Effects of levo- and dextrosimendan on NF-kappaB-mediated transcription, iNOS expression and NO production in response to inflammatory stimuli.

BACKGROUND AND PURPOSE: Levosimendan is used in the treatment of decompensated heart failure. It increases the contractility of the myocardium by sensitizing troponin C to calcium. In addition, levosimendan has been reported to have beneficial effects in experimental models of septic shock. Because heart failure and sepsis have been associated with excessive nitric oxide (NO) production through inducible NOS (iNOS), we investigated the effects of the simendans on NO production and iNOS expression and on generation of pro-inflammatory cytokines. EXPERIMENTAL APPROACH: Macrophages and fibroblasts were exposed to inflammatory stimuli to induce iNOS expression. Proteins were measured by western blot and mRNA expression was determined by quantitative RT-PCR. Promoter activity and nuclear factor-kappaB (NF-kappaB) and the gamma-activated site (GAS; binding site for signal transducer and activator of transcription 1; STAT1)-mediated transcription were investigated using luciferase reporter constructs. Cytokines tumour necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) were measured by ELISA. KEY RESULTS: Levosimendan and dextrosimendan decreased NO production in a dose-dependent manner in cells exposed to inflammatory stimuli. The simendans decreased iNOS protein and mRNA expression but did not affect iNOS mRNA decay. These compounds decreased iNOS promoter activity and inhibited NF-kappaB-mediated transcription but not that mediated by STAT1/GAS. The simendans reduced IL-6 production slightly but they had no effect on TNF-alpha synthesis. CONCLUSIONS AND IMPLICATIONS: The simendans downregulated NF-kappaB-dependent transcription and decreased iNOS promoter activity, iNOS expression and NO production. These mechanisms may contribute to their beneficial clinical effects.

The COMT inhibitor, entacapone, reduces levodopa-induced elevations in plasma homocysteine in healthy adult rats.

Levodopa treatment has been shown to increase plasma homocysteine levels in Parkinson's disease (PD) patients and this may lead to an increased risk for coronary arterial diseases. Levodopa is metabolised via O-methylation by catechol-O-methyltransferase (COMT) using S-adenosyl-L-methionine (SAM) as the methyl donor, this leading to the subsequent formation of homocysteine. In this study, the effects of the COMT inhibitor, entacapone, on levodopa-induced hyperhomocysteinaemia were studied in rats. Using a single dose acute treatment paradigm, entacapone (10 or 30 mg/kg) prevented the levodopa (30 or 100 mg/kg) induced rise in plasma homocysteine levels in a dose-dependent manner. Five-day sub-chronic treatment with levodopa (3 x 100 mg/kg per day) resulted in a marked rise in plasma homocysteine levels when measured 2 hours post-treatment on Day 5. These levels fell but remained greater than baseline at 8 hours post-treatment on Day 5. Consistent with findings in the acute treatment test paradigm, the co-administration of entacapone (30 mg/kg) significantly (p<0.001) reduced levodopa-induced hyperhomocysteinaemia for up to 2 hours post-treatment on Day 5 of the sub-chronic study. These results suggest that entacapone may reduce levodopa-induced hyperhomocysteinaemia in PD patients.

Different toxicological profile of two COMT inhibitors in vivo: the role of uncoupling effects.

The toxicity profiles of entacapone and tolcapone, novel catechol- O-methyl-transferase (COMT) inhibitors, have been reported to differ from each other. It has also been shown that tolcapone, but not entacapone, is a potent uncoupler of oxidative phosphorylation in vitroat low micromolar concentrations. Signs of hepatotoxicity induced by tolcapone treatment have been previously reported in toxicological studies and in clinical use. The present study was designed to investigate the mechanism of hepatotoxicity of tolcapone and its possible relationship to uncoupling of oxidative phosphorylation in vivo. A 15-day oral toxicity study with entacapone or tolcapone (300 and 500 mg/kg/day) was carried out, and 2, 4-dinitrophenol (DNP), a known uncoupling agent of oxidative phosphorylation, served as a positive reference substance (20 mg/kg/day). No treatment related findings were observed in entacapone-treated rats. Clinical chemistry parameters regarding hepatocellular damage were increased in tolcapone and DNP-treated animals. The energy status measured as ATP/ADP ratio from the liver samples and energy charge (EC) in liver cell mitochondria were diminished both in tolcapone- and in DNP-treated rats. These signs together with clinical symptoms consisting of increased respiration, decreased activity and drowsiness, and elevation of the rectal body temperature observed in tolcapone- and DNP-treated animals suggest a relationship between the treatment and uncoupling of oxidative phosphorylation in vivo.

Nitecapone inhibits myeloperoxidase in vitro and enhances functional performance after 8 h of ischemia in experimental heart transplantation.

Nitecapone (NC) has been shown to have beneficial effects on the functional recovery of rat hearts in Langendorff-preparation. The present study was executed to evaluate the effect of NC on preservation of grafts in heart transplantation and the role of NC in the inhibition of granulocyte infiltration. Donor hearts were perfused and stored at +4 degrees C for 8 h in either Ringer solution in the control-group (C-group, n = 26) or in NC (50 microM) added Ringer solution (NC-group, n = 18). The heterotopic heart transplantation was performed. The rats in both groups were killed at either 10 min or 60 min after release of the aortic clamp and tissue samples were obtained for antioxidative capacity, myeloperoxidase activity, and lipid peroxidation measurements. In vitro studies were performed using sodium azide or nitecapone to inhibit myeloperoxidase (MPO) activity of isolated human leukocytes. A total of 61% of the grafts began to beat in the NC-group, compared to 46% in the control group. Using an arbitrary scale of functional performance, only 33% (4/12) of the grafts were classified as well functioning in the control group, compared to 82% (9/11) in the NC-group (P<0.05). MPO activity was equal in both groups after 10 min but significantly lower after 60 min in the NC-group as compared to the control group (P<0.05). In vitro studies demonstrated that NC inhibits 50% of purified MPO activity at a concentration of 10 microM. NC did not significantly affect lipid peroxidation or the preservation of endogenous antioxidants. Since NC inhibited myeloperoxidase both in vitro and in vivo, it seems that the positive effects of NC on graft preservation may be mediated via the inhibition of granulocyte infiltration.

The Effect of Nitecapone, a New Antioxidant, on Myocardial Function After Aortic Cross-clamping in Experimental Heart Ischemia.

During aortic cross-clamping, the myocardium suffers from global ischemia, which is followed by reperfusion after declamping. The generation of free oxygen radicals increases during reperfusion, resulting in arrhythmias and impaired cardiac function. This study was conducted to evaluate the effect of a novel antioxidant nitecapone (NC) on cardiac reperfusion injury in vivo. Twelve pigs were anesthetized and after sternotomy the aorta and the right atrium were cannulated for cardiopulmonary bypass. The heart was arrested with either +4 degreesC crystalloid cardioplegia alone in the control group (n = 6) or cardioplegia with NC (50 µM) added in the NC group (n = 6). Cardioplegia was added every 20 minutes. After 1 hour of aortic cross-clamping, blood samples for oxidative stress analysis were taken, and hemodynamic profile surveillance continued for 90 minutes. Heart rate (p = 0.04) and left ventricular end diastolic pressure (LVEDP) (p = 0.04) were significantly lower in the NC group than in the C group after aortic declamping. Cardiac output and myocardial contractility (dP/dtmax) were also enhanced in the group receiving NC, but the difference was not statistically significant. At 30 minutes after reperfusion, the coronary production (coronary sinus-aorta) of thiobarbituric acid reactive substances correlated inversely with cardiac output (r = -0.90, p = 0.001) and stroke volume (r = -0.82, p = 0.007). The effect of NC on lipid peroxidation seems to be modest and therefore the target of NC is unclear. NC would appear, however, to be a beneficial additive in the crystalloid cardioplegia in terms of functional recovery.

Nitecapone is of benefit to functional performance in experimental heart transplantation.

In heart transplantation, global ischemia of a graft is followed by reperfusion injury. The formation of oxygen free radicals induces arrhythmias and impairs functional recovery of the graft. This study was executed to evaluate the effect of the new antioxidant, nitecapone, on ischemia-reperfusion injury in heart transplantation in rats. Donor hearts were perfused and stored at +4 degrees C for 2 h in either Ringer's solution in the control group (C-group, n = 26) or Ringer's solution with nitecapone (NC) added (NC-group, n = 18). The donor aorta was anastomosed to the recipient's abdominal aorta and the pulmonary artery to the recipient's inferior vena cava. The grafts were classified into three categories based on the functional recovery. The rats in both groups were killed at 10, 30, or 60 min after release of the aortic clamp. Tissue samples for chemiluminescence were obtained from the left ventricle, the right ventricle, and the septum of the heart. All grafts in the NC-group (18/18) began beating after release of the aortic clamp, whereas only 50% (13/26) of the grafts in the C-group recovered (P < 0.0004). Chemiluminescence analysis showed lipid peroxidation values to be higher in the C-group than the NC-group up to 1 h after reperfusion. Also, the right ventricle samples showed lower chemiluminescence values in the NC-group than in the C-group. In conclusion, our results do not support the theory that different regions of the heart have different vulnerability to ischemia-reperfusion injuries. Nitecapone has a beneficial effect on the preservation of the grafts in terms of functional recovery.

Entacapone, a novel catechol-O-methyltransferase inhibitor for Parkinson's disease, does not impair mitochondrial energy production.

Entacapone, a novel mainly peripherally acting catechol-O-methyltransferase inhibitor used in the treatment of Parkinson's disease, was evaluated for its possible uncoupling activity in cell culture, in rat liver mitochondria, and in isolated guinea-pig heart. Entacapone did not stimulate respiration in the L1210 murine T cell lymphoma cell line at the concentrations studied (5-40 microM). Furthermore, entacapone neither increased mitochondrial respiration nor impaired cardiac function at pharmacologically relevant concentrations (< 10 microM). In fact, the threshold concentration for increased mitochondrial oxygen consumption was 20 microM and half-maximal stimulation of respiration was not detected until 58 microM. Surprisingly, tolcapone, another catechol-O-methyltransferase inhibitor, which acts both peripherally and centrally, stimulated respiration in L1210 cells at the lowest concentration studied (5 microM). In addition, 1 microM tolcapone increased mitochondrial respiration, indicating that it caused uncoupling at a much lower concentration than that of 2,4-dinitrophenol, a well-known uncoupler of oxidative phosphorylation. Tolcapone also impaired the mechanical function and oxygen consumption of the isolated guinea-pig heart at 1 microM. These results show that peripherally acting entacapone, unlike the brain-penetrating tolcapone, is a safe catechol-O-methyltransferase inhibitor for the treatment of Parkinson's disease, since it does not interfere with mitochondrial energy metabolism at pharmacologically effective concentrations.

Cardiac troponin C as a target protein for a novel calcium sensitizing drug, levosimendan.

The role of cardiac troponin C (cTnC) as a target protein for the calcium sensitization by levosimendan, pimobendan, MCI-154 and EMD 53998 was evaluated using purified recombinant human cTnC. For determination of calcium- and magnesium-dependent binding of the compounds to cTnC a new type of cTnC-HPLAC column was used. Furthermore, dansylated cTnC was utilized to study the effect of the calcium sensitizing compounds on calcium-induced conformation of cTnC. Only levosimendan showed calcium-dependent and to a lesser extent magnesium-dependent retention in the cTnC column. The findings indicate that levosimendan binds both to the N-terminal and C-terminal domains of cTnC. In agreement with this, only levosimendan shifted the calcium-induced fluorescence curve of dansylated cTnC to the left. In the control experiments Ca50 and KCa2+ were calculated to be 2.73 microM and 4 x 10(5) M-1, respectively. Levosimendan at 3 microM decreased the value of Ca50 to 1.19 microM. In conclusion, it is suggested that the mechanism of calcium sensitizing effect of levosimendan, unlike that of the other calcium sensitizers, is based on calcium-dependent binding to the N-terminal domain of cTnC. This is proposed to amplify the trigger of contraction induced by cTnC in the cardiac muscle.

Troponin C-mediated calcium sensitization induced by levosimendan does not impair relaxation.

Levosimendan is a novel positive inotropic drug targeted to increase contraction force of the heart through its calcium-dependent binding to troponin C (cTnC). We investigated the calcium-sensitizing effect of levosimendan on contractile proteins as well as its positive inotropic and lusitropic effects in paced guinea pig papillary muscle. We also studied the effect on energy consumption of myosin-actin crossbridges in a myosin ATPase assay. The calcium sensitization induced by levosimendan in fibers skinned with saponin was dependent on the perforation velocity of cell membranes. Levosimendan was almost ineffective in slowly perforated fibers, but was the most potent calcium sensitizer in fibers with rapidly perforated cells. The perforation-dependent calcium sensitization was probably due to changes in phosphorylation state of contractile proteins during the slow dissection of fibers. It is noteworthy that the calcium-sensitizing effect of levosimendan was not affected by acidic pH. Levosimendan at therapeutically relevant (0.3-10 microM) concentrations markedly increased calcium sensitivity both at pH 6.7 and 7.0, being more potent than EMD 53998, pimobendan, and MCI-154. The lack of effect of levosimendan on maximum tension supports the hypothesis that levosimendan increases calcium sensitivity through its action on cTnC. Unlike EMD 53998, levosimendan did not increase myosin ATPase activity, indicating that it did not increase the cycling rate of myosinactin crossbridges. In paced papillary muscles, levosimendan induced positive inotropic effect without changing relaxation time. Thus, levosimendan was devoid of the main negative factors described for calcium sensitizers.

Antioxidant properties of nitecapone are potentiated by glutathione.

The effect of nitecapone (3[(3,4-dihydroxy-5-nitrophenyl) methylene]-2,4-pentanedione) on lipid peroxidation in guinea pig liver microsomes was assessed with the thiobarbituric acid assay. Nitecapone inhibited dose-dependently hydroxyl and peroxyl radical induced lipid peroxidation with IC50-values of 11.1 microM and 16.2 microM, respectively. Nitecapone was an effective antioxidant in native microsomes, where it potentiated glutathione-dependent protection against oxidative stress. Nitecapone provided dose-dependent reduction in lipid peroxidation lasting up to three hours in the presence of glutathione. The long-lasting effect was lost, when the microsomes were boiled. These data suggest that nitecapone is an effective scavenger of oxygen derived free radicals, and that its antioxidant properties are potentiated by glutathione.

Binding of a new Ca2+ sensitizer, levosimendan, to recombinant human cardiac troponin C. A molecular modelling, fluorescence probe, and proton nuclear magnetic resonance study.

The binding of a new calcium sensitizer, levosimendan, to human cardiac troponin C (cTnC) is described. Fluorescence studies done on dansylated recombinant human cTnC and a site-directed mutant showed that levosimendan modulated the calcium-induced conformational change in cTnC, and revealed the role of Asp-88 in the binding of the drug to the NH2-terminal domain of cTnC. Furthermore, NMR studies performed on the NH2-terminal fragment of cTnC showed a spatial proximity between levosimendan and Met81, Met85, and Phe77 in the drug-protein complex. These data were used to build an optimized model of the drug-protein complex, in which levosimendan binds cTnC at the hydrophobic pocket of the NH2-terminal domain. The role of the binding of levosimendan to cTnC in the pharmacological action of this drug in vivo is discussed.

Protection by nitecapone against sodium taurocholate-induced damage to cultured gastric cells.

The goal of this study was to observe if nitecapone protected against taurocholate-induced damage in primary cultured rat gastric mucosal cells, as well as in a well-differentiated human gastric epithelial cell line (MKN 28). Prostaglandins were measured to analyze the protection mechanism. In primary rat gastric mucosal cell culture, nitecapone 125-250 microM protected the cells significantly against damage induced by sodium taurocholate, increasing cell viability by 31-38%. In the human gastric epithelial cell line, in which mitochondrial activity was measured as an indication of cell viability, nitecapone (62.5-250 microM) protected the cells against sodium taurocholate-induced damage by 12-20%. Prostaglandin E2, thromboxane B2, and 6-keto-prostaglandin F1 alpha measurements in the primary cultured rat gastric mucosal cells showed that nitecapone (125 microM and 250 microM) significantly stimulated prostaglandin E2 production (84.7% and 61.0%, respectively), and inhibited thromboxane B2 formation (50% at 250 microM), while the 6-keto-prostaglandin F1 alpha formation was unaffected. Nitecapone had no effect on prostaglandin E2 production in the MKN 28 epithelial cell line. Indomethacin or aspirin, at concentrations that did not affect cell viability, antagonized the stimulative effect of nitecapone on prostaglandin E2 formation in the primary cultured rat gastric mucosal cells. Although the prostaglandin E2 synthesis was blocked, nitecapone still protected against cell damage induced by taurocholate. These results demonstrated the direct and efficacious protection of nitecapone on gastric cell level and suggest that the "cytoprotection" by nitecapone against taurocholate may not be mediated through the mechanism of stimulated synthesis of prostaglandin E2.

Biochemical and pharmacological properties of a peripherally acting catechol-O-methyltransferase inhibitor entacapone.

Entacapone, OR-611, was found to be a potent peripherally acting inhibitor of catechol-O-methyltransferase (COMT). IC50 values of 10 nmol/l and 160 nmol/l were obtained for rat duodenum and liver-soluble COMT, respectively. There were no effects on other catecholamine metabolizing enzymes. Entacapone showed reversible, tight-binding type of inhibition of soluble rat liver COMT with a Ki-value of 14 nmol/l and it also caused 50% inhibition of rat duodenal, erythrocyte, liver and striatal COMT activity 1 h after oral dosing with 1.1, 5.4, 6.7 and 24.2 mg/kg, respectively. However, penetration of entacapone into the brain was poor, since the formation of homovanillic acid (HVA), the O-methyl metabolite of dopamine in the striatum, was not reduced, even after the highest dose of 30 mg/kg. In rat blood serum, the concentration of 3-O-methyldopa (3OMD), the O-methylated product of L-dopa, was reduced in a dose-dependent manner, and the concentration of L-dopa was increased after the administration of entacapone (3-30 mg/kg p.o.) together with L-dopa + carbidopa. These changes were reflected, in the striatum, by a significant rise in the dopamine concentration and a reduction in the 3OMD concentration. Consequently, when entacapone was added to the treatment with L-dopa + carbidopa, the dose of L-dopa could be lowered from 50 mg/kg to 15 mg/kg in order to produce the same striatal dopamine concentrations as with 50 + 50 mg/kg of L-dopa + carbidopa alone.

PLS modelling of structure-activity relationships of catechol O-methyltransferase inhibitors.

Quantitative structure-activity analysis was carried out for in vitro inhibition of rat brain soluble catechol O-methyltransferase by a series (N = 99) of 1,5-substituted-3,4-dihydroxybenzenes using computational chemistry and multivariate PLS modelling of data sets. The molecular structural descriptors (N = 19) associated with the electronics of the catecholic ring and sizes of substituents were derived theoretically. For the whole set of molecules two separate PLS models have to be used. A PLS model with two significant (crossvalidated) model dimensions describing 82.2% of the variance in inhibition activity data was capable of predicting all molecules except those having the largest R1 substituent or having a large R5 substituent compared to the NO2 group. The other PLS model with three significant (crossvalidated) model dimensions described 83.3% of the variance in inhibition activity data. This model could not handle compounds having a small R5 substituent, compared to the NO2 group, or the largest R1 substituent. The predictive capability of these PLS models was good. The models reveal that inhibition activity is nonlinearly related to the size of the R5 substituent. The analysis of the PLS models also shows that the binding affinity is greatly dependent on the electronic nature of both R1 and R5 substituents. The electron-withdrawing nature of the substituents enhances inhibition activity. In addition, the size of the R1 substituent and its lipophilicity are important in the binding of inhibitors. The size of the R1 substituent has an upper limit. On the other hand, ionized R1 substituents decrease inhibition activity.

Inhibition of human erythrocyte and gastroduodenal catechol-O-methyltransferase activity by nitecapone.

The effect of increasing single oral doses of the novel catechol-O-methyltransferase (COMT) inhibitor, nitecapone, on enzyme activity in red cells (RBC) and gastroduodenal COMT activity has been studied in healthy male volunteers. A dose-dependent decrease in RBC COMT activity was seen in all cases after 1 to 150 mg of the drug. The highest dose of 300 mg did not produce much more inhibition of COMT than 150 mg. The inhibition was not complete; at the highest doses the COMT activity was reduced by 50-60%. The effect and the duration of the inhibition in RBC COMT was strongly correlated with plasma nitecapone concentrations in the dose range up to 150 mg. RBC COMT activity recovered fully in 4 h after medication. Gastric mucosal COMT activity was several-fold higher than that in RBCs. It was also dose-dependently inhibited at the two doses (25 and 100 mg) studied. The inhibition of gastric and duodenal COMT was greater than that in RBCs. This also indicates that nitecapone is locally active in the gastroduodenal tract. The results confirm nitecapone as a potent COMT inhibitor in human tissues. New COMT inhibitors may provide a valuable approach to the treatment of Parkinson's disease in combination with L-dopa and dopa decarboxylase inhibitor therapy.

Effect of a novel catechol-O-methyltransferase inhibitor, nitecapone, on the metabolism of L-dopa in healthy volunteers.

A new catechol-O-methyltransferase (COMT) inhibitor, nitecapone, was given in increasing doses of 0-100 mg concomitantly with L-Dopa/carbidopa (100/25 mg or 100/100 mg) to healthy male volunteers. Plasma concentrations of L-Dopa, 3-O-methyldopa (3-OMD), 3,4-dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA), as well as the excretion of catecholamine metabolites in urine were followed to evaluate the changes in the metabolism of L-Dopa after nitecapone. Plasma concentrations of nitecapone and the soluble COMT activity in erythrocytes were also measured. The area under the plasma concentration-time curves (AUC) values for plasma nitecapone, L-Dopa and its metabolites were calculated. Nitecapone dose-dependently inhibited the soluble COMT activity in erythrocytes at 30 min after drug intake. Nitecapone slightly but significantly increased the relative bioavailability of L-Dopa. The AUC values of plasma 3-OMD decreased dose-dependently after nitecapone, and those of HVA decreased less, whereas the AUC values of DOPAC increased significantly. The elevation of the dose of carbidopa from 25 to 100 mg increased the AUC value of L-Dopa, but the effect of nitecapone was not clearly modified. Nitecapone decreased the excretion of the methylated dopamine metabolites 3-methoxytyramine (3-MT) and HVA at an L-Dopa/carbidopa dose of 100/25 mg. At a dose of 100/100 mg, the excretion of metanephrine, in addition to 3-MT and HVA, was also significantly decreased by nitecapone. The biochemical changes in L-Dopa metabolism and erythrocyte COMT activity indicate that nitecapone is an active COMT inhibitor in humans, when given orally in single doses. The changes in L-Dopa metabolism by COMT inhibitor warrant further clinical studies in Parkinson's disease.

Inhibition of rat liver and duodenum soluble catechol-O-methyltransferase by a tight-binding inhibitor OR-462.

The inhibition kinetics of rat liver and duodenum soluble catechol-O-methyltransferase (COMT) with a disubstituted catechol OR-462 was studied. After preincubation of the enzyme and inhibitor in the presence of magnesium and S-adenyosylmethionine, an inhibition about thirty times greater than that without preincubation was observed. Reversible tight-binding inhibition was demonstrated with Ki values of 0.7 nM and 1.0 nM for liver and duodenum enzyme, respectively. Km values of 53.4 microM and 56.9 microM for substrate 3,4-dihydroxybenzoic acid and 23.0 microM and 17.5 microM for S-adenosylmethionine were calculated for liver and duodenum enzyme, respectively. A catalytic number of 24/min for liver soluble COMT was calculated.

Purification of rat liver soluble catechol-O-methyltransferase by high performance liquid chromatography.

The soluble form of catechol-O-methyltransferase (EC 2.1.1.6) from rat liver was purified to homogeneity by high-performance anion-exchange chromatography and high-performance gel-filtration chromatography. The specific activity of the final pool was 270 U/mg protein. The purification was 1180-fold and recovery of the enzyme activity was 15%. During this rapid and gentle purification there were no problems with loss of activity, and the estimated half of the final purified enzyme pool was 5.5 days at +4 degrees C. The only additive used was phenylmethylsulfonylfluoride in the homogenizing buffer.

Synthesis of some novel potent and selective catechol O-methyltransferase inhibitors.

A series of disubstituted catechol derivatives was synthesized and tested as potential COMT inhibitors. The most active compounds were more than 1000 times more potent (IC50 = 3-6 nM) in vitro than the known COMT inhibitor, 3',4'-dihydroxy-2-methylpropiophenone (U 0521, IC50 = 6000 nM). The new compounds were also highly selective COMT inhibitors with no activity against other essential enzymes involved in the synthesis and metabolism of catecholamines.