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1.
Acta Physiol Scand ; 177(4): 449-58, 2003 Apr.
Article in English | MEDLINE | ID: mdl-12648162

ABSTRACT

AIM: In failing myocardium the mechanical response to beta-adrenoceptor stimulation is attenuated. Alternative signalling systems might provide inotropic support when the beta-adrenoceptor system is dysfunctioning. Accordingly, the inotropic responses to alpha 1- and beta-adrenoceptor stimulation by the endogenous adrenoceptor agonist noradrenaline in non-failing and failing rat hearts were compared. METHODS: Chronic heart failure was induced in male Wistar rats by coronary artery ligation. Corresponding sham groups were prepared. After 6 weeks, papillary muscles from non-failing and failing hearts were isolated. Receptor binding studies were performed in the corresponding myocardium. The alpha 1-adrenoceptor-mediated inotropic response was not changed while the beta-adrenoceptor-mediated response was substantially reduced in failing compared with non-failing myocardium. RESULTS: No change in potency for the agonists was observed at the alpha 1-adrenoceptors, while an increased potency for the agonists at the beta-adrenoceptors was found during heart failure. The lusitropic response to beta-adrenoceptor stimulation was intact during heart failure. No over all change in affinity or number of either adrenoceptor type was observed in receptor binding studies. The alpha 1-adrenoceptor-mediated inotropic response became dominating compared with the beta-adrenoceptor-mediated one in failing rat myocardium in contrast to the dominating role of the latter in non-failing myocardium. The attenuation of the beta-adrenoceptor-mediated inotropic response in rat failing myocardium was not because of a reduced number of receptors. CONCLUSION: Increasing contractility through stimulation of alpha 1-adrenoceptors in situ by the endogenous agonist may be an alternative way of inotropic support during heart failure and even more so during beta-adrenoceptor blockade.


Subject(s)
Adrenergic alpha-Agonists/pharmacology , Heart Failure/physiopathology , Myocardial Contraction/drug effects , Norepinephrine/pharmacology , Receptors, Adrenergic/physiology , Vasoconstrictor Agents/pharmacology , Adrenergic beta-Agonists/pharmacology , Animals , Cardiotonic Agents/pharmacology , Dose-Response Relationship, Drug , Isoproterenol/pharmacology , Male , Phenylephrine/pharmacology , Rats , Rats, Wistar , Receptors, Adrenergic, alpha-1/physiology , Receptors, Adrenergic, beta/physiology
2.
J Cell Physiol ; 184(1): 27-36, 2000 Jul.
Article in English | MEDLINE | ID: mdl-10825231

ABSTRACT

It was previously found that pertussis toxin (PTX) pretreatment inhibits the activation of extracellular signal-regulated kinases ERK1 (p44(mapk)) and ERK2 (p42(mapk)) in hepatocytes in response to either agonists that bind to heptahelical receptors or epidermal growth factor (EGF), suggesting a role of G(i) proteins in stimulatory mechanisms for ERK1/2. The present work shows that ERK1/2 is activated in a PTX-sensitive way not only by vasopressin, angiotensin II, prostaglandin (PG) F(2alpha), alpha(1)-adrenergic stimulation, and EGF but also by agents whose actions bypass receptors and stimulate protein kinase C (PKC) and/or elevate intracellular Ca(2+), such as 12-O-tetradecanoyl phorbol-13-acetate (TPA), exogenous phosphatidylcholine-specific phospholipase C (PC-PLC, from Bacillus cereus), thapsigargin, and the Ca(2+) ionophore A23187. Under the same conditions, PTX did not affect agonist stimulation of phosphoinositide-specific phospholipase C (PI-PLC) (IP(3) generation), and did not reduce the activation by these agents of phospholipase D (PLD). The results suggest that in hepatocytes a PTX-sensitive mechanism, presumably involving G(i) proteins, exerts a stimulatory effect on ERK at a level distal to receptor coupling, acting either as an integral part of the signaling pathway(s) or by a permissive, synergistic regulation.


Subject(s)
Angiotensin II/pharmacology , ErbB Receptors/physiology , GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , Liver/drug effects , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinases/metabolism , Pertussis Toxin , Vasopressins/pharmacology , Virulence Factors, Bordetella/pharmacology , Animals , Calcimycin/pharmacology , Cell Division/drug effects , Cells, Cultured , Dinoprost/pharmacology , Enzyme Activation , Epidermal Growth Factor/pharmacology , ErbB Receptors/drug effects , Inositol 1,4,5-Trisphosphate/metabolism , Liver/cytology , Liver/metabolism , Male , Mitogen-Activated Protein Kinase 3 , Norepinephrine/pharmacology , Phosphatidylinositol Diacylglycerol-Lyase , Phosphoinositide Phospholipase C , Phospholipase D/metabolism , Rats , Rats, Wistar , Receptors, Adrenergic, alpha-1/physiology , Thapsigargin/pharmacology , Type C Phospholipases/metabolism
3.
Tidsskr Nor Laegeforen ; 120(20): 2397-9, 2000 Aug 30.
Article in Norwegian | MEDLINE | ID: mdl-11475224

ABSTRACT

BACKGROUND: Bisphosphonates are potent inhibitors of osteoclast-mediated bone resorption and effective in preventing osteoporotic fractures, but they can occasionally cause oesophageal adverse events. MATERIAL AND METHODS: We report on seven patients who developed severe oesophagitis or oesophageal ulceration during treatment with bisphosphonates. They were registered at our endoscopy unit during a 31-month period. RESULTS: Six of the patients took alendronate (Fosamax) and one etidronate (Didronate). The oesophageal lesions heal on discontinuation of the bisphosphonate. Institution of gastric acid suppression treatment may enhance the healing process. INTERPRETATION: In order to minimise the risk of serious side-effects, it is important to give detailed instructions regarding medication and to ensure that the instructions are properly understood. The risk of serious complications can be reduced by early recognition of oesophageal symptoms and appropriate intervention.


Subject(s)
Alendronate/adverse effects , Diphosphonates/adverse effects , Esophageal Stenosis/chemically induced , Esophagitis/chemically induced , Esophagus/drug effects , Etidronic Acid/adverse effects , Aged , Esophagoscopy , Esophagus/pathology , Female , Humans
4.
J Cell Physiol ; 180(2): 203-14, 1999 Aug.
Article in English | MEDLINE | ID: mdl-10395290

ABSTRACT

The role of diacylglycerol (DAG) in hormonal induction of S phase was investigated in primary cultures of rat hepatocytes. In this model, several agonists that bind to G protein-coupled receptors act as comitogens when added to the cells soon after plating (i.e., in Go/early Gl phase), while the cells are most responsive to the mitogenic effect of epidermal growth factor (EGF) at 24-48 h of culturing (i.e., mid/late Gl). It was found that the cellular concentration of DAG rose markedly and progressively during the first 24 h of culturing. Exposure of the hepatocytes at 3 h to alpha1-adrenergic stimulation (norepinephrine with timolol), vasopressin, or angiotensin II further increased this rise, producing a sustained increase in the DAG level. Norepinephrine, which was the most efficient comitogen, produced the most prolonged DAG elevation. In contrast, no significant increase of DAG was found in response to EGF, neither at 3 nor at 24 h, using concentrations that markedly stimulated the ERK subgroup of the mitogen-activated protein kinases (MAPK) and DNA synthesis. Addition of Bacillus cereus phosphatidylcholine-specific phospholipase C (PC-PLC) strongly elevated DAG, while Streptomyces phospholipase D (PLD) increased phosphatidic acid (PA) but not DAG. B. cereus PC-PLC and the protein kinase C (PKC) activator tetradecanoyl phorbol-acetate (TPA), like norepinephrine, vasopressin, and angiotensin II, stimulated MAPK and enhanced the stimulatory effect of EGF on DNA synthesis. The PKC inhibitor GF109203X did not diminish the effect of EGF on MAPK or DNA synthesis, but strongly inhibited the effects of norepinephrine, vasopressin, angiotensin II, TPA and B. cereus PC-PLC on MAPK and almost abolished the enhancement by these agents of EGF-stimulated DNA synthesis. These results suggest that although generation of DAG is not a direct downstream response mediating the effects of the EGF receptor in hepatocytes, a sustained elevation of DAG with activation of PKC markedly increases the responsiveness to EGF. Mechanisms involving DAG and PKC seem to play a role in the comitogenic effects of various agents that bind to G protein-coupled receptors and activate the cells early in Gl, such as norepinephrine, angiotensin II, and vasopressin.


Subject(s)
Diglycerides/metabolism , Epidermal Growth Factor/pharmacology , Neuropeptides/pharmacology , Protein Kinase C/metabolism , S Phase/physiology , Angiotensin II/pharmacology , Animals , Bacterial Proteins/pharmacology , Calcium-Calmodulin-Dependent Protein Kinases/antagonists & inhibitors , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Carcinogens/pharmacology , Cells, Cultured , DNA/biosynthesis , Enzyme Activation/physiology , Enzyme Inhibitors/pharmacology , G1 Phase/drug effects , G1 Phase/physiology , GTP-Binding Proteins/metabolism , Indoles/pharmacology , Liver/cytology , Liver/enzymology , Male , Maleimides/pharmacology , Norepinephrine/pharmacology , Phospholipase D/metabolism , Phospholipase D/pharmacology , Protein Kinase C/antagonists & inhibitors , Protein Kinase C/pharmacology , Rats , Rats, Wistar , S Phase/drug effects , Tetradecanoylphorbol Acetate/pharmacology , Vasopressins/pharmacology
5.
Pharmacol Toxicol ; 84(5): 234-40, 1999 May.
Article in English | MEDLINE | ID: mdl-10361980

ABSTRACT

Previous studies have shown that while vasopressin and angiotensin II are markedly more effective than norepinephrine and prostaglandin F2alpha in eliciting an acute elevation of inositol 1,4,5-trisphosphate (IP3), norepinephrine and prostaglandin F2alpha produce larger enhancement of DNA synthesis. This suggests that the initial activation of phosphoinositide-specific phospholipase C is not a common factor for the growth response to these agonists, but does not exclude a role of the integral of phospholipase C activity over a prolonged part of the prereplicative period, during which agonist-specific changes in responsiveness might occur. We show that vasopressin and angiotensin II also cause a prolonged elevation of cellular IP3 levels. which remain elevated for at least 60 min., while norepinephrine and prostaglandin F2alpha elevate IP3 levels slightly and transiently For vasopressin the dose-effect curves for IP3 accumulation and stimulation of DNA synthesis were closely parallel, while this was not the case for angiotensin II, norepinephrine, or prostaglandin F2alpha. After cultivation of the hepatocytes, hormone-stimulated IP3 accumulation rapidly declined, particularly in response to norepinephrine and prostaglandin F2alpha. When the IP3 response to norepinephrine and prostaglandin F2alpha was completely down-regulated, these agonists still enhanced the DNA synthesis. These results suggest that other mechanisms in addition to IP3 accumulation and Ca2+ release are likely to be involved in the growth stimulatory effects of the Ca2+-mobilizing agonists studied here, in particular for angiotensin II, norepinephrine, and prostaglandin F2alpha.


Subject(s)
Calcium Signaling/drug effects , Inositol 1,4,5-Trisphosphate/metabolism , Liver/drug effects , Phosphatidylinositols/metabolism , Type C Phospholipases/metabolism , Angiotensin II/pharmacology , Animals , Cell Division/drug effects , Cells, Cultured , DNA/biosynthesis , DNA/drug effects , Dinoprost/pharmacology , Down-Regulation , Enzyme Activation , Liver/cytology , Liver/metabolism , Male , Norepinephrine/pharmacology , Rats , Rats, Wistar , Time Factors , Vasopressins/pharmacology
6.
Pharmacol Toxicol ; 83(1): 23-8, 1998 Jul.
Article in English | MEDLINE | ID: mdl-9764422

ABSTRACT

The aim of the present study was to investigate the accumulation of inositol-1,4,5-trisphosphate (IP3) in isolated adult rat ventricular cardiomyocytes after alpha 1- and beta-adrenoceptor stimulation, separate and in combination, in order to elucidate a possible influence of concomitant beta-adrenoceptor stimulation on the alpha 1-adrenoceptor stimulated response. IP3 was measured by a radioligand binding assay based on an (1,4,5)IP3-specific binding protein from bovine adrenal cortex. The basal IP3 content was 4.06 +/- 0.31 pmol/mg protein (N = 56). alpha 1-Adrenoceptor stimulation resulted in a rapid increase in the IP3 level, which reached a plateau, 50-80% above basal level, at 10-30 sec. The plateau lasted at least up to 120 sec., while at 300 sec. there was no significant difference between control values and values after alpha 1-adrenoceptor stimulation. Li+ did not affect either the basal IP3 level, or the magnitude or time course of alpha 1-adrenoceptor-stimulated IP3 accumulation. Combined adrenoceptor stimulation gave a similar response as separate alpha 1-adrenoceptor stimulation, whereas there was no significant change in the IP3 level after beta-adrenoceptor stimulation. No inhibitory influence of simultaneous beta-adrenoceptor stimulation on the alpha 1-adrenoceptor-stimulated increase of IP3 mass was revealed.


Subject(s)
Inositol 1,4,5-Trisphosphate/metabolism , Myocardium/metabolism , Receptors, Adrenergic, alpha-1/metabolism , Receptors, Adrenergic, beta/metabolism , Adrenergic alpha-Agonists/pharmacology , Animals , Cattle , Cells, Cultured , Heart Ventricles/drug effects , Male , Myocardium/cytology , Norepinephrine/pharmacology , Rats , Rats, Wistar
7.
J Biol Chem ; 273(27): 16639-42, 1998 Jul 03.
Article in English | MEDLINE | ID: mdl-9642213

ABSTRACT

Activation of the epidermal growth factor receptor (EGFR) kinase was analyzed in cells conditionally defective for clathrin-dependent endocytosis by overexpression of mutant dynamin (K44A). EGF-induced autophosphorylation of the EGFR on ice was strongly reduced in cells overexpressing mutant dynamin, and consistently, binding analyses showed that high-affinity EGFRs were lost. In the absence of mutant dynamin the cells displayed both high- and low-affinity EGFR. At 4 degreesC EGF-EGFR localized mainly outside coated pits regardless of expression of mutant dynamin. However, also low-affinity EGFR efficiently moved to coated pits upon incubating cells at 37 degreesC. Thus, expression of mutant dynamin disrupts high-affinity binding of EGF, but not ligand-induced recruitment of EGFR to clathrin-coated pits.


Subject(s)
Epidermal Growth Factor/metabolism , ErbB Receptors/metabolism , GTP Phosphohydrolases/metabolism , Dynamins , GTP Phosphohydrolases/genetics , HeLa Cells , Humans , Isoenzymes/metabolism , Microscopy, Immunoelectron , Phospholipase C gamma , Protein Binding , Recombinant Proteins/metabolism , Signal Transduction/genetics , Subcellular Fractions/metabolism , Type C Phospholipases/metabolism
8.
J Cell Physiol ; 175(3): 348-58, 1998 Jun.
Article in English | MEDLINE | ID: mdl-9572480

ABSTRACT

Several agents that act through G-protein-coupled receptors and also stimulate phosphoinositide-specific phospholipase C (PI-PLC), including angiotensin II, vasopressin, norepinephrine, and prostaglandin (PG) F2alpha, activated the ERK1 (p44mapk) and ERK2 (p42mapk) members of the mitogen-activated protein (MAP) kinase family in primary cultures of rat hepatocytes, measured as phosphorylation of myelin basic protein (MBP) by a partially purified enzyme, immunoblotting, and in-gel assays. All these agonists induced a peak activation (two to threefold increase in MBP-phosphorylation) at 3-5 min, followed by a brief decrease, and then a sustained elevation or a second increase of the MAP kinase activity that lasted for several hours. Although all the above agents also stimulated PI-PLC, implicating a Gq-dependent pathway, the elevations of the concentration of inositol (1,4,5)-trisphosphate did not correlate well with the MAP kinase activity. Furthermore, pretreatment of the cells with pertussis toxin markedly reduced the MAP kinase activation by angiotensin II, vasopressin, norepinephrine, or PGF2alpha. In addition, hepatocytes pretreated with pertussis toxin showed a diminished MAP kinase response to epidermal growth factor (EGF). The results indicate that agonists acting via G-protein-coupled receptors have the ability to induce sustained activation of MAP kinase in hepatocytes, and suggest that Gi-dependent mechanisms are required for full activation of the MAP kinase signal transduction pathway by G-protein-coupled receptors as well as the EGF receptor.


Subject(s)
Dinoprost/pharmacology , GTP-Binding Protein alpha Subunits, Gi-Go/physiology , Liver/enzymology , Mitogen-Activated Protein Kinase 1/metabolism , Neuropeptides/pharmacology , Norepinephrine/pharmacology , Animals , Cells, Cultured , DNA/biosynthesis , Enzyme Activation , Epidermal Growth Factor/pharmacology , Inositol 1,4,5-Trisphosphate/analysis , Liver/cytology , Male , Pertussis Toxin , Rats , Rats, Wistar , Time Factors , Virulence Factors, Bordetella/pharmacology
9.
J Cell Physiol ; 175(1): 10-8, 1998 Apr.
Article in English | MEDLINE | ID: mdl-9491776

ABSTRACT

The epidermal growth factor (EGF) receptor mediates the effects of both EGF and transforming growth factor alpha (TGFalpha). Recent data suggested that EGF acts as a partial agonist/antagonist in hepatocytes, TGFalpha exerting a larger maximal stimulation of DNA synthesis than EGF. To further study the mechanisms involved in mediating the different effects of EGF and TGFalpha, we have examined receptor binding of the two growth factors and their action on the p42/p44 mitogen-activated protein (MAP) kinase activity in hepatocytes. Single-ligand concentration curves and competition experiments showed that the binding affinity to a common population of surface binding sites was about 20-fold lower for TGFalpha than for EGF. MAP kinase activity responded to EGF and TGFalpha with different kinetics. While the two agents produced almost identical acute (5 min) stimulation (peak about fivefold), TGFalpha produced a more sustained MAP kinase activity than EGF. The difference between EGF and TGFalpha was still detectable 24 h after growth factor addition. The results show that in hepatocytes a lower receptor affinity of TGFalpha, as compared to EGF, is associated with a more sustained activation of the MAP kinase and a greater efficacy in the stimulation of DNA synthesis. This suggests that differential interaction of these two agents with the EGF receptor results in differences in the downstream events elicited at a given level of receptor occupancy. The data also are compatible with a role of a prolonged MAP kinase activity in the mitogenic effects of EGF and TGFalpha.


Subject(s)
Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Epidermal Growth Factor/pharmacology , ErbB Receptors/physiology , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinases , Transforming Growth Factor alpha/pharmacology , Animals , Binding, Competitive/drug effects , Cells, Cultured , DNA/biosynthesis , Dose-Response Relationship, Drug , Drug Interactions , Enzyme Activation , Liver/chemistry , Liver/cytology , Liver/enzymology , Male , Mitogen-Activated Protein Kinase 3 , Rats , Rats, Wistar , Time Factors
10.
Eur J Pharmacol ; 323(2-3): 269-75, 1997 Apr 04.
Article in English | MEDLINE | ID: mdl-9128849

ABSTRACT

The aim of the present study was to identify the receptor subtypes involved in the alpha 1-adrenoceptor-mediated increase in 86Rb+ influx rate and in inositol 1,4,5-trisphosphate (IP3) accumulation in isolated ventricular cardiomyocytes from adult rat heart, in order to identify a possible response pattern compatible with a causative relationship. Subtype-selective receptor antagonists used were: 5-methylurapidil (alpha 1A), WB 4101 [2([2,6-dimethoxyphenoxy-ethyl]aminomethyl)-1,4-benzodioxane] (alpha 1A), chloroethylclonidine (alpha 1B) and BMY 7378 [8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4,5]dec ane-7,9-dione] (alpha 1D). The basal 86Rb+ influx rate was 0.22 +/- 0.01 ml/g protein x min. At 15 min, 5 x 10(-5) mol/l noradrenaline in the presence of 3 x 10(-5) mol/l timolol increased the 86Rb+ influx rate by 33 +/- 1%. This response was not affected by either chloroethylclonidine or BMY 7378 at concentrations up to 10(-5) mol/l. 5-Methylurapidil dose dependently inhibited the response to 5 x 10(-5) mol/l noradrenaline with a -logIC50 value of 5.27 +/- 0.12 and 5.61 +/- 0.27 in the presence and absence of 10(-5) mol/l chloroethylclonidine, respectively. WB 4101 in the presence of 10(-5) mol/l chloroethylclonidine dose dependently inhibited the response to 5 x 10(-5) mol/l noradrenaline with a -logIC50 value of 6.10 +/- 0.14. Noradrenaline in the presence of 10(-5) mol/l chloroethylclonidine dose dependently increased the 86Rb+ uptake rate with a -logEC50 value of 6.19 +/- 0.35. The basal IP3 level was 2.15 +/- 0.19 pmol/mg protein. Incubation with 10(-5) mol/l noradrenaline for 2 min increased this by 65 +/- 7% of control levels. 10(-5) mol/l chloroethylclonidine and 10(-4) mol/l 5-methylurapidil reduced the response to 27 +/- 6% and 18 +/- 9% of control level, respectively. BMY 7378 dose dependently inhibited the IP3 response at relatively high concentrations, and it was completely eliminated at 10(-5) mol/l BMY 7378. The combination of chloroethylclonidine and 5-methylurapidil or 3 x 10(-6) mol/l prazosin alone completely abolished the hormone-induced effect. We conclude that whereas the alpha 1-adrenoceptor-stimulated increase in 86Rb+ influx rate is mediated via the alpha 1A-adrenoceptor subtype only, both alpha 1A- and alpha 1B-adrenoceptor subtypes are involved in the increase in IP3 mass. Furthermore, a contribution from the alpha 1D-adrenoceptor in the IP3 response cannot be excluded. Thus there does not appear to be a simple causative relationship between an increase in 86Rb+ influx rate and an increase in IP3.


Subject(s)
Adrenergic alpha-1 Receptor Antagonists , Inositol 1,4,5-Trisphosphate/metabolism , Myocardium/metabolism , Rubidium Radioisotopes/metabolism , Adrenergic alpha-2 Receptor Antagonists , Adrenergic alpha-Antagonists/pharmacology , Animals , Cells, Cultured , Clonidine/analogs & derivatives , Clonidine/pharmacology , Dioxanes/pharmacology , Heart Ventricles/metabolism , Myocardium/cytology , Piperazines/pharmacology , Rats , Receptors, Adrenergic, alpha-1 , Receptors, Adrenergic, alpha-2
11.
J Cell Physiol ; 168(3): 608-17, 1996 Sep.
Article in English | MEDLINE | ID: mdl-8816915

ABSTRACT

Although several hormones that promote hepatocyte proliferation also activate phosphoinositide-specific phospholipase C (PI-PLC) and mobilize Ca2+, the role of PI-PLC in the growth-stimulating effect of these agents is not clear. We have investigated this issue further, by exposing freshly isolated adult rat hepatocytes to vasopressin, angiotensin II, norepinephrine (in the presence of the beta-adrenoceptor blocker timolol) or PGF2 alpha, and examined both acute responses and the subsequent DNA synthesis when the cells were grown in monolayer culture. All the agonists elevated the level of inositol 1,4,5-trisphosphate (InsP3) and enhanced the DNA synthesis, amplifying the response to epidermal growth factor (EGF), and this comitogenic effect could be exerted by a single exposure of the cells 24 h prior to the addition of EGF. The acute activation of PI-PLC, measured as the early rise (peak 15-60 s) in InsP3, was 8-10-fold with vasopressin or angiotensin II, 3-4-fold with norepinephrine, and approximately 2-fold with PGF2 alpha. For all the agonists, a rise in cytosolic free Ca2+ in 100% of the cells and a maximal increase in glycogen phosphorylase activity were evoked at concentrations that approximately doubled the level of InsP3. However, the growth-stimulatory effects of these agonists showed a different order of efficacy as compared to the activation of PI-PLC; in terms of the maximal stimulation of DNA synthesis, the effects were: norepinephrine approximately PGF2 alpha > angiotensin II > vasopressin. Also, norepinephrine, PGF2 alpha, and angiotensin II, but not vasopressin, further enhanced the DNA synthesis when their concentrations were increased above those yielding maximal elevation of InsP3. In experiments where vasopressin and angiotensin II were combined, their effects on the DNA synthesis were additive while the InsP3 responses were not. The results show that the extent of the initial activation of PI-PLC is not the determinant for the magnitude of the growth effects of Ca(2+)-mobilizing hormones in hepatocytes. This suggests either (a) that the proliferative response to these agents is determined by the activity of PI-PLC at a later time, or its integral over an extended part of the prereplicative period, rather than by the acute activation, or (b) that additional, PI-PLC-independent, mechanisms are required.


Subject(s)
Angiotensin II/pharmacology , Calcium/physiology , Cell Division , Dinoprost/pharmacology , Inositol 1,4,5-Trisphosphate/metabolism , Liver/cytology , Norepinephrine/pharmacology , Phosphoric Diester Hydrolases/metabolism , Vasopressins/pharmacology , Animals , Cell Division/drug effects , DNA/biosynthesis , Enzyme Activation/drug effects , Epidermal Growth Factor/pharmacology , Male , Phosphatidylinositol Diacylglycerol-Lyase , Phosphoinositide Phospholipase C , Phosphorylases/metabolism , Rats , Rats, Wistar , Second Messenger Systems , Time Factors
12.
Pharmacol Toxicol ; 79(1): 15-22, 1996 Jul.
Article in English | MEDLINE | ID: mdl-8841091

ABSTRACT

Addition of 8-bromo-adenosine 3',5'-cyclic monophosphate (8-bromo-cAMP) or 8-(4-chlorophenylthio)-adenosine 3',5'-cyclic monophosphate (8-CPT-cAMP) to hepatocytes at the time of plating enhanced the acquisition of beta-adrenoceptors that occurs spontaneously upon culturing as primary monolayers. This effect was partially suppressed by the phosphodiesterase inhibitor isobutyl methylxanthine, and was mimicked by 8-bromo-AMP, 8-bromo-adenosine, and the adenosine kinase inhibitor 5'-amino-5'-deoxyadenosine. Agents that elevated the intracellular level of cAMP, such as glucagon and forskolin, and Sp-8-bromo-adenosine 3',5'-monophosphorothioate (Sp-8-bromo-cAMPS), a cAMP analogue that is resistant towards metabolic breakdown, did not significantly enhance beta-adrenoceptor expression when used alone, but glucagon enhanced the effect of 8-bromo-adenosine. 8-bromo-cAMP and 8-bromo-adenosine decreased cellular ATP-levels. These observations suggest that the enhanced beta-adrenoceptor acquisition was mediated mainly through the action of metabolites of 8-bromo-cAMP and 8-CPT-cAMP, although there may be a cAMP-mediated component in the effect. Several mechanisms, including depletion of ATP, are probably involved, and might affect beta-adrenoceptor degradation.


Subject(s)
8-Bromo Cyclic Adenosine Monophosphate/pharmacology , Cyclic AMP/analogs & derivatives , Liver/drug effects , Receptors, Adrenergic, beta/drug effects , Thionucleotides/pharmacology , 1-Methyl-3-isobutylxanthine/metabolism , 1-Methyl-3-isobutylxanthine/toxicity , 8-Bromo Cyclic Adenosine Monophosphate/metabolism , Adenylyl Cyclases/metabolism , Animals , Binding, Competitive , Cell Survival , Cells, Cultured , Cyclic AMP/metabolism , Cyclic AMP/pharmacology , Liver/cytology , Liver/metabolism , Male , Phosphodiesterase Inhibitors/metabolism , Phosphodiesterase Inhibitors/toxicity , Rats , Rats, Wistar , Receptors, Adrenergic, beta/metabolism , Thionucleotides/metabolism
13.
J Cell Physiol ; 164(3): 465-73, 1995 Sep.
Article in English | MEDLINE | ID: mdl-7650056

ABSTRACT

While many observations indicate that prostaglandins may act as positive regulators of hepatocyte proliferation, the underlying mechanisms are not known. We have examined some of the signal pathways in the growth response induced by prostaglandins in hepatocytes, with particular focus on adenylyl cyclase and phosphoinositide-specific phospholipase C. Adult rat hepatocytes were cultured as primary monolayers in serum-free medium in the presence of EGF and insulin. PGE2 or PGF2 alpha (added 0-3 h after plating) enhanced the incorporation of [3H]-thymidine into DNA (measured at 50 h); at 100 microM the stimulation was about threefold PGI2 and PGD2 also showed significant but smaller stimulatory effects. No significant increase in the level of cyclic AMP (cAMP) was detected in response to any of the prostaglandins. Low concentrations of glucagon (0.1-10 nM), a potent activator of hepatic adenylyl cyclase, or 8-bromo-cAMP (0.1-10 microM) enhanced the DNA synthesis. When 8-bromo-cAMP was used in maximally effective concentrations, no further stimulation was obtained by combining it with glucagon, whereas the effects of PGE2 and 8-bromo-cAMP were completely additive. All the prostaglandins also showed additivity with the effect of glucagon on the DNA synthesis. PGE2, PGF2 alpha, PGI2, and PGD2 increased intracellular inositol-1,4,5-trisphosphate (InsP3), with a relative order of efficacy roughly corresponding to their activity as stimulators of DNA synthesis. Increases in cytosolic free Ca2+, as measured in single cells, were elicited in a majority of the hepatocytes by all these prostaglandins at 1 microM. Supramaximal concentrations of vasopressin, a strong activator of phospholipase C in hepatocytes, acted additively with PGE2 on the DNA synthesis. Pretreatment of the hepatocytes with a concentration of pertussis toxin that prevented the inhibitory effect of PGE2 on glucagon-induced cAMP accumulation did not abolish the ability of PGE2 to stimulate the DNA synthesis. The results do not support a role for adenylyl cyclase activation in the stimulatory effect of prostaglandins on hepatocyte growth. While the data are compatible with an involvement of phosphoinositide-specific phospholipase C in the growth-promoting effect of prostaglandins in cultured rat hepatocytes, they suggest this may not be the sole mechanism.


Subject(s)
Adenylyl Cyclases/physiology , Cell Division/drug effects , Liver/cytology , Phosphoric Diester Hydrolases/physiology , Prostaglandins/pharmacology , Adenylate Cyclase Toxin , Animals , Calcium/metabolism , Cells, Cultured , Cyclic AMP/physiology , DNA/biosynthesis , Dinoprost/pharmacology , Dinoprostone/pharmacology , Epoprostenol/pharmacology , GTP-Binding Proteins/physiology , In Vitro Techniques , Male , Pertussis Toxin , Phosphatidylinositol Diacylglycerol-Lyase , Phosphatidylinositols/physiology , Prostaglandin D2/pharmacology , Rats , Rats, Wistar , Signal Transduction , Virulence Factors, Bordetella/pharmacology
14.
Eur J Pharmacol ; 246(2): 163-9, 1993 Jul 15.
Article in English | MEDLINE | ID: mdl-8397096

ABSTRACT

Long-term effects of cAMP on the surface expression of beta-adrenoceptors and adenylyl cyclase activity were investigated in primary cultures of rat hepatocytes. beta-Adrenoceptor density and catecholamine-responsive adenylyl cyclase activity increased during culturing in a biphasic manner, with a plateau of 10-20 h duration occurring approximately 10 h after plating. Treatment of hepatocyte cultures with 8-bromo-cAMP during the plateau period did not affect the density of beta-adrenoceptors. In contrast, addition of 8-bromo-cAMP, 8-chlorophenylthio-cAMP, forskolin or glucagon during a period of active recruitment of surface beta-adrenoceptors resulted in a suppression of the acquisition of beta-adrenoceptors. In both experimental situations there was a partial decrease in hormone-stimulated and basal adenylyl cyclase activity. The results suggest that cAMP exerts at least two types of long-term regulation of adenylyl cyclase in hepatocytes: a suppressive effect on beta-adrenoceptor acquisition, and a partial, nonselective decrease in adenylyl cyclase activity not involving beta-adrenoceptor down-regulation.


Subject(s)
Adenylyl Cyclases/metabolism , Cyclic AMP/pharmacology , Liver/metabolism , Receptors, Adrenergic, beta/drug effects , 8-Bromo Cyclic Adenosine Monophosphate/pharmacology , Animals , Cells, Cultured , Colforsin/pharmacology , Cyclic AMP/analogs & derivatives , Down-Regulation/drug effects , Glucagon/pharmacology , Iodocyanopindolol , Isoproterenol/pharmacology , Liver/cytology , Liver/drug effects , Male , Pindolol/analogs & derivatives , Rats , Rats, Wistar , Receptors, Adrenergic, beta/metabolism , Thionucleotides/pharmacology
15.
Pharmacol Toxicol ; 71(3 Pt 1): 165-72, 1992 Sep.
Article in English | MEDLINE | ID: mdl-1438037

ABSTRACT

The mutagenicity of senna glycosides and extracts of senna folium and senna fructus was investigated in the Salmonella typhimurium reversion assay. Senna glycosides were inactive in all strains, except for a slight, but significant increase in mutant frequency in TA102 in the absence and presence of liver microsomes. Extracts of senna fructus and senna folium demonstrated weak activity in TA97a, TA100 and TA102 in the presence of liver microsomes, and in TA97a and TA102 in the absence of liver microsomes. A strong increase in mutant frequency (3- to 5-fold above background frequency) was observed with all extracts in TA98 in the presence of liver microsomes. This activity increased further following enzymatic hydrolysis with hesperidinase of extracts of senna fructus from one source, and could be correlated to the release of the flavonol aglycones kaempferol and quercetin. The weak or lacking activity of anthraquinone aglycones in the tested strains of Salmonella typhimurium indicates that mutagenicity can not be attributed solely to the anthraquinone content of these plant materials. The chemical nature of other mutagenic components has not been elucidated.


Subject(s)
Anthraquinones/toxicity , Flavonoids , Kaempferols , Mutagens/toxicity , Senna Extract/toxicity , Animals , Biotransformation , Chromatography, High Pressure Liquid , Emodin/toxicity , Glycoside Hydrolases/metabolism , Microsomes, Liver/metabolism , Quercetin/analogs & derivatives , Quercetin/toxicity , Rats , Salmonella typhimurium/drug effects , Salmonella typhimurium/genetics
16.
J Cell Physiol ; 151(1): 164-71, 1992 Apr.
Article in English | MEDLINE | ID: mdl-1313818

ABSTRACT

Previous studies suggest that catecholamines may be involved in the regulation of liver growth. Considerable evidence implicates alpha 1-adrenergic mechanisms in the initiation of hepatocyte proliferation, while the role of beta-adrenoceptors is less clear. We have examined further the adrenergic regulation of hepatocyte DNA synthesis, using primary monolayer cultures. In hepatocytes that were also treated with epidermal growth factor and insulin, epinephrine or norepinephrine added early after the seeding strongly accelerated the rate of S phase entry. The beta-adrenergic agonist isoproterenol and the alpha-adrenergic agonist phenylephrine also stimulated the DNA synthesis, but were less efficient than epinephrine and norepinephrine. Experiments with the alpha 1-receptor blocker prazosine and the beta-receptor blocker timolol showed that the stimulatory effect of norepinephrine consisted of both an alpha 1- and a beta-adrenergic component. The alpha 1-component was most prominent in terms of maximal response at high concentrations of the agonist, but the beta-component contributed significantly and predominated at low concentrations (less than 0.1 microM) of norepinephrine. At later stages (about 40 h) of culturing norepinephrine strongly but reversibly inhibited the cells, acting at a point late in the G1 phase. This inhibition was mimicked by isoproterenol and abolished by timolol but was unaffected by prazosine, suggesting a beta-adrenoceptor-mediated effect. The results confirm the alpha 1-adrenoceptor-mediated stimulatory effect, but also show that beta-adrenoceptors may contribute to the growth stimulation by catecholamines. Furthermore, catecholamines, via beta-adrenoceptors and cyclic AMP, inhibit the G1-S transition, and may thus play a role in the termination of hepatic proliferation.


Subject(s)
Catecholamines/pharmacology , DNA/biosynthesis , Liver/cytology , Liver/metabolism , Receptors, Adrenergic, alpha/physiology , Receptors, Adrenergic, beta/physiology , Animals , Cell Division/drug effects , Cells, Cultured , Cyclic AMP/physiology , Dose-Response Relationship, Drug , Epidermal Growth Factor/pharmacology , Epinephrine/pharmacology , G1 Phase/physiology , Insulin/pharmacology , Isoproterenol/pharmacology , Liver/ultrastructure , Male , Norepinephrine/pharmacology , Phenylephrine/pharmacology , Prazosin/pharmacology , Rats , Rats, Inbred Strains , Receptors, Adrenergic, alpha/drug effects , Receptors, Adrenergic, beta/drug effects , S Phase/physiology , Time Factors , Timolol/pharmacology
17.
Pharmacol Toxicol ; 65(4): 249-51, 1989 Oct.
Article in English | MEDLINE | ID: mdl-2587507

ABSTRACT

Diuretics have been reported to alter arteriolar smooth muscle responses to adrenergic stimulation as well as lymphocyte beta-adrenoceptor density. We have investigated the effect of four days treatment with hydrochlorothiazide or furosemide (10 mg/kg and day) injected subcutaneously in the rat on alpha 1-,alpha 2- and beta-adrenoceptors in different tissues. alpha 1-Adrenoceptor density and affinity in whole kidney and heart ventricle as determined by 3H-prazosin binding (in the absence and presence of 10(-5) M phentolamine) were unaltered by diuretic treatment, as were beta-adrenoceptor density and affinity in whole kidney, heart ventricle and skeletal muscle as determined by 125I-iodocyanopindolol binding (in the absence and presence of 10(-7) M (-)-propranolol). The density of alpha 2-adrenoceptors in particulate fraction from whole kidney homogenate as determined by 3H-rauwolscine binding was 12.8% higher in the hydrochlorothiazide-treated group (P less than 0.05) and 15.0% higher in the furosemide-treated group (P less than 0.01) than in the control group. The receptor affinity was similar in the different groups. Thus diuretic-induced alterations in adrenoceptors reported in previous studies were not observed in this study.


Subject(s)
Diuretics/pharmacology , Receptors, Adrenergic/drug effects , Animals , Binding, Competitive/drug effects , Furosemide/pharmacology , Heart/drug effects , Hydrochlorothiazide/pharmacology , In Vitro Techniques , Iodine Radioisotopes , Kidney/drug effects , Kidney/metabolism , Kinetics , Male , Muscles/drug effects , Muscles/metabolism , Myocardium/metabolism , Radioligand Assay , Rats , Rats, Inbred Strains
18.
Pharmacol Toxicol ; 62(4): 199-202, 1988 Apr.
Article in English | MEDLINE | ID: mdl-2898776

ABSTRACT

The binding characteristics of 125I-iodocyanopindolol (125I-ICYP) and 3H-CGP-12177 to intact hepatocytes and a particulate fraction prepared from hepatocytes from sham-operated and partially hepatectomized rats were compared. In the particulate fraction, the beta-adrenoceptor number increased 4-10-fold after partial hepatectomy. 125I-ICYP binding to intact cells demonstrated a high fraction of non-specific binding, although phentolamine was included in the assay to reduce non-specific binding. 3H-CGP-12177 binding to intact cells also demonstrated a higher fraction of non-specific binding than in most cell types. In intact cells, the beta-adrenoceptor number increased 5-6-fold after partial hepatectomy. The number of receptors was comparable in intact cells and the broken cell preparation, indicating that receptors are conserved during the preparation of the particulate fraction.


Subject(s)
Liver/metabolism , Receptors, Adrenergic, beta/metabolism , Adrenergic beta-Antagonists/pharmacology , Animals , Hepatectomy , Iodocyanopindolol , Liver/cytology , Liver/drug effects , Male , Pindolol/analogs & derivatives , Pindolol/pharmacology , Propanolamines/pharmacology , Radioligand Assay , Rats , Rats, Inbred Strains
19.
Pharmacol Toxicol ; 61(4): 265-70, 1987 Oct.
Article in English | MEDLINE | ID: mdl-2893368

ABSTRACT

Inhibition of total 125I-ICYP binding to intact human mononuclear leucocytes at 32 degrees by propranolol and (+/-) CGP-12177 was biphasic. The high affinity component of 125I-ICYP binding, representing approximately 30% of total, was stereospecific, while the low affinity binding site was inhibited without stereospecificity. (-) Isoproterenol inhibited the high affinity component of 125I-ICYP binding only, with low affinity. By performing binding studies in intact cells at 4 degrees or in broken cell preparations at 37 degrees, the fraction of total 125I-ICYP binding representing specific binding was increased, and agonist affinity was high. Inhibition of 3H-CGP-12177 binding to intact cells at 32 degrees demonstrated a high fraction of specific binding and high agonist affinity. Computer-assisted analysis of total radioligand binding determined over a broad concentration range revealed two populations of saturable 125I-ICYP binding sites in intact cells as well as in broken cell preparations, while 3H-CGP-12177 binding demonstrated only one saturable binding site. The number of high affinity 125I-ICYP binding sites was comparable to the number of saturable 3H-CGP-12177 binding sites. Receptor numbers determined by analysis of total radioligand binding were comparable to receptor numbers determined by subtraction of non-specific binding, determined in the presence of a high concentration of competing ligand. Analysis of total radioligand binding was found to be a better procedure because it eliminates the use of an arbitrary concentration of unlabelled ligand and improves the accuracy of the assay.


Subject(s)
Neutrophils/analysis , Receptors, Adrenergic, beta/analysis , Adolescent , Adrenergic beta-Antagonists , Adult , Female , Humans , Iodine Radioisotopes , Iodocyanopindolol , Male , Middle Aged , Pindolol/analogs & derivatives , Propanolamines , Subcellular Fractions/drug effects , Subcellular Fractions/metabolism , Temperature
20.
Biochem Pharmacol ; 36(8): 1303-11, 1987 Apr 15.
Article in English | MEDLINE | ID: mdl-3036165

ABSTRACT

Incubation of human mononuclear leukocytes (MNL) in vitro with isoproterenol resulted in a rapid loss of surface beta-adrenoceptors, determined by radioligand binding at 4 degrees. Isoproterenol concentrations in the range of 10 nM to 100 microM resulted in significant down-regulation of beta-adrenoceptors. At a concentration of 1 microM isoproterenol, the time-dependent loss of surface beta-adrenoceptors closely paralleled the loss in isoproterenol-stimulated adenylate cyclase activity. If receptor number in intact cells was determined at 32 degrees, hardly any loss in receptor number was observed, due to reversal of down-regulation during the incubation period. When beta-adrenoceptor number in broken cell preparations was determined by [125I]cyanopindolol binding at 37 degrees no significant loss was observed, even after 2 hr of isoproterenol treatment, while [3H]CGP-12177 binding resulted in a similar reduction in binding sites as in intact cells. Reversal of loss in surface beta-adrenoceptors was rapid after 1 hr pretreatment with isoproterenol, but followed a biphasic time course after 4 hr pretreatment, with an initial rapid return of about 40% of the down-regulated receptors, followed by a slow, gradual reappearance of receptors. The results indicate that catecholamine exposure leads to a rapid sequestration of MNL surface beta-adrenoceptors away from the cell surface, to a compartment where they are inaccessible to the hydrophilic ligand [3H]CGP-12177 as well as to the lipophilic ligand [125I]cyanopindolol at 4 degrees. Up to 2 hr of isoproterenol treatment does not lead to any breakdown of sequestered beta-adrenoceptors, as they are still recognized by [125I]cyanopindolol binding in broken cell preparations.


Subject(s)
Isoproterenol/pharmacology , Leukocytes/analysis , Receptors, Adrenergic, beta/analysis , Adenylyl Cyclases/analysis , Adolescent , Adult , Dose-Response Relationship, Drug , Female , Humans , Iodocyanopindolol , Male , Middle Aged , Pindolol/analogs & derivatives , Pindolol/metabolism , Propanolamines/metabolism , Receptors, Adrenergic, beta/drug effects , Time Factors
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