but prazosin (10^?6 M) did not. Methoxamine did not enhance this immunological release reaction at concentrations up to 10^?6 M. PGE₁ (2×10^?8?2×10^?5 M), isoproterenol (10^?10?10^?8 M), dopamine (4×10^?8?4×10^?8 M) and aminophylline (6×10^?6?6×10^?4 M) caused dose-related inhibition of this mediator release 1 min after antigen challenge. Clonidine (10^?13?10^?12 M), but not methoxamine (10^?8?10^?6 M), reversed dose-dependently this inhibition of mast cells by PGE₁ (2×10^?6 M), isoproterenol (10^?8 M), dopamine (4×10^?6 M); yohimbine (10^?8 M) antagonized this reversing action of clonidine (10^?12 M), but prazosin (10^?10 M) did not. Neither clonidine (10^?14?10^?11 M) nor methoxamine (10^?8?10^?6 M) reversed the inhibitory action of aminophylline (2×10^?4 M). These results suggest that clonidine enhances IgE-mediated¹⁴C-serotonin release from rat mast cells and also antagonizes the inhibition of mast cells by PGE₁, isoproterenol and dopamine, but not by aminophylline in this immunological reaction through α₂-adrenergic receptors, and that the inhibition of adenylate cyclase of mast cells is one of the biochemical actions of α₂-adrenergic mechanisms.     

Regional difference in lipolysis caused by a β-adrenergic agonist as determined by the microdialysis technique

We have investigated the difference in lipolysis caused by a β-adrenergic agent between visceral and abdominal subcutaneous adipose tissues in vivo. Glycerol levels (lipolysis index) were continuously monitored in mesenteric and abdominal subcutaneous adipose tissues of anaesthetized Wistar rats using the microdialysis technique. During microdialysis, increasing concentrations of the lipolytic agent, isoproterenol (10^?8, 10^?7, 10^?6, 10^?5 mol L^?1), were added to the perfusion. Glycerol concentrations in dialysate at each isoproterenol concentration, blood glucose concentrations during the experiment, and plasma insulin concentrations before and immediately after the experiment were measured. The effect of isoproterenol on local blood flow was investigated using the ethanol technique. The clearance rate of ethanol from the perfusion medium was used as the index of local blood flow. There was no significant change in blood glucose or plasma insulin concentrations during the study. Glycerol levels in dialysate were significantly higher in mesenteric than in abdominal subcutaneous adipose tissues at all isoproterenol concentrations. The percentage change of baseline ethanol ratio was not altered by increasing isoproterenol concentrations in both mesenteric and subcutaneous adipose tissues. There was also no significant difference in percentage change of the baseline ethanol ratio between mesenteric and abdominal subcutaneous adipose tissues. These results suggest that mesenteric adipose tissue is characterized by an even higher β-adrenergic agonist-induced lipolysis than abdominal subcutaneous adipose tissue.     

Interactions of morphine with PGE1, isoproterenol,dopamine and aminophylline in rat mast cells; their effect on IgE-mediated14C-serotonin release

The formaldehyde method was used to examine the interactions of morphine with PGE₁, isoproterenol, dopamine and aminophylline in rat mast cells by their effects on IgE-mediated¹⁴C-serotonin release. PGE₁ (2×10^–8–2×10^–5 M), isoproterenol (10^–10–10^–8 M), dopamine (4×10^–8–4×10^–6 M) and aminophylline (6×10^–6–6×10^–4 M) caused dose-related inhibition of the mediator release 1 min after an antigen challenge, and propranolol (10^–7 M) blocked the inhibition by isoproterenol (10^–8 M) but not that by dopamine (4×10^–6 M), while haloperidol (4×10^–6 M) blocked that by dopamine (4×10^–6 M) but not that by isoproterenol (10^–8 M). Morphine (3×10^–7–3×10^–5 M) reversed the inhibitory effects of PGE₁ (2×10^–6 M), isoproterenol (10^–8 M) and dopamine (4×10^–6 M) dose-dependently and stereospecifically; naloxone (2×10^–4 M) antagonized these reversing actions of morphine (3×10^–5 M). Morphine (10^–6–10^–4 M) did not reverse the inhibitory action of aminophylline (6×10^–4 M). These results suggest that the inhibitory responses of mast cells to PGE₁, isoproterenol and dopamine but not to aminophylline in immunological mediator release were reversed by morphine through opioid receptors, and that the inhibition of adenylate cyclase in mast cells is one of the biochemical actions of morphine.     

Failure of catecholamines to inhibit epidermal mitosis in vitro

Skin samples were obtained from the forearm and leg of normal volunteers and patients with atopic eczema. The samples were incubated with ³H-thymidine, and the radioactivity incorporated was measured at 48 hours. Isoproterenol, epinephrine, or norepinephrine was added in concentration of 10^?5M to 10^?10M. There was inhibition of deoxyribonuclear acid (DNA) synthesis in control samples with the catecholamines. In contrast, the catecholamines failed to inhibit DNA synthesis in the skin from patients with atopic eczema. It was concluded that inhibition of DNA synthesis is a beta adrenergic effect, and the cells of patients with atopic eczema are insensitive to beta stimulation.     

Cyclic AMP metabolism in asthma: studies with leukocytes and lymphocytes

The effect of isoproterenol on cyclic AMP (cAMP) levels in lymphocytes and leukocytes from asthmatic and normal individuals has been studied. Lymphocyte cAMP levels rose in response to 10^?8 to 10^?2 M isoproterenol; the dose-response curve was biphasic with a maximum response at concentrations of 10^?6 to 10^?4 M. At all drug concentrations the response of cells from asthmatic individuals was less than the response of cells from normal control subjects. The difference between the two groups was not statistically significant, however. Similarly, basal cAMP levels were lower in the cells of asthmatic as compared with normal individuals, but again the difference was not significant. When these two parameters were combined and the results expressed as absolute cAMP levels after isoproterenol treatment, a significant difference was observed. Both the unstimulated. cAMP levels and the response to isoproterenol of leukocytes from normal and asthmatic subjects were influenced by the incubation medium. Use of a medium buffered with tris as compared with bicarbonate-phosphate resulted in lower basal cAMP levels and increased responsiveness to isoproterenol. Treatment of normal leukocytes with serum from asthmatic individuals did not alter their response to isoproterenol.     

Inhibition of ACTH-induced differentiation of cortical cells and their mitochondria by corticosterone in tissue culture of fetal rat adrenals

Tissue cultures of fetal rat adrenals were used to study the effects of corticosterone on the ACTH-induced ultrastructural differentiation of cortical cells and their mitochondria. Corticosterone in dosages of 0.2, 2.0, 5.0, 10, and 20 μg/ml (corresponding to concentrations of 6 × 10^?7, 6 × 10^?6, 1.5 × 10^?5, 3 × 10^?5, and 6 × 10^?5 molar) was added alone or together with 100 mU/ml of ACTH to the culture medium, daily from the sixteenth day of cultivation up to and including the twenty-first day. Corticosterone alone induced no ultrastructural changes in cortical cells. Corticosterone in concentrations of 6 × 10^?7 to 3 × 10^?5 M given with ACTH induced hypertrophy of Golgi apparatus. Corticosterone in concentrations of 6 × 10^?5 M inhibited the ACTH-induced differentiation of cortical cells. However, the nuclear chromatin increased and Golgi apparatus was strikingly hypertrophied. Mitochondria often aggregated adjacent to the nuclear envelope but their ultrastructure remained undifferentiated with tubular or tubulovesicular cristae. Ribosomes appeared as single particles. A marked increase of smooth surfaced endoplasmic reticulum was noted also in cortical cells treated with 6 × 10^?5 M of corticosterone. The present observations suggest that corticosterone acts as an intracellular inhibitor in cortical cells. It appears to inhibit cytoplasmic protein synthesis at the ribosomal level and prevents synthesis of cytoplasmic mitochondrial protein synthesis stimulating factor and the latter, in turn, inhibits the activation of mitochondrial protein synthesis. A new model is presented to explain the regulation of growth and secretion in the adrenal cortex.     

The effect of sympathomimetic drugs on immediate skin reactions and metabolic responses in asthmatic patients

The varied effects of intradermal injection of isoproterenol and propranolol on the immediate skin reaction were studied in relation to the metabolic responses to the intravenous injection of epinephrine. In asthmatic patients whose skin reaction was not suppressed with 10(-7) M isoproterenol, the hyperglycemic response after the injection of epinephrine was significantly reduced.     

17β‐Estradiol Exposure Accelerates Skeletal Development in Xenopus laevis Tadpoles

Although it is well established that estrogen regulates skeletal growth and ossification in mammals, the effects of estrogen on skeletal development in amphibians are relatively uncharacterized. This study was conducted to characterize the impact of 17β‐estradiol exposure on skeletal development in Xenopus laevis tadpoles. On day 48 postfertilization, tadpoles were placed in tanks containing 50% Holtfreter's Solution ±17β‐estradiol at one of four concentrations (10^?11, 10^?10, 10^?9, and 10^?8 M). At 7–11 day intervals until day 91, 7–10 tadpoles per group were killed, fixed, measured, and staged. Specimens were then cleared and double‐stained for cartilage and bone, and 34 skeletal elements were analyzed for ossification. Results from the study indicate that both low (10^?11 M) and high (10^?8 M) concentrations of 17β‐estradiol have a significant stimulatory effect on tadpole development. Both the larval stage and ossification index of tadpoles exposed to 10^?11 or 10^?8 M 17β‐estradiol were significantly greater than those observed in control animals by day 91 postfertilization. These results are consistent with the hypothesis that endogenous and exogenous estrogen could play a role in the regulation of bone ossification in amphibians. Anat Rec, 2010. © 2010 Wiley‐Liss, Inc.     

Exhaustive exercise reduces TNF-α and IFN-α production in response to R-848 via toll-like receptor 7 in mice

Stressful exercise results in temporary immune depression. However, the impact of exercise on the immune responses via toll-like receptor (TLR) 7, which recognizes the common viral genomic feature, single-stranded RNA, remains unclear. To clarify the effect of stressful exercise on immune function in response to viral infection, we measured the changes in the plasma concentration of tumor necrosis factor (TNF)-α and interferon (IFN)-α, which are induced downstream from the TLR–ligand interaction, in exhaustive-exercised mice immediately after treatment with the imidazoquinoline R-848, which can bind to and activate TLR7. Both exhaustive-exercised (EX) and non-exercised (N-EX) male C3H/HeN mice were injected with R-848 (5 mg kg^?1), and blood samples were collected. In addition, RAW264 cells, which are mouse macrophage cells, were cultured 30 min after epinephrine (10 μM) or norepinephrine (10 μM) treatments, and were then stimulated with R-848 (10 μg ml^?1). In addition, the effect of propranolol (10 mg kg^?1) as blockade of β-adrenergic receptors on R-848-induced TNF-α and IFN-α production in the exercised mice was examined. Both the TNF-α and IFN-α concentrations in the plasma of EX were significantly lower than those in the plasma of N-EX after R-848 injection (P < 0.05 and P < 0.01, respectively), although the R-848 treatment increased the plasma TNF-α and IFN-α concentrations in both groups (P < 0.01, respectively). The R-848-induced TNF-α production in RAW264 cells was significantly inhibited by epinephrine and norepinephrine pre-treatment, although IFN-α was not detected. The propranolol treatment completely inhibited exercise-induced TNF-α and IFN-α suppression in response to R-848 in the mice. These data suggest that EX induces a reduction in TNF-α and IFN-α production in response to R-848, and that these phenomena might be regulated by an exercise-induced elevation of the systemic catecholamines.     

Characterization of granulocyte beta adrenergic receptors in atopic eczema

The binding of (?)-³H-dihydroalprenolol (³H-DHA) and ¹²⁵I-hydroxybenzylpindolol (¹²⁵I-HYP) to the β-adrenergic receptors in homogenized granulocyte preparations from control subjects and patients with atopic eczema was characterized. No difference was found for the affinity (K_D = 2 × 10^?9 M) or the total number of high-affinity binding sites (1,200 to 1,600 per cell) with ³H-DHA or ¹²⁵I-HYP (K_D 1.6 × 10^?10 M) in granulocytes from the two study populations. Scatchard plots of the data obtained from DHA binding isotherms suggested that negative cooperativity may exist as a property of the β₂-receptor. Granulocyte preparations from control subjects and patients with atopic eczema also showed propranolol protectable ³H-DHA binding with a K_D of 10^?7 M. It is not clear whether these ³H-DHA binding sites represent physiologically significant β-adrenergic receptors at these concentrations of radioligand. It was found that 15,000 to 20,000 binding sites of this lower affinity for DHA exist per cell in granulocytes from the two populations of subjects. These data suggest that the reduced isoproterenol responsiveness of granulocytes from patients with atopic eczema is not the result of abnormal or reduced numbers of β-adrenergic receptors.     

Human Fetal/Neonatal Suppressor Activity: Relation Between OKT Phenotypes and Sensitivity to Prostaglandin E2 in Maternal and Neonatal Lymphocytes

ABSTRACT: T lymphocytes from human fetuses and newborns strongly and spontaneously suppress various adult cell functions (i.e. T-cell proliferation, B-cell differentiation, and Ig synthesis). The precise phenotype of the suppressor cell is controversial. In this investigation we use cord T-cell subsets negatively selected by the panning technique or by complement-mediated lysis using the monoclonal antibodies OKT4 and OKT8. Cord T cells deprived of the OKT4⁺ subpopulation exerted only a marginal suppressor activity (12 ± 7 as compared to 73 ± 4% of unfractionated T cells) on the proliferation of maternal cells in our PHA-stimulated co-culture assay using sex chromosomes as markers for dividing cord (male) and maternal cells. The suppressive effect was direct, i.e. not mediated by induction of maternal OKT8⁺ suppressor effector cells. Cord and maternal T-cell subsets were also tested for their sensitivity to exogenous prostaglandin E₂ (PGE₂) at doses varying between 1.4 × 10^?5 and 1.4 × 10^?9 M. Both maternal OKT4^? and OKT8^? T-cell subsets were highly sensitive to suppression by PGE₂. In contrast, cord OKT8^? T cells were essentially nonsensitive at all doses of PGE₂ used, whereas cord OKT4^? T cells were significantly suppressed at four out of five concentrations tested (1.4 × 10^?6 through 1.4 × 10^?9). Our results suggest a direct correlation between the phenotypes of the cord-suppressor and maternal-target T cells and their sensitivity to PGE₂.     

Reversible restriction of vesicular stomatitis virus in permissive cells treated with inhibitors of prostaglandin biosynthesis

Indomethacin, a potent nonsteroidal inhibitor of prostaglandin synthetase (cyclooxygenase) reduced yields of infectious vesicular stomatitis virus in HEp-2 cells more than 99% if added to cultures at levels of 10^?3M either before or after infection. Other permissive cell lines differed according to the treatment period and drug level required for restricting productive infections. The inhibitory effect of indomethacin was progressively reduced if infection of cells was delayed for increasing times after drug removal. Strong inhibition of viral replication also occurred in cells treated with the cyclooxygenase antagonists naproxen, phenylbutazone, and oxyphenylbutazone whereas phenacetin, which does not block cyclooxygenase function, was inactive. Enhanced viral replication occurred in indomethacin-treated HEp-2 cultures when these cells were subsequently exposed to such substances as prostaglandin El, cyclic AMP, or insulin. Conversely, indomethacin-treated cells remained restrictive for VSV if they were subsequently exposed to metabolic inhibitors of functional DNA (actinomycin D or mitomycin C), messenger RNA synthesis (α-amanitin), or protein synthesis (cycloheximide) at concentrations that normally do not compromise viral replication. Pretreatment of HEp-2 cells with mitomycin C markedly shifted the dose response for indomethacin-mediated inhibition of VSV from a 90% inhibitory dose of about 10^?4 M to one of 10^?9 M or lower. These findings suggest that preexisting host factors essential for replication of VSV, although rendered nonfunctional by the drug indomethacin, can be replenished unless their synthesis is blocked by various classes of metabolic inhibitors.     

Regulatory effect of hydrocortisone on the in vitro synthesis of IgE by human lymphocytes

The effect of different concentrations of hydrocortisone (HC) on the in vitro, pokeweed mitogen-driven synthesis of IgG and IgE by human lymphocytes was studied. HC had the same modulatory action on the production of both immunoglobulins. Two different effects were observed: the first is enhancement of Ig synthesis by low concentrations of HC (10(-7)-10(-4) M), and the second is inhibition of synthesis by higher concentrations due to lymphocytotoxic effect.     

Cardiac effects of the new H2-receptor antagonists

A series of new H₂-receptor antagonists were tested for their effects on different isolated heart preparations. In the guinea-pig atria and papillary muscle the inhibitory effect on histamine H₂-receptors was evaluated. In the perfused rabbit heart and in strips of human atria the effect of the H₂-antagonists on the spontaneous or electrically-stimulated contractions was evaluated. In the first two preparations some main quantitative differences were pointed out, tiotidine and compound SKF 93479 being the most potent antagonists, cimetidine, metiamide and ranitidine the less effective. In the rabbit heart and in human atria results were quite different: cimetidine and ranitidine were virtually ineffective up to the maximum concentration tested (3×10^?3 M), oxmetidine and compound SKF 93479 had a negative inotropic and chronotropic effect starting from concentrations of 3×10^?6?10^?5 M. On the basis of the behaviour of other compounds endowed with negative cardiac effects (propranolol, anaesthetic-like compounds, verapamil) and of that of compounds capable of counteracting the effect of oxmetidine (increased concentration of calcium ions and isoproterenol) it was hypothesized that oxmetidine may interfere in the transport of calcium ions. Our data emphasize the importance of the different structure of the H₂-antagonists in determining non-specific effects absolutely independent of the primary action that is the H₂-receptor blockade.     

Detection of opiate-enhanced increases in DNA damage,HPRT mutants,and the mutation frequency in human HUT-78 cells

In previous studies we have shown highly significant increases in chromosome damage and sister chromatid exchanges in heroin addicts, particularly when caffeine and metabolic inhibitors are added to the medium. Using human HUT-78 T-cell cultures, we now find direct in vitro evidence of opiate-induced or opiate-promoted mutagenesis via several assay systems. First, with microgel electrophoresis (MGE), we observed graded, dose-dependent, significant increases (P < .0001) in the frequency of comet tails of fragmented DNA when cells were treated with morphine alone (5 × 10^?9M up to 10^?7M) or when co-treated with the more potent mutagen, ethylmethanesulfonate (EMS). There were also dose-dependent increases in the lengths and densities of the comet tails observed. These findings were confirmed by a series of MGE experiments in which several days of morphine exposure preceded a 2-hr pulse of EMS. Second, mutant frequency (MF) assays also indicated significant opiate effects. These studies required separate assessment of cloning efficiencies and the frequencies of TG-resistant, HPRT-deficient mutant clones under four test conditions: no treatment, morphine alone for 4 days, morphine plus EMS, and EMS alone. Prior to the treatment phase, aminopterin was used to eliminate background HPRT mutations. The medium was changed after the treatment phase, the cells were allowed to express mutant pheno-types, and then TG was added and resistant mutant clones counted after 16 days. The background MF level for controls and for cells treated with EMS alone were negligible at 5.12 × 10^?8 and 7.25 × 10^?8, respectively. In the cells treated with morphine alone or morphine plus EMS, MF levels increased very significantly (P < .001) by >100-fold to 5.1 × 10^?6 and 7.0 × 10^?6, respectively. Cloning efficiency also decreased significantly with both morphine-exposed conditions. Preliminary analysis with the single strand conformational polymorphism (SSCP) procedure following 6-thioguanine (TG) selection, also confirmed the occurrence of Exon 3 mutants of the HPRT gene in cells exposed to morphine plus EMS. It appears that brief EMS exposure can be repaired, whereas, if morphine exposure persists through one or more cell cycles, direct or indirect mutagenesis is initiated. © 1994 Wiley-Liss, Inc.     

Specific uptake of tritiated serotonin in the adult rat pancreas: Evidence for the presence of serotonergic fibers

Fragments of adult rat pancreas were incubated in vitro with tritiated serotonin at concentrations from 10^?8 to 10^?7 M. The pancreas exhibited an uptake of serotonin which was saturable, with an uptake constant (Km) of 8.75 × 10^?7 M, and a Vmax of 873 pmoles per gram. Specificity was determined by the addition of fluoxetine or norepinephrine to the reaction mixture, both at 10^?5 M. Fluoxetine significantly reduced the ³H-5HT uptake, whereas norepinephrine was without effect. Metergoline (10^?6 M), a specific 5-HT postsynaptic receptor blocker, similarly had no effect on the serotonin uptake in the pancreas. Radioautography of the fragments following uptake of tritiated serotonin (5 × 10^?8 M) revealed silver- grain aggregates dispersed along blood vessels in the interstitial spaces of the exocrine and endocrine pancreas, areas known to be traversed by nerve fibers. There were no silver- grain aggregates over the exocrine or islet parenchymal cells. These data support the hypothesis that the pancreas is innervated by serotonergic fibers. Further evidence for this hypothesis was provided by a preliminary study demonstrating the presence of tryptophan hydroxylase in pancreatic homogenates. These serotonergic fibers may be involved in the regulation of pancreatic secretion.     

Stimulation of protein phosphatases as a mechanism of the muscarinic-receptor-mediated inhibition of cardiac L-type Ca2+ channels

Acetylcholine decreases currents through cardiac L-type Ca²⁺ channels after stimulation with agents which elevate levels of cyclic adenosine monophosphate, such as isoproterenol, but there is still a controversy over the mechnisms of this muscarinic effect. We tested the hypothesis of whether, after isoproterenol stimulation, protein phosphatases are activated by acetylcholine. Whole-cell currents were recorded from guinea-pig ventricular myocytes. The effect of 10^–5 M acetylcholine on currents induced by 10^–8 M isoproterenol was studied in the absence or presence of protein phosphatase inhibitors. Three agents reduced the acetylcholine response: okadaic acid (3 or 9 · 10^–6 M) and cantharidin (3 · 10^–6 M) added to the pipette solution, and bath-applied fluoride (3 mM). In contrast, pipette application of other phosphatase inhibitors, namely the inhibitor PPI₂ (1000 U/ml), ciclosporin (10^–5 M), or calyculin A (10^–6 M) did not significantly diminish the acetylcholine effect. Interestingly, there was no correlation between the effects of the compounds on basal Ca²⁺ current and their interference with the muscarinic response. An activation of type 2A phosphatases by acetylcholine would explain these findings. Indeed, okadaic acid is 3 orders of magnitude more potent in vitro in its inhibition of this isoform (purified from cardiac myocytes) than is calyculin A, while type-1 phosphatases are inhibited equally. The data support the attractive possibility that stimulation of protein phosphatases is part of the signal transduction cascade of Ca²⁺ channel inhibition by acetyl-choline.     

Steroid Sex Hormones and Macrophage Function: Modulation of Reactive Oxygen Intermediates and Nitrite Release

PROBLEM: In general, females have a more active immune response than do males. The effects of female sex hormones on lymphocytes have been studied extensively but their effects on macrophages are poorly understood. METHOD: In this study, peritoneal macrophages (M0) obtained from male rats were treated in vitro with estradiol (E₂), progesterone (P), testosterone (TS), or hydrocortisone (HC) and their effects on superoxide, hydrogen peroxide, and nitrite release determined. RESULTS: At concentrations between 10^?10 and 10^?9 M, female and male sex hormones had no significant effect on superoxide release but, at concentrations above or below that range, these hormones stimulated the release of these reactive oxygen intermediates (ROI). In contrast, M0 treated with HC generally exhibited either unaltered or reduced ROI release. CONCLUSIONS: These findings suggest that female sex hormones regulate ROI release by Mø in a manner not entirely shared by other steroid hormones. At most concentrations used, E₂, P, TS, and HC significantly inhibited nitrite release by Mø. However, with 10^?10 M of E₂ or 10^?9M of P, nitrite release by Mø was not affected. In the presence of anti-TNF antibody, the amounts of superoxide and hydrogen peroxide release were moderately reduced but nitrite release was dramatically inhibited. The sensitivity of Mø to variations in the concentrations of female sex hormones may contribute to gender-related differences in the immune response.