Brain pharmacokinetics and biodistribution of 11C-labeled isoproterenol in rodents.

INTRODUCTION: Isoproterenol is a non-selective ß receptor agonist, which is a drug approved for bradycardia and bronchial asthma in many countries. Recently, isoproterenol has been reported to have the potential as a drug for the treatment of Alzheimer's disease by inhibiting the aggregation of tau protein. Isoproterenol is a highly potent drug causing increases in heart rates even when its plasma concentration is very low. Thus, it is critical to know if potentially effective therapeutic levels of isoproterenol can be achieved, maintaining safe plasma levels without any untoward pharmacological effects. The purpose of the study is to investigate the brain pharmacokinetics and biodistribution of 11C-labeled isoproterenol in rodents. METHODS: We performed positron emission tomography (PET) brain imaging and biodistribution studies of [11C]isoproterenol. 120-min scans with arterial blood sampling were performed in rats. Additionally, plasma and brain homogenates were analyzed with radio-HPLC to characterize its metabolite profiles. As a measure of [11C]isoproterenol brain uptake, total distribution volumes were determined by a pharmacokinetic compartment model. Biodistribution of [11C]isoproterenol was investigated in mice at six-time points from 1-min to 90-min after injection. RESULTS: We found a modest brain uptake of [11C]isoproterenol. Its brain pharmacokinetics showed that the concentration of isoproterenol in the brain at equilibrium was about two-fold higher than in the plasma (total distribution volumes 2.0 ± 0.2 cm3/mL). Only unmetabolized isoproterenol was detected in the brain at 30 min after injection, although isoproterenol was rapidly metabolized in plasma. The biodistribution study showed that isoproterenol and its metabolite are excreted mainly via the urinary system. CONCLUSIONS, ADVANCES IN KNOWLEDGE AND IMPLICATIONS FOR PATIENT CARE: In this study, we have shown that rat brain concentrations of isoproterenol are only two-fold of that in plasma at equilibrium. If the brain pharmacokinetics are similar in the human brain, it may be difficult to achieve potentially therapeutic levels of this drug safely in humans. Further studies appear warranted to investigate the brain pharmacokinetics in humans with PET using [11C]isoproterenol.

Influence of perfusate temperature on nasal potential difference.

Nasal potential difference (NPD) quantifies abnormal ion transport in cystic fibrosis. It has gained acceptance as an outcome measure for the investigation of new therapies. To quantify the effect of solution temperature on NPD, we first examined the effect of switching from room temperature (20-25°C) to warmed (32-37°C) solutions and vice versa during each perfusion step. Secondly, standard protocols were repeated at both temperatures in the same subjects. Changing solution temperature did not alter NPD during perfusion with Ringer's solution (<1 mV) (p>0.1). During perfusion with zero chloride solution, changing from room temperature to warmed solutions tended to decrease absolute NPD (i.e. it became less negative) by 0.9 mV (p>0.1); changing from warmed to room temperature increased NPD by 2.1 mV (p<0.05). During isoprenaline perfusion, changing from room temperature to warmed solutions increased NPD by 1.5 mV (p<0.01) and from warmed to room temperature decreased NPD by 1.4 mV (p<0.05). For full protocols at room temperature or warmed in the same subjects, mean values were similar (n = 24). During warmed perfusion, group results for total chloride response had a larger standard deviation. As this increased variability will probably decrease the power of trials, this study suggests that solutions at room temperature should be recommended for the measurement of NPD.

Comparison of electrophysiological and antiarrhythmic effects of vernakalant, ranolazine, and sotalol in canine pulmonary vein sleeve preparations.

BACKGROUND: Vernakalant (VER) is a relatively atrial-selective antiarrhythmic drug capable of blocking potassium and sodium currents in a frequency- and voltage-dependent manner. Ranolazine (RAN) is a sodium-channel blocker shown to exert antiarrhythmic effects in pulmonary vein (PV) sleeves. dl-Sotalol (SOT) is a ß-blocker commonly used in the rhythm-control treatment of atrial fibrillation. This study evaluated the electrophysiological and antiarrhythmic effects of VER, RAN, and SOT in canine PV sleeve preparations in a blinded fashion. METHODS: Transmembrane action potentials were recorded from canine superfused PV sleeve preparations exposed to VER (n = 6), RAN (n = 6), and SOT (n = 6). Delayed afterdepolarizations were induced in the presence of isoproterenol and high-calcium concentrations by periods of rapid pacing. RESULTS: In PV sleeves, VER, RAN, and SOT (3-30 µM) produced small (10-15 ms) increases in action potential duration. The effective refractory period, diastolic threshold of excitation, and the shortest S(1)-S(1) cycle length permitting 1:1 activation were significantly increased by VER and RAN in a rate- and concentration-dependent manner. VER and RAN significantly reduced V(max) in a concentration- and rate-dependent manner. SOT did not significantly affect the effective refractory period, V(max), diastolic threshold of excitation, or the shortest S(1)-S(1) cycle length permitting 1:1 activation. All 3 agents (3-30 µM) suppressed delayed afterdepolarization-mediated triggered activity induced by isoproterenol and high calcium. CONCLUSIONS: In canine PV sleeves, the effects of VER and RAN were similar and largely characterized by concentration- and rate-dependent depression of sodium-channel-mediated parameters, which were largely unaffected by SOT. All 3 agents demonstrated an ability to effectively suppress delayed afterdepolarization-induced triggers of atrial arrhythmia.

Ureteral selectivity of intravenous ß-adrenoceptor agonists in pig model of acute ureteral obstruction: comparison of KUL-7211, a selective ß2/ß3 agonist, with isoproterenol, terbutaline, and CL-316243.

OBJECTIVES: To compare the potency and ureteral selectivity of the selective ß(2)/ß(3)-adrenoceptor agonist KUL-7211 with those of the nonselective ß-adrenoceptor agonist isoproterenol, selective ß(2)-adrenoceptor agonist terbutaline, and selective ß(3)-adrenoceptor agonist CL-316243, we performed the study using an isolated porcine ureter and a porcine model of acute unilateral ureteral obstruction. METHODS: The effects of the drugs on the 80-mM KCl-induced contraction of the ureteral segments isolated from male pigs were evaluated using a functional experimental technique. Anesthetized male miniature pigs with complete obstruction of the left lower ureter were used to evaluate the effects of the cumulative intravenous drug administration on the elevated intraureteral pressure and mean blood pressure. RESULTS: The KCl-induced contractions in the isolated ureter were concentration-dependently attenuated by KUL-7211, isoproterenol, terbutaline, and CL-316243, with a rank order of potency of 6.26, 6.98, 5.41, and 5.41, respectively. In the anesthetized pigs, all 4 drugs reduced the unilateral ureteral obstruction-induced elevated intraureteral pressure in a dose-dependent manner, with KUL-7211 reducing it with a lower hypotensive effect than either isoproterenol or terbutaline. The ureteral selectivity (defined as the ratio of the effective dose to decrease the mean blood pressure by 25% to the effective dose to decrease the intraureteral pressure by 50%) of KUL-7211 (1.5) was significantly greater than that of isoproterenol (0.04) or terbutaline (0.43). CONCLUSIONS: The present results have demonstrated that in pigs, KUL-7211 is a potent ureteral relaxant with a relatively small hypotensive effect. A selective ß(2)/ß(3)-adrenoceptor agonist, such as KUL-7211, warrants additional investigation as a potentially useful drug for the promotion of stone passage in patients with urolithiasis.

Influence of plasma protein binding on pharmacodynamics: Estimation of in vivo receptor affinities of beta blockers using a new mechanism-based PK-PD modelling approach.

The objective of this investigation was to examine in a systematic manner the influence of plasma protein binding on in vivo pharmacodynamics. Comparative pharmacokinetic-pharmacodynamic studies with four beta blockers were performed in conscious rats, using heart rate under isoprenaline-induced tachycardia as a pharmacodynamic endpoint. A recently proposed mechanism-based agonist-antagonist interaction model was used to obtain in vivo estimates of receptor affinities (K(B,vivo)). These values were compared with in vitro affinities (K(B,vitro)) on the basis of both total and free drug concentrations. For the total drug concentrations, the K(B,vivo) estimates were 26, 13, 6.5 and 0.89 nM for S(-)-atenolol, S(-)-propranolol, S(-)-metoprolol and timolol. The K(B,vivo) estimates on the basis of the free concentrations were 25, 2.0, 5.2 and 0.56 nM, respectively. The K(B,vivo)-K(B,vitro) correlation for total drug concentrations clearly deviated from the line of identity, especially for the most highly bound drug S(-)-propranolol (ratio K(B,vivo)/K(B,vitro) approximately 6.8). For the free drug, the correlation approximated the line of identity. Using this model, for beta-blockers the free plasma concentration appears to be the best predictor of in vivo pharmacodynamics.

Time course and predictors of autonomic dysfunction after ablation of the slow atrioventricular nodal pathway.

Withdrawal of parasympathetic tone has been reported after ablation in the posteroseptal right atrium and has been attributed to injury of vagal efferent fibers. The purpose of this study was to assess the time course and predictors of autonomic dysfunction after slow pathway ablation. In 30 patients with AV nodal reentrant tachycardia, time- and frequency-domain measures of heart rate variability (HRV) were measured before, 30 minutes after, and 1 day after slow pathway ablation. There were significant reductions in mean RR interval (724 +/- 163 vs 836 +/- 164 ms, P < 0.05), SD of RR intervals (29 +/- 17 vs 40 +/- 18 ms, P < 0.05), root mean squared difference (15 +/- 8 vs 29 +/- 17 ms, P < 0.05), and high frequency power (4.1 +/- 0.4 vs 4.5 +/- 0.6 log10ms2, P < 0.05) 30 minutes after ablation. However, these parameters returned to baseline 1 day after ablation. Multivariate regression identified isoproterenol dose during the diagnostic study (P = 0.02) and radiofrequency duration (P = 0.02) as statistically significant predictors of heart rate change (R2= 0.45). These findings suggest that changes in autonomic tone after ablation in the posteroseptal right atrium are transitory and resolve within 1 day of the procedure. These short-term changes may be related to procedural variables rather than direct injury to vagal efferent fibers.

In vivo and in vitro characterization of the novel antiarrhythmic agent SSR149744C: electrophysiological, anti-adrenergic, and anti-angiotensin II effects.

SSR149744C (SSR, 2-butyl-3-[4-[3-(dibutylamino)pro-pyl]benzoyl]-1-benzofuran-5-carboxylate isopropyl fumarate), is a new non-iodinated benzofuran derivative. The aim of this study was to evaluate in vivo its electrophysiological, hemodynamic, and anti-adrenergic properties and to determine its mechanism of action using in vitro studies. In chloralose-anesthetized dogs, SSR149744C (1-10 mg/kg i.v.) prolonged the sinus cycle length, A-H interval, Wenckebach cycle length, atrial effective refractory period (ERP), and atrio-ventricular node ERP in a dose-dependent manner without change of ventricular ERP and HV, QRS, or QTc intervals. Arterial blood pressure and ventricular inotropism were slightly decreased. SSR149744C, which has no or low affinity for alpha 1 and beta 1 adrenergic and angiotensin II AT1 receptors, reduced isoproterenol-induced tachycardia and phenylephrine- or angiotensin II-induced hypertension in anaesthetized dogs. In guinea pig papillary muscle, SSR149744C did not modify the resting potential, action potential amplitude and duration, but reduced the dV/dt max of the depolarization phase in a frequency-dependent manner. In isolated guinea pig cardiomyocytes and transfected CHO cells, SSR149744C (0.01-30 microM) inhibited several potassium currents: IKr (IC50 approximately 10 microM), IKs (IC50 approximately 30 microM), IK(ACh) (IC50 = 0.09 microM), and IKv1.5 (IC50 = 2.7 microM), the L-type calcium current: ICa(L) (IC50 approximately 5 microM) and also the amplitude of [Ca2+]i transient and cell shortening. Therefore, SSR149744C appears to have a multifactorial mechanism of action, which combines the blockade of several ion channels with the inhibition of responses of alpha 1 and beta 1 adrenergic as well as AT1 receptor stimulation. Like amiodarone, SSR149744C possesses the pharmacological effects of class I, II, III, and IV antiarrhythmic agents, which may confer upon this new drug a strong antiarrhythmic potential without ventricular proarrhythmia and iodine-related amiodarone-like side-effects.

Chronic beta-adrenoreceptor stimulation in vivo decreased Bcl-2 and increased Bax expression but did not activate apoptotic pathways in mouse heart.

Prolonged activation of the sympathetic nervous system is deleterious to heart function. In vitro beta1-adrenergic activation promotes apoptosis, whereas beta2-adrenergic activation reduces apoptosis in cultured adult cardiomyocytes. To determine the effect of chronic catecholamine infusion in vivo, we measured apoptosis marker expression in C57Bl/6 and catecholamine-sensitive Egr-1 deficient mice after treatment with the nonspecific beta-adrenergic agonist, isoproterenol, the beta1-specific agonist, dobutamine, or the beta2-specific agonist, metaproterenol. Antiapoptotic and proapoptotic protein expression, cytochrome c release and caspases 3, 9, and 12 activation products were measured on immunoblots. Catecholamine-treated mice had decreased Bcl-2 and increased Bax and BNIP1 expression, suggesting mitochondria-dependent apoptosis pathway activation. However, cytosolic cytochrome c or caspase 3 or 9 activation products were not detected. In mice, increased molecular chaperone expression and caspase 12 activation characterize endoplasmic-reticulum-driven apoptosis. Clusterin expression was increased in catecholamine-treated mice, but GRP78 expression was not increased, and caspase 12 activation products were not detected. Thus, neither the mitochondrial nor the endoplasmic apoptotic pathway was fully activated. Further, Egr-1 deficiency did not increase cardiac apoptosis. We conclude that although chronic in vivo infusion of beta1- or beta2-adrenergic receptor agonists partially activates the apoptosis program, full activation of the caspase cascade requires more, or other, cardiac insults.

Endothelin-1 limits vascular smooth muscle beta-adrenergic receptor sensitivity by a PKC-dependent pathway.

Endothelin-1 reduces the chronotropic and inotropic effects of the beta-adrenoceptor agonist isoproterenol in rabbit isolated atria. Vascular interactions between endothelin-1 and isoproterenol have not been reported. Rings of the rabbit aorta without endothelium were mounted on myographs to measure isometric tension. Vessels were precontracted to similar levels with phenylephrine (30 micromol/L) or endothelin-1 (30 nmol/L). Relaxation to isoproterenol and forskolin were obtained. Vascular sensitivity (pD2) to isoproterenol was not different in the presence of endothelin-1 (7.6 +/- 0.3; n = 13) and phenylephrine (7.5 +/- 0.3; n = 11). The maximal relaxation (Emax) however, was doubled (P < 0.05) by endothelin-1 (42 +/- 5%), as compared with phenylephrine (23 +/- 4%). In the presence of endothelin-1, chelerythrine (protein kinase C inhibitor; 10 micromol/L) increased (P < 0.05) vascular sensitivity to isoproterenol (8.6 +/- 0.4, n = 7), but had no influence on the Emax. In contrast, in the presence of phenylephrine, pD2 was unaffected by chelerythrine, whereas the Emax to isoproterenol was increased (P < 0.05; 50 +/- 4%, n = 8). Vascular sensitivity and Emax to forskolin were similar in the presence of endothelin-1 and phenylephrine. In conclusion, endothelin-1 reduces vascular sensitivity to isoproterenol in a PKC-dependent pathway. The permissive effect of endothelin-1 appears to directly target the beta-adrenoceptor/G protein complex upstream of adenylate cyclase.

Beta-adrenergic stimulation of pig myocytes with decreased cytosolic free magnesium prolongs the action potential and enhances triggered activity.

INTRODUCTION: Heart failure results in chronic beta-adrenergic stimulation, repolarization lability, and arrhythmias associated with early afterdepolarizations (EADs) and delayed afterdepolarizations (DADs). Having described a significant reduction in intracellular free magnesium ([Mg2+]i) in experimental heart failure, we asked whether a reduction in [Mg2+]i would delay repolarization or facilitate EADs and/or DADs. METHODS AND RESULTS: Left ventricular myocytes were isolated from Yorkshire swine. Cytosolic free [Mg2+] was set at 0.12 mM (LoMg) or 1.2 mM (HiMg) through pipette dialysis. Action potentials (AP), Ca current (I(Ca)), and sodium/calcium exchange current (I(NCX)) were measured in the presence or absence of isoproterenol (2 microM) at 37 degrees C. Under basal conditions (0.1-Hz stimulation, 2 mM external [Ca2+]), reducing [Mg2+]i had no effect on AP duration and I(Ca) but did significantly enhance I(NCX). In contrast, during superfusion with isoproterenol, reduced [Mg2+]i caused a significant increase in AP duration at both 50% and 90% repolarization (APD50 and APD90) compared with HiMg (P < 0.05). LoMg cells manifested a high incidence of triggered activities, including spontaneous AP, EADs, and DADs (83.3% in LoMg, n = 12 vs 38.3% in HiMg, n = 13; P < 0.05). I(Ca) and I(NCX) were significantly increased in LoMg cells compared with HiMg cells (P < 0.05). CONCLUSION: Decreased cytosolic free magnesium prolongs AP duration and increases the incidence of triggered activity during beta-adrenergic stimulation. These effects may be due to increased I(Ca) and I(NCX) in the presence of reduced intracellular [Mg2+]. A magnesium-dependent increase in triggered activity coupled with delayed repolarization during beta-adrenergic stimulation could contribute to the arrhythmogenic substrate in heart failure.

Interaction between FK 506 and isoproterenol in the modulation of glutamate release from cerebrocortical nerve terminals.

We have examined the interaction between FK 506 and isoproterenol in their modulation of glutamate release from cerebrocortical nerve terminals (synaptosomes). Application of FK 506, an inhibitor of protein phosphatase 2B (calcineurin), resulted in a concentration-dependent potentiation of 4AP-evoked glutamate release. The beta-adrenergic receptor agonist isoproterenol and the membrane-permeable activator of protein kinase A Sp-cAMP also caused a significant increase in evoked glutamate release, which was occluded by FK 506 pretreatment. By studying the voltage-dependent Ca2+ influx with fura-2, we show that, while FK 506 and isoproterenol alone produced a potentiation of the 4AP-evoked increase in intracellular Ca2+, the addition of FK 506 abolished the isoproterenol-mediated potentiation of Ca2+ influx. Based on these results, we suggest that the interaction between the two substances in their potentiating effect occurs, at least in part, at the level of the voltage-dependent Ca2+ entry that affects cell excitability and glutamate release.

Drug-induced hemodynamic perturbations alter the disposition of markers of blood volume, extracellular fluid, and total body water.

Recirculatory pharmacokinetic models for indocyanine green (ICG), inulin, and antipyrine facilitate description of intravascular mixing and tissue distribution following intravenous administration. These models characterized physiologic marker disposition in four awake dogs under control conditions and during phenylephrine, isoproterenol, and nitroprusside infusions. Systemic vascular resistance was more than doubled by phenylephrine and was decreased more than 50% by both isoproterenol and nitroprusside. Dye (ICG) dilution cardiac output (CO) was decreased nearly one-third by phenylephrine, was more than doubled by isoproterenol, and was largely unaffected by nitroprusside. Although phenylephrine reduced CO, the fraction of CO represented by nondistributive blood flow nearly doubled at the expense of blood flow to rapidly equilibrating tissues. The area under the blood antipyrine concentration versus time relationship for 3 min after administration (AUC(0-3 min)) during the phenylephrine infusion was nearly 75% larger than control due to both increased first-pass AUC and an increased fraction of CO represented by nondistributive blood flow. The large increase in CO produced by isoproterenol increased blood flow to rapidly equilibrating tissues and relatively decreased blood flow to slowly equilibrating tissues, because some appeared to equilibrate rapidly. Antipyrine AUC(0-3 min) during the isoproterenol infusion decreased more than 30%, due to decreased first-pass AUC. Nitroprusside changed antipyrine intercompartmental clearances in proportion to CO and, hence, had little effect on antipyrine AUC(0-3 min). These data provide further evidence that changes in antipyrine (a lipophilic drug surrogate) blood flow-dependent distribution after rapid i.v. administration are not proportional to changes in CO but depend on both CO and its distribution.

beta-adrenergic stimulation restores rat lung ability to clear edema in ventilator-associated lung injury.

Mechanical ventilation with high tidal volume (HVT) causes lung injury and decreases the lung's ability to clear edema in rats. beta-adrenergic agonists increase active Na(+) transport and lung edema clearance in normal rat lungs by stimulating apical Na(+) channels and basolateral Na,K-ATPase in alveolar epithelial cells. We studied whether beta-adrenergic agonists could restore lung edema clearance in rats ventilated with HVT (40 ml/kg, peak airway pressure of 35 cm H(2)O) for 40 min. The ability of rat lungs to clear edema decreased by approximately 50% after 40 min of HVT ventilation. Terbutaline (TERB) and isoproterenol (ISO) increased lung edema clearance in control nonventilated rats (from 0.50 +/- 0. 02 ml/h to 0.81 +/- 0.04 ml/h and 0.99 +/- 0.05 ml/h, respectively) and restored the lung's ability to clear edema in HVT ventilated rats (from 0.25 +/- 0.03 ml/h to 0.64 +/- 0.02 ml/h and 0.88 +/- 0. 09 ml/h, respectively). Disruption of cell microtubular transport system by colchicine inhibited the stimulatory effects of ISO in HVT ventilated rats, whereas beta-lumicolchicine did not affect beta-adrenergic stimulation. The Na,K-ATPase alpha(1)- and beta(1)-subunit mRNA steady state levels were not affected by incubation with ISO for 60 min in alveolar type II cells isolated from control and HVT ventilated rats. The data suggest that beta-adrenergic agonists increased alveolar fluid reabsorption in rats ventilated with HVT by promoting recruitment of ion-transporting proteins from intracellular pools to the plasma membrane of alveolar epithelial cells.

Agonist-induced sensitization of beta-adrenoceptor signaling in neonatal rat heart: expression and catalytic activity of adenylyl cyclase.

Agonist stimulation of neonatal cardiac beta-adrenoceptors produces heterologous sensitization of adenylyl cyclase (AC) signaling, rather than desensitization, as seen in adults. We examined the ontogenetic patterns of AC expression and activity, and evaluated isoproterenol effects on this pattern. [(3)H]Forskolin binding showed an increase in AC concentration across the period (birth to 25 days of age) in which agonist-induced sensitization is replaced by desensitization; binding affinity also increased, suggesting a shift in conformation and/or isoform. Indeed, catalytic properties of AC changed substantially with development, as evaluated by AC responses to forskolin versus Mn(2+). In contrast, there were only minor changes in the levels of mRNAs encoding the two major isoforms. Neonates given repeated isoproterenol treatment showed an enhancement of [(3)H]forskolin binding B(max) and a precocious shift to the mature affinity state and corresponding catalytic properties. Although isoproterenol caused significant increases in AC mRNAs, the effects were small and showed no isoform preference. Thus, a primary mode for ontogenetic increases in cardiac cellular responsiveness to adrenergic stimulation is the increase in AC activity attendant upon an absolute increase in the membrane concentration of AC molecules, along with changes in the catalytic properties of AC. The lack of correlation between mRNA and AC protein suggests that the primary regulatory events are post-transcriptional. The induction of AC by beta-adrenoceptor stimulation in the fetus and neonate accounts for heterologous, agonist-induced sensitization, a phenomenon that preserves cellular responses during the period of the perinatal transition.

Catecholamine handling in the porcine heart: a microdialysis approach.

Experimental findings suggest a pronounced concentration gradient of norepinephrine (NE) between the intravascular and interstitial compartments of the heart, compatible with an active neuronal reuptake (U1) and/or an endothelial barrier. Using the microdialysis technique in eight anesthetized pigs, we investigated this NE gradient, both under baseline conditions and during increments in either systemic or myocardial interstitial fluid (MIF) NE concentration. At steady state, baseline MIF NE (0.9 +/- 0.1 nmol/l) was higher than arterial NE (0.3 +/- 0.1 nmol/l) but was not different from coronary venous NE (1.5 +/- 0.3 nmol/l). Local U1 inhibition raised MIF NE concentration to 6.5 +/- 0.9 nmol/l. During intravenous NE infusions (0.6 and 1.8 nmol. kg(-1). min(-1)), the fractional removal of NE by the myocardium was 79 +/- 4% to 69 +/- 3%, depending on the infusion rate. Despite this extensive removal, the quotient of changes in MIF and arterial concentration (DeltaMIF/DeltaA ratio) for NE were only 0.10 +/- 0.02 for the lower infusion rate and 0.11 +/- 0.01 for the higher infusion rate, whereas U1 blockade caused the DeltaMIF/DeltaA ratio to rise to 0.21 +/- 0.03 and 0.36 +/- 0.05, respectively. From the differences in DeltaMIF/DeltaA ratios with and without U1 inhibition, we calculated that 67 +/- 5% of MIF NE is removed by U1. Intracoronary infusion of tyramine (154 nmol. kg(-1). min(-1)) caused a 15-fold increase in MIF NE concentration. This pronounced increase was paralleled by a comparable increase of NE in the coronary vein. We conclude that U1 and extraneuronal uptake, and not an endothelial barrier, are the principal mechanisms underlying the concentration gradient of NE between the interstitial and intravascular compartments in the porcine heart.

Affinity constants and beta-adrenoceptor reserves for isoprenaline on cardiac tissue from normotensive and hypertensive rats.

To determine whether there are differences in cardiac beta-adrenoceptor responsiveness, isoprenaline affinity constants and fractional beta-adrenoceptor occupancy-response relationships for isoprenaline in the early stages of established hypertension, we studied the effects of bromoacetylalprenololmenthane (BAAM) and ([3,5-diamino-6-chloro-N-(1[N-beta-(2-hydroxyl-3-alpha-naphthoxypropy lamino)ethylcarbamoyl]-1-methylethyl)-pyrazine-2-carboxamide (ICI 147 798), slowly reversible beta-adrenoceptor antagonists, on the isoprenaline responses of the left ventricular papillary muscle and the left and right atria of 6-month-old Wistar Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). The papillary muscles, but not the right and left atria, of the SHR were less responsive to isoprenaline than those of the WKY. The isoprenaline pD2 values (the negative logarithms of the molar concentrations of agonist producing 50% of the maximum response) were 7.72 and 8.00 on the SHR and WKY papillary muscles, respectively. On the WKY papillary muscle the isoprenaline KA values were 2-3 x 10(-6) M, which is as expected for isoprenaline at beta1 or beta2-adrenoceptors. Isoprenaline had 100-fold greater affinity on the WKY and SHR left atria than on the papillary muscles; the isoprenaline KA values were 2-4 x 10(-8) M. On the WKY papillary muscle and left atrium, isoprenaline had to occupy 3-4% of the beta-adrenoceptors to produce a 50% maximum response; on the WKY papillary muscle and left atrium isoprenaline had to occupy 25-35% and 55%, respectively, of the beta-adrenoceptors to produce a 90% maximum response. The SHR papillary muscles and left atrium had smaller beta-adrenoceptor reserves for isoprenaline than did the WKY tissues. We were unable to obtain isoprenaline KA values on the WKY right atrium. The isoprenaline KA value on the SHR right atrium was 1-4 x 10(-8) M. Because the isoprenaline KA values for the left and right atria are markedly different from those previously reported for isoprenaline at beta1 or beta2-adrenoceptors, we suggest that atypical beta-adrenoceptors might be present on the atria of WKY and SHR. We have also demonstrated a lower beta-adrenoceptor reserve on SHR papillary muscle and atria in the early stages of established hypertension.

Sympathomimetic enantiomers and asthma.

Airways of asthma patients can become hyperresponsive to airway spasmogens following regular use of isoprenaline or beta 2-selective sympathomimetics. Hyper-reactivity that results from acute exposure of animals to these drugs is pre-empted by vagal section (a procedure which does not influence spasmolytic efficacy of sympathomimetics), is not diminished by antagonism of beta 2-adrenoceptors and is not associated with loss of responsivity of beta 2-adrenoceptors in the airways. Since activation, modulation, or blockade of beta 2-adrenoceptors does not determine this form of hyperreactivity, the possibility that distomers may induce hyperreactivity must be considered. Ocular and vascular responses to distomers of sympathomimetics have long been recognised and, more recently, comparable observations have been made for the airways. Thus, reactivity of guinea-pig airways to spasmogens was increased following exposure to S-isoprenaline, S-salbutamol, or S-terbutaline and exposure to S-isoprenaline or S-salbutamol can intensify symptoms in asthmatics. Regular exposure to the racemate, especially during or following an allergic reaction, predisposes to expression of hyper-reactivity, which is nullified, acutely, by the eutomer. These observations imply that biological effects of sympathomimetic distomers may contribute to morbidity and mortality in asthma patients.

A match between binding to beta-adrenoceptors and stimulation of adenylyl cyclase parameters of (-)isoproterenol and salbutamol on rat brain.

Inhibition experiments of (-)[3H]CGP 12177 binding by (-)isoproterenol and salbutamol were performed on synaptosomes prepared from rat brain cortex and cerebellum. Adenylyl cyclase (AC) stimulation experiments on slices of these structures were also performed, with measuring [14C]cAMP obtained from [14C]adenine. Studying beta 1- and beta 2-adrenoceptors (beta 2AR) separately, dissociation constants of (-)isoproterenol for the high- (KH) and low- (KL) affinity states are 8 and 206 nM, respectively, for beta 1AR, vs 20 and 900 nM for beta 2AR. With salbutamol, KH and KL for beta 2AR are 37 and 1250 nM, respectively, vs 430 and 8500 nM for beta 1AR. In any case, the proportion of high-affinity state (RH) of beta 2AR in the cerebellum (59% and 35% for (-)isoproterenol and salbutamol, respectively) is twice that of beta 1AR (30% and 18%). Surprisingly, the RH of cortex beta 2AR with (-)isoproterenol (30%) is significantly lower than in the cerebellum, but not with salbutamol (35%). To allow meaningful comparisons of potencies in stimulating [14C]cAMP production, we define the coupling efficiency (CE), applicable to specified beta AR subtype and agonist, and expressed as the maximal production of mol cAMP.mol-1 beta AR.min-1. The order of CE was always in favor of (-)isoproterenol vs salbutamol on cerebellum beta 2AR > on cortex beta 2AR > on cortex beta 1AR. This order indicates the partial agonism of salbutamol for both beta AR subtypes, and an intrinsic better coupling of beta 2AR vs beta 1AR in rat brain. Moreover, this order corresponds roughly to that of RH. Hence, CE is directly correlated with RH at least for these two agonists. EC50 for cAMP production for each subtype and agonist is in the same order than the respective KL, and might only reflect the rapid return of receptor to low-affinity state after the activation of Gs protein. In binding experiments on the whole beta AR in both areas, the pseudo-Hill coefficient did not reach 1 with 0.3 mM guanosine 5'-(beta, gamma-imido)triphosphate (GppNHp). This dysfunction of GppNHp in rat brain structures might be caused by a major difference in the regulation of coupling in the ternary complex as compared with peripheral tissues.

Simultaneous determination of the elimination profiles of the individual enantiomers of racemic isoproterenol using capillary electrophoresis and microdialysis sampling.

Microdialysis sampling and capillary electrophoresis with electrochemical detection (CE-EC) were used in combination to simultaneously define the elimination profile of each enantiomer of isoproterenol (ISP) administered as a racemic mixture to Sprague-Dawley rats. Resolution of the enantiomers of ISP was accomplished using a running buffer containing methyl-O-beta-cyclodextrin as a chiral recognition reagent. The CE-EC system provided a concentration limit of detection of 0.63 ng ml-1, allowing monitoring of the elimination of ISP for up to six half-lives. Microdialysis sampling was capable of continuously monitoring the concentration of ISP with 60 s resolution. The concentration versus time data for the elimination of (+) and (-) ISP were fit to a biphasic first order elimination model yielding average apparent distribution half-lives of 0.52 +/- 0.07 min and 0.55 +/- 0.08 min and average apparent elimination half-lives of 9.8 +/- 2.2 and 8.8 +/- 2.0 min for (-) and (+) ISP, respectively (n = 3 rats). No statistically significant difference in the average half-lives was found. However, because each enantiomer was simultaneously determined in each animal a paired two-sample t-Test could also be done. This statistical analysis demonstrated that a difference in the elimination half-lives of the enantiomers of ISP does exist.

Actividad de la 6-fosfofructo-2-quinasa y contenido de fructosa-2.6-bifosfato en glándulas parotidas de ratas despues del tratamiento con isoproterenol / Activity of 6-phosphofructo-2-quinase (PFK-2) and contents of fructose-2.6-biphosphate in parotid glands of the rats after treatment of isoproterenol

La sialadenosis en animales de laboratorio puede ser provocada por la administración de un agonista betadrenérgico conocido como isoproterenol. Su efecto produce hipertrofia e hiperplasia de las glándulas salivales, especialmente de la glándula parótida. La 6-fosfofructo-2-quinasa (PKF-2) es una enzima bifuncional que cataliza tanto la síntesis como la degradación de la fructosa-2,6-difosfato. este metabolito es un potente activador de la 6-fosfofructo-1-quinasa (PKF-1) enzima clave en el proceso glucolítico. En este trabajo determinamos la actividad de la PKF-2 y el contenido de fructosa-2,6-bifosfato en glándulas parótidas de ratas, al ser estimuladas con varias dosis de isoproterenol