μM Propranolol Research Articles

Cardiotonic and sedative effects of Cecropia pachystachya Mart. (ambay) on isolated rat hearts and conscious mice

Cecropia pachystachya Mart. is popularly called “ambay” and extensively used in herbal medicine of South America for cough and asthma. In Argentina it grows in neotropical rainforest ( Ntr C.p.) and in a temperate region ( Tp C.p.). In a previous work we showed their hypotensive properties with different potency and toxicity, and now we studied the Tp C.p. effects in isolated heart from rats and central effects of both plants on the open-field test for mice. Tp C.p. produced a positive inotropic effect on isolated rat hearts, which was not affected by 1 μM propranolol, suggesting that it is not due to a β-adrenergic effect. In contrast, it was prevented by pretreatment with high [ K] o media, which stimulates the Na,K-ATPase pump, suggesting an inhibition of the pump by “ambay”, as digital do. In the open-field test, both Ntr C.p. and Tp C.p. similarly decreased the spontaneous locomotion and exploratory behavior of mice at doses between 180 and 600 mg/kg. Ntr C.p. potentiated the effect of 3 mg/kg diazepam to one similar to 10 mg/kg diazepam, but was not antagonized by 0.5 mg/kg flumazenil. Amphetamine at 5 mg/kg prevented the sedative effect of Ntr C.p. Chromatographic analysis showed that both plants have a qualitatively similar fingerprint but quantitatively differed in at least three components. Although the purpose was not to identify them, both plants have at least 10 compounds. Two of them were in higher amount in Tp C.p. than in Ntr C.p., and then, they could be responsible for the cardiovascular toxicity of Tp C.p. In conclusion, the results suggest that ambay has cardiotonic and sedative properties. The sedative effect could be useful in cough treatment. The extract resulted additive to benzodiazepines but it did not bind to the same site on GABA-A receptor, and it was interfered by the dopamine release produced with amphetamine.

β-Adrenergic receptor blockade mimics ischemic preconditioning in protecting against myocardial sympathetic activity during global ischemia

Study objectives: Cardiac preconditioning is associated with improved cardiac function after global ischemia. During global cardiac ischemia, developed pressure and heart rate typically decrease. We have previously suggested that ischemic preconditioning results from norepinephrine release from adrenergic nerve termini in the heart. The study objectives are to assess cardiac function and energy expenditure after cyclic ischemia and β-adrenergic blockade. Methods: Isolated rat hearts (n=12) were subject to 4 cycles of ischemia (5 minutes) followed by reperfusion (5 minutes). Developed pressure and heart rate were assessed using high-resolution time-series analysis, and adenosine triphosphate (ATP) expenditure was calculated. Results: Immediately after the onset of transient ischemia, there was a brief period when developed pressure (Δ +13±3 mm Hg; P<.05 versus baseline) and heart rate (Δ +51±0.12 beats/min) increased. This inotropic and chronotropic reflex occurred within 10 seconds of ischemia. Subsequent cycles of transient ischemia and reperfusion differentially reduced the inotropic (Δ −30±15%; P<.05 fourth versus first cycle) and chronotropic (Δ −53±23%; P<.05 versus baseline) aspects of the reflex, with eventual diminution of both reflexes after 4 cycles. Attenuation of the compensatory reflex resulted in decreased contractile work and energy expenditure during repeated episodes of global ischemia, with a net ATP savings of 4.3 μmol/g wet weight. The inotropic and chronotropic responses during all cycles of ischemia were attenuated by 10 μM propranolol. Conclusion: This study reports a novel, locally mediated adrenergic sympathetic response that results in a desperate energy expenditure during global cardiac ischemia. Ischemic preconditioning curtails this reflex after the onset of global ischemia, and the preconditioning effect may be replicated using β-blockade. Blocking this adrenergic response may prevent energy expenditure on potentially futile cardiac contractile work during prolonged global ischemia and prove beneficial in protecting cardiac function during acute ischemic states, such as coronary syndromes, arrest, shock, and trauma. Study objectives: Cardiac preconditioning is associated with improved cardiac function after global ischemia. During global cardiac ischemia, developed pressure and heart rate typically decrease. We have previously suggested that ischemic preconditioning results from norepinephrine release from adrenergic nerve termini in the heart. The study objectives are to assess cardiac function and energy expenditure after cyclic ischemia and β-adrenergic blockade. Methods: Isolated rat hearts (n=12) were subject to 4 cycles of ischemia (5 minutes) followed by reperfusion (5 minutes). Developed pressure and heart rate were assessed using high-resolution time-series analysis, and adenosine triphosphate (ATP) expenditure was calculated. Results: Immediately after the onset of transient ischemia, there was a brief period when developed pressure (Δ +13±3 mm Hg; P<.05 versus baseline) and heart rate (Δ +51±0.12 beats/min) increased. This inotropic and chronotropic reflex occurred within 10 seconds of ischemia. Subsequent cycles of transient ischemia and reperfusion differentially reduced the inotropic (Δ −30±15%; P<.05 fourth versus first cycle) and chronotropic (Δ −53±23%; P<.05 versus baseline) aspects of the reflex, with eventual diminution of both reflexes after 4 cycles. Attenuation of the compensatory reflex resulted in decreased contractile work and energy expenditure during repeated episodes of global ischemia, with a net ATP savings of 4.3 μmol/g wet weight. The inotropic and chronotropic responses during all cycles of ischemia were attenuated by 10 μM propranolol. Conclusion: This study reports a novel, locally mediated adrenergic sympathetic response that results in a desperate energy expenditure during global cardiac ischemia. Ischemic preconditioning curtails this reflex after the onset of global ischemia, and the preconditioning effect may be replicated using β-blockade. Blocking this adrenergic response may prevent energy expenditure on potentially futile cardiac contractile work during prolonged global ischemia and prove beneficial in protecting cardiac function during acute ischemic states, such as coronary syndromes, arrest, shock, and trauma.

Bromoenol Lactone Promotes Cell Death by a Mechanism Involving Phosphatidate Phosphohydrolase-1 Rather than Calcium-independent Phospholipase A2

Originally described as a serine protease inhibitor, bromoenol lactone (BEL) has recently been found to potently inhibit Group VI calcium-independent phospholipase A2 (iPLA2). Thus, BEL is widely used to define biological roles of iPLA2 in cells. However, BEL is also known to inhibit another key enzyme of phospholipid metabolism, namely the magnesium-dependent phosphatidate phosphohydrolase-1 (PAP-1). In this work we report that BEL is able to promote apoptosis in a variety of cell lines, including U937, THP-1, and MonoMac (human phagocyte), RAW264.7 (murine macrophage), Jurkat (human T lymphocyte), and GH3 (human pituitary). In these cells, long term treatment with BEL (up to 24 h) results in increased annexin-V binding to the cell surface and nuclear DNA damage, as detected by staining with both DAPI and propidium iodide. At earlier times (2 h), BEL induces the proteolysis of procaspase-9 and procaspase-3 and increases cleavage of poly(ADP-ribose) polymerase. These changes are preceded by variations in the mitochondrial membrane potential. All these effects of BEL are not mimicked by the iPLA2 inhibitor methylarachidonyl fluorophosphonate or by treating the cells with a specific iPLA2 antisense oligonucleotide. However, propranolol, a PAP-1 inhibitor, is able to reproduce these effects, suggesting that it is the inhibition of PAP-1 and not of iPLA2 that is involved in BEL-induced cell death. In support of this view, BEL-induced apoptosis is accompanied by a very strong inhibition of PAP-1-regulated events, such as incorporation of [3H]choline into phospholipids and de novo incorporation of [3H]arachidonic acid into triacylglycerol. Collectively, these results stress the role of PAP-1 as a key enzyme for cell integrity and survival and in turn caution against the use of BEL in studies involving long incubation times, due to the capacity of this drug to induce apoptosis in a variety of cells.

Calmodulin mediates α 1-adrenergic receptor-stimulated hypertrophy in adult rat ventricular myocytes via a calcium-independent pathway

We previously reported that α1-adrenergic receptor (α1-AR) stimulation induces hypertrophy via activation of MEK1/2-ERK1/2 cascade in adult rat ventricular myocytes (ARVM). However, the upstream regulatory molecules of MEK1/2-ERK1/2 cascade in α1-AR signaling pathway are still unclear. In this study, we thus investigated the role of an important upstream signaling molecule, calmodulin, in α1-AR-stimulated hypertrophy in isolated ARVM. In primary cultured ARVM, α1-AR-stimulation (1 μM norepinephrine in the presence of 2 μM propranolol) induced cardiac myocyte hypertrophy (48 h) was determined by the increase in [3H]leucine incorporation, cell size and expression of the fetal gene atrial natriuretic factor (ANF).

Comparison of Fonsecaea pedrosoi sclerotic cells obtained in vivo and in vitro: ultrastructure and antigenicity

The parasitic form of Fonsecaea pedrosoi from the hyperkeratotic layer of the skin was obtained from four patients with chromoblastomycosis. Primary cultures containing hyphae and conidia were successfully converted into sclerotic cells in the presence of 800 μM propranolol and low pH as described before [1]. The morphology of sclerotic cells of F. pedrosoi obtained in vivo and in vitro was analyzed by light and electron microscopy. Their antigenicity was also compared by immunofluorescence microscopy and ELISA assays, using serum samples from untreated patients infected with F. pedrosoi. Due to the similarity of the sclerotic cells obtained in vivo and in vitro, the latter can be more adequately in studies of host–parasite interactions in chromoblastomycosis.

Inhibition of type 1 protein phosphatase activity by activation of β-adrenoceptors in ventricular myocardium

The regulation of protein phosphatase (PP) activity by cardiac β-adrenergic receptor stimulation with isoproterenol (ISO) was studied in four groups of guinea pigs consisting of seven animals each. Group 1 received the vehicle solution only intraperitoneally; group 2, 6 μg/kg of ISO; group 3, 60 μg/kg of ISO; and group 4, 600 μg/kg of ISO. Total PP activity (consisting of both type 1 and type 2A PP), activity of each PP subtype, the cAMP-dependent protein kinase activity ratio (−cAMP/+cAMP), the phosphorylation of PP inhibitor 1, and the phosphorylation of phospholamban were measured in ventricular tissue. PP activity was also studied in ventricular cardiomyocytes isolated from guinea pigs treated with and without 1 μM ISO or 1 μM ISO plus 10 μM propranolol, an antagonist of the β-adrenoceptor. PP activity decreased significantly in membrane vesicles, but not in cytosolic fractions, of guinea pigs treated with 60 and 600 μg/kg of ISO compared with untreated animals. The PKA activity ratio, PLB phosphorylation, and PP inhibitor 1 phosphorylation increased in ventricles of guinea pigs treated with 60 and 600 μg/kg of ISO compared with vehicle-treated animals. The decrease in overall PP activity was due primarily to a reduction in type 1 but not type 2A PP activity. In isolated ventricular cardiomyocytes, PP activity was decreased significantly after treatment with 1 μM ISO, and this inhibition was reversed by treatment with 10 μM propranolol. The membrane vesicles of group 1 animals did not release any catalytic subunit of type 1 PP upon phosphorylation by exogenous PKA. These results indicate that activation of cardiac β-adrenoceptors inhibits type 1 PP activity via phosphorylation of PP inhibitor 1 in the ventricles. This effect is associated with the well-known effect of ISO on increases in the PKA activity ratio and PLB phosphorylation. Inhibition of type 1 PP activity could be one possible mechanism, in addition to activation of adenylate cyclase, by which ISO mediates enhanced contractility of the heart.

Effect of Ethanol on Spontaneous Phasic Contractions of Cat Gastric Smooth Muscle

Background: Ethanol is generally believed to inhibit extracellular Ca 2+ influx, thereby inhibiting gastric muscle contraction. Recently, we observed that verapamil inhibited only the amplitude of spontaneous phasic contractions, whereas ethanol inhibited both amplitude and frequency. In our objective to investigate the mechanism of ethanol's inhibition of gastric motility, the involvement of various protein kinases in ethanol-inhibited spontaneous phasic contractions of the stomach muscle strips was tested. Methods: Circular muscle strips (2.0 × 0.2 cm) were prepared from the corpus of cat stomach in order to measure isometric contraction in a chamber filled with Krebs-Ringer solution (pH 7.4, temperature 36 °C) bubbled with 5% CO 2 in O 2. Results: Spontaneous phasic contraction was not affected by various receptor antagonists (1 μM atropine, 1 μM hexamethonium, 1 μM phentolamine and 1 μM propranolol) or 1 μM tetrodotoxin. EGTA and verapamil dose-dependently inhibited only the amplitude of spontaneous phasic contractions and not the frequency. Ethanol dose-dependently inhibited both the amplitude and frequency of phasic contractions. The amplitude and frequency of spontaneous phasic contractions were significantly inhibited by protein kinase C and tyrosine kinase inhibitors. However, neither protein kinase C activator nor various phosphatase inhibitors blocked the inhibitory effect of ethanol. Conclusions: Ethanol appears to inhibit spontaneous phasic contractions by a mechanism other than the inhibition of protein kinase C or tyrosine kinase or the inhibition of extracellular Ca 2+ influx.

Sustained norepinephrine stimulation induces different regulation of expression in three α 1-adrenoceptor subtypes

The norepinephrine (NE) -induced regulation of α 1-adrenoceptors (ARs) expression in human embryonic kidney (HEK) 293 cells stably expressing cloned α 1-AR subtypes with similar receptor densities was investigated. In the presence of 10 μM propranolol, the treatment of cells with 10 μM NE for 4 –72 h down-regulated α 1A- and α 1D-AR, but increased α 1B-AR expression in a time-dependent manner. The down-regulation of α 1A-AR reached maximum of 40.3 ± 14.7 % at 48h. The down-regulation of α 1D-AR reached maximum of 51.3 ± 3.7 % at 24h. With the stimulation of NE, α 1B-AR density was increased maximally by 112.4 ± 43.4 % at 48h. The protein kinase C (PKC) inhibitor calphostin C or Ro-31-8220 abolished the NE-induced down-regulation of α 1A- and α 1D-AR, but showed no effect on the up-regulation of α 1B-AR. The PKC agonist PMA not only mimicked the NE-induced down-regulation of α 1A- and α 1D-AR, but also induced a down-regulation of α 1B-AR. The endoplasmic reticulum Ca 2+-ATPase inhibitor cyclopiazonic acid (CPA) or thapsigargin, or the calcium chelator BAPTA/AM did not affect the down-regulation of α 1A-AR, but inhibited the up-regulation of α 1B-AR induced by NE. Calmodulin antagonist W-7, tyrosine kinase inhibitor genistein or tyrphostin A25 had no effect on NE-induced up-regulation of α 1B-AR. The results suggest that three α 1-AR subtypes are differently regulated by sustained NE stimulation with different signal transduction pathways.

Relaxant effect of Pimpinella anisum on isolated guinea pig tracheal chains and its possible mechanism(s)

We have studied the relaxant effect of Pimpinella anisum on isolated guinea pig tracheal chains and its possible mechanism(s). The bronchodilatory effects of aqueous and ethanol extracts and essential oil were examined on precontracted isolated tracheal chains of the guinea pig by 10 μM methacholine in two different conditions including: non-incubated tissues (group 1) and incubated tissues with 1 μM propranolol and 1 μM chlorpheniramine (group 2). In addition, the anticholinergic effects of essential oil and 10 nM atropine were tested by comparing the cumulative log concentration–response curves of methacholine induced contraction of tracheal chains and the effective concentration of methacholine, causing 50% of maximum response (EC 50) in the presence of essential oil or atropine. Aqueous and ethanol extracts, essential oil and theophylline (1 mM) showed significant relaxant effects compared to those of controls. Although relaxant effect of essential oil was lower than theophylline, there was no significant difference between the effect of aqueous and ethanol extracts and that of theophylline. There was also no significant difference between the relaxant effects obtained in group 1 and 2 experiments. The results also showed parallel rightward shifts of methacholine–response curves and significant increase in EC 50 with the presence of atropine or essential oil. These results indicated bronchodilatory effects of essential oil, aqueous, and ethanol extracts from P. anisum. The results also showed that the relaxant effect of this plant is not due to an inhibitory effect of histamine (H 1) or stimulatory effect of β 2-adrenergic receptors, but due to inhibitory effects on muscarinic receptors.

Inhibitory effect of Carum copticum on histamine (H 1) receptors of isolated guinea-pig tracheal chains

In a previous study, the relaxant and anti-cholinergic (functional antagonism) effects of Carum copticum were demonstrated on guinea-pig tracheal chains. To elucidate the other mechanisms responsible for this relaxant effect, the inhibitory effect of this plant on histamine H 1 receptors was examined in this study. The anti-histaminic effects of extracts, essential oil, 5 nM chlorpheniramine, and saline were tested by performing the cumulative log concentration–response curves of histamine induced contraction of isolated guinea-pig tracheal chains incubated with three different conditions: 1.4 μM indomethacin (group 1, n=9); indomethacin, 1 μM propranolol, and 10 nM atropine (group 2, n=8); and indomethacin and propranolol (group 3, n=7). The results showed clear rightward shifts in histamine–response curves obtained in the presence of extracts, essential oil, and chlorpheniramine in all three sets of experiments compared with the curves obtained in the presence of saline. The effective concentrations of histamine causing 50% of maximum response (EC 50) obtained in the presence of extracts, essential oil, and chlorpheniramine in all three sets of experiments were significantly higher than those of saline ( P<0.05–<0.001) except EC 50 of macerated extract in group 1. However, maximum response to histamine obtained in the presence of extracts and essential oil were lower ( P=0.005–<0.001) except maximum response of essential oil in group 2. In addition, the maximum responses obtained in the presence of extracts in group 2 experiments compared to the other two sets of experiments were improved. Comparison of the slope of histamine–response curves showed parallel shifts of the curves obtained in the presence of all extracts and essential oil in group 2 experiments. The results of this study indicated a competitive antagonism effect of C. copticum at histamine H 1 receptors. A β-adrenergic stimulatory effect of essential oil and an anti-cholinergic property of the plant were also suggested.

β 3-Adrenergic stimulation and insulin inhibition of non-selective cation channels in white adipocytes of the rat

Single-channel currents were recorded from the plasma membrane of white adipocytes of 6–8-week-old male Sprague–Dawley rats. In outside-out patches (high K +, no Ca 2+ in pipette), a voltage-dependent K-channel (delayed rectifier) with a single-channel conductance ( γ) of 16 pS (24°C) in modified Ringer’s was active at a density of 0.5/μm 2. It was blocked by TEA (IC 50=1.5 mM). A Ca 2+-activated non-selective cation channel (NSC-channel) appeared at a mean density of 1/μm 2 in inside-out patches ([Ca 2+] i=1.2 mM). γ was 28 pS (24°C). The NSC showed weak voltage dependence and was blocked by mefenamic acid and by internal ATP. In the cell-attached mode spontaneous activity could be blocked reversibly by 100 nM insulin. Noradrenaline (NA, 100 nM) induced a flickering activity of the NSC-channels. Isoproterenol (100 nM) caused activity of the NSC-channel as well. After 1 μM propranolol even 1 μM NA did not induce any activity. The α-antagonist phentolamine had no effect on isoproterenol- or on NA-induced currents. The β 3-agonists BRL 37344 and BRL 35135A induced activity of the NSC-channel at 100 nM as well. We conclude that white adipocytes express ion channels which are comparable to those in brown adipocytes and that β-receptor activation opens NSC-channels thus allowing for Na + entry into white adipocytes.

Superoxide anion and K + channels mediate electrical stimulation-induced relaxation in the rat basilar artery

Electrical field stimulation (a single pulse, 0.2 ms) caused a rapid relaxation of rat basilar artery segments precontracted with different agents, but not with 30 mM KCl. This relaxation was not modified by endothelium removal, 10 μM tetrodotoxin, 1 μM propranolol, 1 μM atropine, 30 μM indomethacin, 10 μM methylene blue, 100 μM N G-nitro- l-arginine methyl ester or 1 μM cimetidine but it was significantly reduced by 50 and 100 U/ml superoxide dismutase. Charybdotoxin (0.1 and 0.2 μM), a blocker of large-conductance Ca 2+-activated K + channels (BK Ca), decreased the relaxation elicited by electrical stimulation, whereas it was unaltered by 10 μM glibenclamide or 1 μM apamin, blockers of ATP-sensitive (K ATP) or small-conductance K Ca channels, respectively. Thapsigargin (0.01 and 0.1 μM), an inhibitor of sarcoplasmic reticulum Ca 2+-ATPase, increased the electrical stimulation-induced relaxation, which was nearly abolished by charybdotoxin. These results show that electrical stimulation induces endothelium-independent and non-neurogenic relaxations in the rat basilar artery. This response appears to involve generation of superoxide anion, increase of cytosolic free Ca 2+ concentration and subsequent activation of BK Ca channels.

Dynamic Complexes of β2-Adrenergic Receptors with Protein Kinases and Phosphatases and the Role of Gravin

Signals mediated by G-protein-linked receptors display agonist-induced attenuation and recovery involving both protein kinases and phosphatases. The role of protein kinases and phosphatases in agonist-induced attenuation and recovery of beta-adrenergic receptors was explored by two complementary approaches, antisense RNA suppression and co-immunoprecipitation of target elements. Protein phosphatases 2A and 2B are associated with the unstimulated receptor, the latter displaying a transient decrease followed by a 2-fold increase in the levels of association at 30 min following challenge with agonist. Protein kinase A displays a robust, agonist-induced association with beta-adrenergic receptors over the same period. Suppression of phosphatases 2A and 2B with antisense RNA or inhibition of their activity with calyculin A and FK506, respectively, blocks resensitization following agonist removal. Recycling of receptors to the plasma membrane following agonist-promoted sequestration is severely impaired by loss of either phosphatase 2B or protein kinase C. In addition, loss of protein kinase C diminishes association of phosphatase 2B with beta-adrenergic receptors. Overlay assays performed with the RII subunit of protein kinase A and co-immunoprecipitations reveal proteins of the A kinase-anchoring proteins (AKAP) family, including AKAP250 also known as gravin, associated with the beta-adrenergic receptor. Suppression of gravin expression disrupts recovery from agonist-induced desensitization, confirming the role of gravin in organization of G-protein-linked signaling complexes. The Ht31 peptide, which blocks AKAP protein-protein interactions, blocks association of beta-adrenergic receptors with protein kinase A. These data are the first to reveal dynamic complexes of beta-adrenergic receptors with protein kinases and phosphatases acting via an anchoring protein, gravin.

Effects of Propranolol on Phosphatidate Phosphohydrolase and Mitogen-Activated Protein Kinase Activities in A7r5 Vascular Smooth Muscle Cells

High doses of propranolol inhibit phosphatidate phosphohydrolase (PAP) activity in intact cells, thus blocking metabolism of phosphatidic acid (PA), product of the phospholipase D (PLD) reaction. Vasopressin and phorbol ester activate PLD and ERK (extracellular signal-regulated protein kinase) mitogen-activated protein kinases in A7r5, a rat vascular smooth muscle cell line. Propranolol increased PA levels in intact A7r5 cells and inhibited cytosolic PAP and membrane calcium-independent phospholipase A 2 but did not activate PLD or enhance agonist-induced PA accumulation. Incubation of cells with 200 μM propranolol for 10–45 min markedly elevated PA but caused only partial acivation of ERKs. Propranolol and other lipophilic amines caused a time- and dose-dependent detachment of cells from their substrate. These results confirm that elevation of PA is not a strong signal for ERK activation and emphasize that caution should be exercised in using propranolol as a PAP inhibitor in intact cells.

Maturation and secretion of rat hepatic lipase is inhibited by alpha1B-adrenergic stimulation through changes in Ca2+ homoeostasis: thapsigargin and EGTA both mimic the effect of adrenaline.

In rats, the daily changes in hepatic lipase (HL) activity in the liver follow the diurnal rhythm of the catecholamines. To study the underlying mechanism, the effect of adrenaline on maturation and secretion of HL was determined in freshly isolated rat hepatocytes. Adrenaline (10 microM) acutely inhibited the secretion of HL. This effect was abolished by 0.1 microM prazosin, but not by 1 microM propranolol, indicating the involvement of the alpha1-adrenergic pathway. Prazosin was at least 1000-fold more potent than WB4101, a selective alpha1A-antagonist. Adrenaline had no effect on HL secretion in hepatocytes pretreated with chloroethylclonidine, an irreversible alpha1B-selective antagonist. Inhibition of HL was not induced by 10 microM methoxamine, a alpha1A-selective agonist. Thus, adrenaline inhibited HL secretion through activation of the alpha1-adrenoceptors subtype B, which have been shown to signal through Ca2+ as well as cAMP. A similar reduction in HL secretion was induced by the Ca2+-mobilizing hormones angiotensin II (100 nM) and vasopressin (12 nM), the Ca2+ ionophore A23187 (2 microM), and by thapsigargin (1 microM), which inhibits the ER Ca2+-ATPase pump. HL secretion was unaffected by elevating cAMP with 10 microM forskolin or 1 microM 8-Br-cAMP. These results suggest that the alpha1B-adrenergic effects on HL expression are mainly mediated through elevation of intracellular Ca2+. Chelation of extracellular Ca2+ and subsequent lowering of intracellular Ca2+ with EGTA also inhibited HL secretion. In pulse-chase experiments, adrenaline was shown to inhibit the maturation of HL from the 53 kDa, Endo H-sensitive precursor to the Endo H-resistant, catalytically active protein of 58 kDa. In addition, adrenaline induced intracellular degradation of newly synthesized HL. Similar post-translational effects, both qualitatively and quantitatively, were observed with A23187, thapsigargin and EGTA. We conclude that the inhibition of HL maturation and increase in intracellular degradation induced by catecholamines, A23187, thapsigargin and EGTA is evoked by changes in Ca2+ homoeostasis, possibly through lowering ER Ca2+.

Involvement of adrenergic system on the cortical granule exocytosis and polyspermic penetration during in vitro fertilization of porcine oocytes

The effects of propranolol, a β-adrenergic blocker, and epinephrine on cortical granule exocytosis and polyspermic penetration in the pig were determined. Pig oocytes precultured in medium containing 1 and 10 μM propranolol had an increased (P < 0.05) percentage of polyspermic penetration. The mean number of spermatozoa penetrating oocytes precultured in 1 and 10 μM propranolol was also higher (P < 0.01) than that in the control group. Cortical granule exocytosis in oocytes after sperm penetration was observed by either transmission electron or epifluorescence microscopy. Preculture of oocytes in media containing 1 and 10 μM propranolol significantly (P < 0.05) decreased the incidence of cortical granule exocytosis and increased abnormal patterns of cortical granule exocytosis. Preculture of pig oocytes with 1 and 10 μM epinephrine did not increase monospermic penetration and complete cortical granule exocytosis. However, combined pretreatment of oocytes with 10 μM propranolol and 10 μM epinephrine decreased polyspermic penetration and incomplete cortical granule exocytosis compared with that of oocytes precultured with 10 μM propranolol alone. These results suggest that the intracellular adrenergic system is involved in cortical granule reaction in the pig and that defects of the adrenergic system may be related to a high incidence of polyspermy in the pig.

HDL 3-signalling in HepG 2 cells involves glycosyl-phospatidylinositol-anchored proteins

In [ 3H]phosphatidylcholine (PC) prelabelled HepG 2 cells, HDL 3 stimulates a biphasic increase in 1,2-diacylglycerol (DAG). The early phase is mediated in part by a phospholipase C which is inhibited by 10 μM D 609, RHC-80267 or U-73122 and less by 100 μM propranolol. A phospholipase D is more likely involved in the late phase, as the DAG peak lags behind phosphatidic acid rise and is blocked by 100 μM propranolol. Cellular preincubation with 200 μg/ml antibodies against the inositolphosphoglycan (IPG) moiety of the GPI-anchor (Ab IPG), or depletion in GPI-anchored proteins by cellular pretreatment with 0.5 U/ml PI-PLC, 1 mM insulin and 2 HU/ml streptolysin-O, or depletion in membrane cholesterol content by filipin (5 μg/ml), digitonin (5 μg/ml) and cholesterol oxidase (0.5 U/ml) decreases the HDL 3-signal, suggesting the involvement of a lipolytic cleavage of GPI-anchored proteins. Inhibition of proteases by 1 mM leupeptin/PMSF improves the response time to HDL 3, with a DAG peak at 2–3 min. In the presence of protease-inhibitors, HDL 3 releases in the culture medium several proteins with a residual IPG that binds Ab IPG after SDS-PAGE analysis and immunoblotting. HDL 3-signalling pathways comprise tyrosine kinases, as preincubation with 100 μg/ml genistein or tyrphostin inhibits the HDL 3-signal. HDL 3 activates PC hydrolysis through a multistep pathway involving the cleavage of GPI-anchored proteins.

Influence of hypothermia on the cardiac effects of propranolol observed in isolated rat atria

1. 1. The purpose was to determine if hypothermia influences cardiac responses to propranolol. 2. 2. Rat atria were used and 11 test groups were created; 3 control groups were maintained at 35, 28 or 20°C. Two additional groups, at each temperature, were exposed to 1.2 or 40 μmol/l propranolol. Developed force and effective refractory period (ERP) were measured. 3. 3. At 35°C, propranolol decreased developed force and lengthened ERP. At 28°C, propranolol did not affect developed force, but ERP was lengthened. At 20°C, 1.2 μM propranolol neither affected developed force or ERP, but 40 μM reduced developed force and lengthened ERP. Conclusion: hypothermia reduced propranolol's usual negative inotropic effect.

P.1.061 - Clinical differences in response to mianserin and fluvoxamine in depressive patients

Serotonin (5-HT, 1 μM) induced an inward current in Xenopus oocytes injected with rat brain mRNA under steady voltage clamp conditions of −60 mV. The 5-HT response was blocked by 0.1 μM mianserin, but neither buspirone, 8-hydroxy-dipropylaminotetralin (8-OH-DPAT), trifluoromethylphenyl piperazine (TFMPP), pindolol, propranolol, spiperone, ketanserin nor ICS 205–930 exerted effects, suggesting that the 5-HTIC subtype of 5-HT receptors is involved in the current. The 5-HT-induced current was inhibited by imipramine or desipramine with IC50 values of 60 nM or 20 μM, respectively. The response was also inhibited by setiptilline, maprotiline or amoxapine at a dose of 10 μM. Imipramine at 10 μM had no effect on the acetylcholine (ACh, 1 mM)-induced current response. It has been reported that inositol triphosphate (IP3) formation and intracellular Ca2+ are involved in the 5-HT- as well as ACh-induced current and that intracellular injection of either 50 pmol IP3 or 50 pmol Ca2+ mimics the 5-HT-induced current. The response induced by intracellular injection of either 50 pmol IP3 or 50 pmol Ca2+ was not affected by 10 μM imipramine. It is suggested that imipramine, and perhaps other antidepressant drugs tested, blocks 5-HTIC receptors, subsequently inhibiting the 5-HT-evoked current.

P.1.063 - Responsiveness of rabbit detrusor, urethra, and trigone strips to imipramine and trazodone

Serotonin (5-HT, 1 μM) induced an inward current in Xenopus oocytes injected with rat brain mRNA under steady voltage clamp conditions of −60 mV. The 5-HT response was blocked by 0.1 μM mianserin, but neither buspirone, 8-hydroxy-dipropylaminotetralin (8-OH-DPAT), trifluoromethylphenyl piperazine (TFMPP), pindolol, propranolol, spiperone, ketanserin nor ICS 205–930 exerted effects, suggesting that the 5-HTIC subtype of 5-HT receptors is involved in the current. The 5-HT-induced current was inhibited by imipramine or desipramine with IC50 values of 60 nM or 20 μM, respectively. The response was also inhibited by setiptilline, maprotiline or amoxapine at a dose of 10 μM. Imipramine at 10 μM had no effect on the acetylcholine (ACh, 1 mM)-induced current response. It has been reported that inositol triphosphate (IP3) formation and intracellular Ca2+ are involved in the 5-HT- as well as ACh-induced current and that intracellular injection of either 50 pmol IP3 or 50 pmol Ca2+ mimics the 5-HT-induced current. The response induced by intracellular injection of either 50 pmol IP3 or 50 pmol Ca2+ was not affected by 10 μM imipramine. It is suggested that imipramine, and perhaps other antidepressant drugs tested, blocks 5-HTIC receptors, subsequently inhibiting the 5-HT-evoked current.