Exercise training normalizes beta-adrenoceptor expression in dogs susceptible to ventricular fibrillation

Previous studies demonstrated an enhanced beta(2)-adrenoceptor (AR) responsiveness in animals susceptible to ventricular fibrillation (VF) that was eliminated by exercise training. The present study investigated the effects of endurance exercise training on beta(1)-AR and beta(2)-AR expression in dogs susceptible to VF. Myocardial ischemia was induced by a 2-min occlusion of the left circumflex artery during the last minute of exercise in dogs with healed infarctions: 20 had VF [susceptible (S)] and 13 did not [resistant (R)]. These dogs were randomly assigned to either 10-wk exercise training [treadmill running; n = 9 (S) or 8 (R)] or an equivalent sedentary period [n = 11 (S) or 5 (R)]. Left ventricular tissue beta-AR protein and mRNA were quantified by Western blot analysis and RT-PCR, respectively. Because beta(2)-ARs are located in caveolae, caveolin-3 was also quantified. beta(1)-AR gene expression decreased ( approximately 5-fold), beta(2)-AR gene expression was not changed, and the ratio of beta(2)-AR to beta(1)-AR gene expression was significantly increased in susceptible compared with resistant dogs. beta(1)-AR protein decreased ( approximately 50%) and beta(2)-AR protein increased (400%) in noncaveolar fractions of the cell membrane in susceptible dogs. Exercise training returned beta(1)-AR gene expression to levels seen in resistant animals but did not alter beta(2)-AR protein levels in susceptible dogs. These data suggest that beta(1)-AR gene expression was decreased in susceptible dogs compared with resistant dogs and, further, that exercise training improves beta(1)-AR gene expression, thereby restoring a more normal beta-AR balance.     

Effects of endurance exercise training on heart rate variability and susceptibility to sudden cardiac death: protection is not due to enhanced cardiac vagal regulation.

Low heart rate variability (HRV) is associated with an increased susceptibility to ventricular fibrillation (VF). Exercise training can increase HRV (an index of cardiac vagal regulation) and could, thereby, decrease the risk for VF. To test this hypothesis, a 2-min coronary occlusion was made during the last min of a 18-min submaximal exercise test in dogs with healed myocardial infarctions; 20 had VF (susceptible), and 13 did not (resistant). The dogs then received either a 10-wk exercise program (susceptible, n=9; resistant, n=8) or an equivalent sedentary period (susceptible, n=11; resistant, n=5). HRV was evaluated at rest, during exercise, and during a 2-min occlusion at rest and before and after the 10-wk period. Pretraining, the occlusion provoked significantly (P<0.01) greater increases in HR (susceptible, 54.9+/-8.3 vs. resistant, 25.0+/-6.1 beats/min) and greater reductions in HRV (susceptible, -6.3+/-0.3 vs. resistant, -2.8+/-0.8 ln ms2) in the susceptible dogs compared with the resistant animals. Similar response differences between susceptible and resistant dogs were noted during submaximal exercise. Training significantly reduced the HR and HRV responses to the occlusion (HR, 17.9+/-11.5 beats/min; HRV, -1.2+/-0.8, ln ms2) in the susceptible dogs; similar response reductions were noted during exercise. In contrast, these variables were not altered in the sedentary susceptible dogs. Posttraining, VF could no longer be induced in the susceptible dogs, whereas four sedentary susceptible dogs died during the 10-wk control period, and the remaining seven animals still had VF when tested. Atropine decreased HRV but only induced VF in one of eight trained susceptible dogs. Thus exercise training increased cardiac vagal activity, which was not solely responsible for the training-induced VF protection.     

Enhanced in vivo and in vitro contractile responses to beta(2)-adrenergic receptor stimulation in dogs susceptible to lethal arrhythmias.

The response to beta-adrenergic receptor (beta-AR) stimulation was evaluated in both isolated cardiomyocytes (video edge detection) and the intact animal (echocardiography) in dogs either susceptible (S) or resistant (R) to ventricular fibrillation induced by a 2-min coronary occlusion during the last minute of exercise. In the intact animal, velocity of circumferential fiber shortening (Vcf) was evaluated both before (n = 27, S = 12 and R = 15) and after myocardial infarction. Before infarction, increasing doses of isoproterenol provoked similar contractile and heart rate responses in each group of dogs. Either beta(1)-AR (bisoprolol) or beta(2)-AR (ICI-118551) antagonists reduced the isoproterenol response, with a larger reduction noted after the beta(1)-AR blockade. In contrast, after infarction, isoproterenol induced a significantly larger Vcf and heart rate response in the susceptible animals that was eliminated by beta(2)-AR blockade. The single-cell isotonic shortening response to isoproterenol (100 nM) was also larger in cells obtained from susceptible compared with resistant dogs and was reduced to a greater extent by beta(2)-AR blockade in the susceptible dog myocytes (S, -48%, n = 6; R, -15%, n = 9). When considered together, these data suggest that myocardial infarction provoked an enhanced beta(2)-AR response in susceptible, but not resistant, animals.     

Effect of endurance exercise training on heart rate onset and heart rate recovery responses to submaximal exercise in animals susceptible to ventricular fibrillation.

Both a large heart rate (HR) increase at exercise onset and a slow heart rate (HR) recovery following the termination of exercise have been linked to an increased risk for ventricular fibrillation (VF) in patients with coronary artery disease. Endurance exercise training can alter cardiac autonomic regulation. Therefore, it is possible that this intervention could restore a more normal HR regulation in high-risk individuals. To test this hypothesis, HR and HR variability (HRV, 0.24- to 1.04-Hz frequency component; an index of cardiac vagal activity) responses to submaximal exercise were measured 30, 60, and 120 s after exercise onset and 30, 60, and 120 s following the termination of exercise in dogs with healed myocardial infarctions known to be susceptible (n = 19) to VF (induced by a 2-min coronary occlusion during the last minute of a submaximal exercise test). These studies were then repeated after either a 10-wk exercise program (treadmill running, n = 10) or an equivalent sedentary period (n = 9). After 10 wk, the response to exercise was not altered in the sedentary animals. In contrast, endurance exercise increased indexes of cardiac vagal activity such that HR at exercise onset was reduced (30 s after exercise onset: HR pretraining 179 +/- 8.4 vs. posttraining 151.4 +/- 6.6 beats/min; HRV pretraining 4.0 +/- 0.4 vs. posttraining 5.8 +/- 0.4 ln ms(2)), whereas HR recovery 30 s after the termination of exercise increased (HR pretraining 186 +/- 7.8 vs. posttraining 159.4 +/- 7.7 beats/min; HRV pretraining 2.4 +/- 0.3 vs. posttraining 4.0 +/- 0.6 ln ms(2)). Thus endurance exercise training restored a more normal HR regulation in dogs susceptible to VF.     

Heart rate response to onset of exercise: evidence for enhanced cardiac sympathetic activity in animals susceptible to ventricular fibrillation

A large heart rate (HR) increase at the onset of exercise has been linked to an increased risk for adverse cardiovascular events, including cardiac death. However, the relationship between changes in cardiac autonomic regulation induced by exercise onset and the confirmed susceptibility to ventricular fibrillation (VF) has not been established. Therefore, a retrospective analysis of the HR response to exercise onset was made in mongrel dogs with healed myocardial infarctions that were either susceptible (S, n = 131) or resistant (R, n = 114) to VF (induced by a 2-min occlusion of the left circumflex artery during the last minute of exercise). The ECG was recorded, and time series analysis of HR variability (vagal activity index, the 0.24-1.04-Hz frequency component of R-R interval variability) was measured before and 30, 60, and 120 s after the onset of exercise (treadmill running). Exercise elicited significantly (ANOVA, P < 0.0001) greater increases in HR in susceptible dogs at all three times (e.g., at 60 s: R, 46.8 +/- 2.3 vs. S, 57.1 +/- 2.2 beats/min). However, the vagal activity index decreased to a similar extent in both groups of dogs (at 60 s: R, -2.8 +/- 0.1 vs. S, -3.0 +/- 0.2 ln ms2). Beta-adrenoceptor blockade (BB, propranolol 1.0 mg/kg iv) reduced the HR increase and eliminated the differences noted between the groups [at 60 s: R (n = 26), 40.4 +/- 3.2 vs. S (n = 31), 37.5 +/- 2.4 beats/min]. After BB, exercise once again elicited similar declines in vagal activity in both groups (at 60 s: R, -3.6 +/- 0.5 vs. S, -3.2 +/- 0.4 ln ms2). When considered together, these data suggest that at the onset of exercise HR increases to a greater extent in animals prone to VF compared with dogs resistant to this malignant arrhythmia due to an enhanced cardiac sympathetic activation in the susceptible dogs.     

Heart rate recovery after exercise: a predictor of ventricular fibrillation susceptibility after myocardial infarction

Heart rate recovery after exercise, thought to be related to cardiac parasympathetic tone, has been shown to be a prognostic tool for all-cause mortality. However, the relationship between this variable and confirmed susceptibility to ventricular fibrillation (VF) has not been established. Therefore, myocardial ischemia was induced with a 2-min occlusion of the left circumflex artery during the last minute of exercise in mongrel dogs with myocardial infarction (n = 105 dogs). VF was induced in 66 animals (susceptible), whereas the remaining 39 dogs had no arrhythmias (resistant). On a previous day, ECG was recorded and a time-series analysis of heart rate variability was measured 30, 60, and 120 s after submaximal exercise (treadmill running). The heart rate recovery was significantly greater in resistant dogs than in susceptible dogs at all three times, with the most dramatic difference at the 30-s mark (change from maximum: 48.1 +/- 3.6 beats/min, resistant dogs; 31.0 +/- 2.2 beats/min, susceptible dogs). Correspondingly, indexes of parasympathetic tone increased to a significantly greater extent in resistant dogs at 30 and 60 s after exercise. These differences were eliminated by atropine pretreatment. When considered together, these data suggest that resistant animals exhibit a more rapid recovery of vagal activity after exercise than those susceptible to VF. As such, postexercise heart rate recovery may help identify patients with a high risk for VF following myocardial infarction.     

Conditioning of beta(1)-adrenoceptor effect via beta(2)-subtype on L-type Ca(2+) current in canine ventricular myocytes

We investigated the roles of beta(1)- and beta(2)-receptors (beta-AR) in adrenergic enhancement of L-type Ca(2+) current (I(CaL)) in canine ventricular myocytes. Isoproterenol and l-norepinephrine produced a monophasic and a biphasic concentration-I(CaL) relationship (CR), respectively. alpha(1)-AR inhibition with prazosin and beta(2)-AR stimulation with zinterol or l-epinephrine shifted the CR of l-norepinephrine leftward. Zinterol (50 nM) and l-epinephrine (10 nM), but not prazosin, altered the biphasic CR of l-norepinephrine to a monophasic CR. Zinterol and l-epinephrine applied after l-norepinephrine had no effect on I(CaL). beta(2)-AR inhibition with ICI-118551 reduced the E(max) of isoproterenol and l-norepinephrine by 60% and abolished the augmentation of l-norepinephrine by zinterol and l-epinephrine. Carbachol (100 nM) modestly reduced the I(CaL) response to beta(1)-AR stimulation but abolished the enhancement via beta(2)-AR. Zinterol augmented the enhancement of I(CaL) by forskolin, IBMX, and theophylline, but not in the presence of CGP-20712A. We conclude that selective beta(2)-AR stimulation does not increase I(CaL) but enhances adenylyl cyclase activity when stimulated via beta(1)-AR and with forskolin. beta(2)-AR activity preconditions adenylyl cyclase for beta(1)-AR stimulation.     

beta2-Adrenergic receptor downregulation and performance decrements during high-intensity resistance exercise overtraining.

Previous research on overtraining due to excessive use of maximal resistance exercise loads [100% 1 repetition maximum (1 RM)] indicates that peripheral muscle maladaptation contributes to overtraining-induced performance decrements. This study examined the cellular and molecular responses of skeletal muscle to performance decrements due to high-relative-intensity (%1 RM) resistance exercise overtraining. Weight-trained men were divided into overtrained (OT, n = 8) and control (Con, n = 8) groups. The OT group performed 10 x 1 at 100% 1 RM daily for 2 wk, whereas the Con group performed normal training 2 days/wk. Muscle biopsies from the vastus lateralis muscle, voluntary static and dynamic muscle performances, and nocturnal urinary epinephrine were assessed before (pre) and after (post) overtraining. Overtraining occurred as indicated by a decrease in 1-RM strength for the OT group (mean +/- SE; OT pre = 159.3 +/- 10.1 kg, OT post = 151.4 +/- 9.9 kg, Con pre = 146.0 +/- 12.9 kg, Con post = 144.9 +/- 13.3 kg), as well as a 36.3% decrease in mean power at 100% 1-RM loads. Normal training could be resumed only after 2-8 wk of training cessation. Muscle beta(2)-adrenergic receptor (beta(2)-AR; fmol/mg protein) density significantly decreased by 37.0% for the OT group and was unchanged for the Con group (-1.8%). Nocturnal urinary epinephrine for the OT group increased by 49%, although this was not significant (effect size = 0.42). The ratio of nocturnal urinary epinephrine to beta(2)-AR density suggested a decreased beta(2)-AR sensitivity for the OT group (2.4-fold increase). Overtraining occurred based on decreased muscular force and power. Desensitization of the beta(2)-AR system suggests that this may be an important contributor to performance decrements due to excessive use of maximal resistance exercise loads.     

Cardiac vagal modulation of heart rate during prolonged submaximal exercise in animals with healed myocardial infarctions: effects of training

The present study investigated the effects of long-duration exercise on heart rate variability [as a marker of cardiac vagal tone (VT)]. Heart rate variability (time series analysis) was measured in mongrel dogs (n = 24) with healed myocardial infarctions during 1 h of submaximal exercise (treadmill running at 6.4 km/h at 10% grade). Long-duration exercise provoked a significant (ANOVA, all P < 0.01, means +/- SD) increase in heart rate (1st min, 165.3 +/- 15.6 vs. last min, 197.5 +/- 21.5 beats/min) and significant reductions in high frequency (0.24 to 1.04 Hz) power (VT: 1st min, 3.7 +/- 1.5 vs. last min, 1.0 +/- 0.9 ln ms(2)), R-R interval range (1st min, 107.9 +/- 38.3 vs. last min, 28.8 +/- 13.2 ms), and R-R interval SD (1st min, 24.3 +/- 7.7 vs. last min 6.3 +/- 1.7 ms). Because endurance exercise training can increase cardiac vagal regulation, the studies were repeated after either a 10-wk exercise training (n = 9) or a 10-wk sedentary period (n = 7). After training was completed, long-duration exercise elicited smaller increases in heart rate (pretraining: 1st min, 156.0 +/- 13.8 vs. last min, 189.6 +/- 21.9 beats/min; and posttraining: 1st min, 149.8 +/- 14.6 vs. last min, 172.7 +/- 8.8 beats/min) and smaller reductions in heart rate variability (e.g., VT, pretraining: 1st min, 4.2 +/- 1.7 vs. last min, 0.9 +/- 1.1 ln ms(2); and posttraining: 1st min, 4.8 +/- 1.1 vs. last min, 2.0 +/- 0.6 ln ms(2)). The response to long-duration exercise did not change in the sedentary animals. Thus the heart rate increase that accompanies long-duration exercise results, at least in part, from reductions in cardiac vagal regulation. Furthermore, exercise training attenuated these exercise-induced reductions in heart rate variability, suggesting maintenance of a higher cardiac vagal activity during exercise in the trained state.     

Expression and functional analysis of beta-adrenoceptor subtypes in rabbit submandibular gland

beta-Adrenoceptors (beta-ARs) mediate important physiological functions in salivary glands. Here we investigated the expression and function of beta-AR subtypes in rabbit submandibular gland (SMG). Both beta(1)- and beta(2)-ARs, but not beta(3)-AR, were strongly expressed in rabbit SMG. beta(1)-AR proteins were widely expressed in acinar and ductal cells whereas beta(2)-AR proteins were mainly detected in ductal cells. A [(3)H]-dihydroalprenolol binding assay revealed that beta-AR B(max) was 186+/-11.9 fmol/mg protein and K(d) was 2.71+/-0.23 nM. A competitive binding assay with CGP 20712A, a beta(1)-AR antagonist, indicated that the proportion of beta(1)-AR and beta(2)-AR was 71.9% and 28.1%, respectively. Gland perfusion with the beta-AR agonist isoproterenol induced a significant increase in saliva secretion which was abolished by pretreatment with the non-selective beta-AR antagonist propranolol. Pretreatment with beta(1)- or beta(2)-AR selective antagonists, CGP 20712A or ICI 118551, diminished isoproterenol-induced increase in saliva secretion by 71.2% and 28.8%, respectively. The expression of alpha-amylase mRNA was significantly stimulated by isoproterenol, which was eliminated by propranolol and CGP 20712A. Perfusion with isoproterenol decreased alpha-amylase protein storage in SMG and increased alpha-amylase activity in saliva. These alterations became less significant after pretreatment with propranolol and CGP 20712A. Our results suggest that both beta(1)- and beta(2)-ARs are expressed in rabbit SMG. beta(1)-AR is the predominant subtype and may play an important role in regulating saliva and alpha-amylase secretion.     

Coupling of beta-adrenergic receptors to cardiac L-type Ca2+ channels: preferential coupling of the beta1 versus beta2 receptor subtype and evidence for PKA-independent activation of the channel.

Beta1- and beta2-adrenergic receptors (beta-ARs) co-exist in mammalian heart, and it is generally accepted that both activate adenylyl cyclase (AC), resulting in increased levels of cAMP and subsequent activation of L-type Ca2+ channels (CaCh). To investigate the contribution of each beta-AR subtype in AC and CaCh coupling, we stably expressed cardiac CaCh alpha1 and beta2 subunits along with either beta1-AR or beta2-AR in CHW fibroblasts. Co-expression of either beta-AR with CaCh subunits conferred responsiveness of AC and CaCh to isoproterenol (ISO), which was not observed in non-transfected cells. ISO-promoted cAMP formation occurred at a lower EC50 through the beta2-AR than through the beta1-AR (0.13 +/- 0.01 vs. 0.6 +/- 0.14 nM). In contrast, activation of CaCh was more efficacious via the beta1-AR than the beta2-AR (EC50 for CaCh activation = 238 +/- 33 vs. 1057 +/- 113 nM). Pre-treatment with pertussis toxin (PTX) had no effect upon the responsiveness of either cAMP formation or CaCh activation through either receptor. We conclude (1) that beta1-ARs exhibit preferential coupling to CaCh activation, versus that observed for the beta2-AR; (2) that this preferential coupling cannot be explained solely by cAMP-dependent processes; and (3) that the relative attenuation of beta2-AR-promoted CaCh activation is not due to receptor coupling to PTX-sensitive G proteins. Thus, it is likely that other subtype-specific, cAMP-independent coupling of the beta-AR to CaCh is present.     

Electrotonic remodeling following myocardial infarction in dogs susceptible and resistant to sudden cardiac death.

Passive electrical remodeling following myocardial infarction (MI) is well established. These changes can alter electrotonic loading and trigger the remodeling of repolarization currents, a potential mechanism for ventricular fibrillation (VF). However, little is known about the role of passive electrical markers as tools to identify VF susceptibility post-MI. This study investigated electrotonic remodeling in the post-MI ventricle, as measured by myocardial electrical impedance (MEI), in animals prone to and resistant to VF. MI was induced in dogs by a two-stage left anterior descending (LAD) coronary artery ligation. Before infarction, MEI electrodes were placed in remote (left circumflex, LCX) and infarcted (LAD) myocardium. MEI was measured in awake animals 1, 2, 7, and 21 days post-MI. Subsequently, VF susceptibility was tested by a 2-min LCX occlusion during exercise; 12 animals developed VF (susceptible, S) and 12 did not (resistant, R). The healing infarct had lower MEI than the normal myocardium. This difference was stable by day 2 post-MI (287 +/- 32 Omega vs. 425 +/- 62 Omega, P < 0.05). Significant differences were observed between resistant and susceptible animals 7 days post-MI; susceptible dogs had a wider electrotonic gradient between remote and infarcted myocardium (R: 89 +/- 60 Omega vs. S: 180 +/- 37 Omega). This difference increased over time in susceptible animals (252 +/- 53 Omega at 21 days) due to post-MI impedance changes on the remote myocardium. These data suggest that early electrotonic changes post-MI could be used to assess later arrhythmia susceptibility. In addition, passive-electrical changes could be a mechanism driving active-electrical remodeling post-MI, thereby facilitating the induction of arrhythmias.     

β(2)-adrenoreceptor mediates the cardioprotection of ischemic preconditioning on myocardial contraction in rats subjected to ischemia/reperfusion injury

The aim of the present study is to investigate the role of beta(2)-adrenoreceptor (beta(2)-AR) in ischemic preconditioning (IP) in isolated rat heart model of ischemia/reperfusion (I/R). Sprague-Dawley rat hearts were quickly removed, mounted on Langendorff apparatus, and perfused with Krebs-Henseleit (KH) solution. After the initial stabilization period, the rats were randomly divided into 6 groups including control group (perfused for an additional 20 min), IP group (4 cycles of 5 min of ischemia followed by 5 min of reflow), isoproterenol (ISO) group (10 nmol/L ISO perfusion for 5 min followed by 5 min washout), IP + ICI118551 group (55 nmol/L ICI118551 perfusion for 5 min before and throughout IP), ISO + ICI118551 group (55 nmol/L ICI118551 perfusion for 5 min before and throughout ISO treatment), ICI118551 group (55 nmol/L ICI118551 perfusion for 20 min). After these treatments, all hearts were followed by 30 min of no-flow ischemia and 30 min of reperfusion. A computer-based electrophysiological recorder system was used to measure changes of the maximal rate of pressure increase in systole phase (+dp/dt(max)), maximal rate of pressure decrease in diastole phase (-dp/dt(max)), and difference of left ventricular pressure (DeltaLVP). Then cardiomyocytes from these hearts were isolated by 5 min of Ca(2+)-free buffer perfusion and 25 min of collagenase perfusion. The ventricles were chopped and filtered. The myocytes were resuspended in KB buffer. The contraction and the viability of cardiomyocytes were measured. Lactate dehydrogenase (LDH) concentration in coronary effluent was assayed with assay kit. The results showed that both IP and ISO significantly increased the values of +/-dp/dt(max), DeltaLVP, the contraction and viability of cardiomyocytes, shortened the time-to-peak contraction (TTP), and decreased the release of LDH in coronary effluent. ICI118551, a selective beta(2)-AR antagonist, blocked these effects. Either the time-to-50% relaxation (R(50)) or the time-to-100% relaxation (R(100)) had no significant differences between groups. Our results indicate that the cardioprotection of IP was mediated by beta(2)-AR in isolated rat hearts subjected to I/R injury.     

The role of beta-adrenergic receptors in the cutaneous water evaporation mechanism in the heat-acclimated pigeon (Columba livia)

The effects of selective and non-selective beta-adrenergic agents on cutaneous water evaporation (CWE) were studied in hand-reared rock pigeons (Columba livia). CWE was measured by the vapor diffusive resistance method, using a transient porometer. Intramuscular and subcutaneous injections of a non-selective beta-adrenergic antagonist (propranolol) or a selective beta(2)-adrenergic antagonist (ICI-118551) to heat-acclimated (HAc) pigeons at ambient temperature (T(a)) of 24 degrees C resulted in intensive CWE. The CWE values that were triggered by propranolol and ICI-118551 (18.59+/-0.73 and 16.48+/-0.70 mg cm(-2) h(-1), respectively) were close to those induced by heat exposure (17.62+/-1.40 mg cm(-2) h(-1)). Subcutaneous administration of propranolol produced local response. Intramuscular injection of salbutamol (selective beta(2)-adrenergic agonist) to HAc pigeons drastically diminished CWE induced by either propranolol, metoprolol or heat exposure. Such manipulations also enhanced panting at relatively low T(a)s (42 degrees C). The inhibition of beta(1)-adrenergic receptors by metoprolol increased CWE, while inhibition by atenolol produced no change from basal values. This difference may be attributed to their distinctive nature in penetrating the blood-brain barrier. Our findings indicate a regulatory pathway for CWE consisting of both beta(1)- and beta(2)-adrenergic receptors. We suggest that the beta(1)-adrenergic effect is restricted mainly to the CNS, while the beta(2)-adrenergic effect takes place at the effector level. We postulate this level to be either the cutaneous microvasculature or the epidermal layer.     

Transgenic overexpression of beta(2)-adrenergic receptors in airway epithelial cells decreases bronchoconstriction

Airway epithelial cells express beta(2)-adrenergic receptors (beta(2)-ARs), but their role in regulating airway responsiveness is unclear. With the Clara cell secretory protein (CCSP) promoter, we targeted expression of beta(2)-ARs to airway epithelium of transgenic (CCSP-beta(2)-AR) mice, thereby mimicking agonist activation of receptors only in these cells. In situ hybridization confirmed that transgene expression was confined to airway epithelium, and autoradiography showed that beta(2)-AR density in CCSP-beta(2)-AR mice was approximately twofold that of nontransgenic (NTG) mice. Airway responsiveness measured by whole body plethysmography showed that the methacholine dose required to increase enhanced pause to 200% of baseline (ED(200)) was greater for CCSP-beta(2)-AR than for NTG mice (345 +/- 34 vs. 157 +/- 14 mg/ml; P < 0.01). CCSP-beta(2)-AR mice were also less responsive to ozone (0.75 ppm for 4 h) because enhanced pause in NTG mice acutely increased to 77% over baseline (P < 0.05) but remained unchanged in the CCSP-beta(2)-AR mice. Although both groups were hyperreactive to methacholine 6 h after ozone exposure, the ED(200) for ozone-exposed CCSP-beta(2)-AR mice was equivalent to that for unexposed NTG mice. These findings show that epithelial cell beta(2)-ARs regulate airway responsiveness in vivo and that the bronchodilating effect of beta-agonists results from activation of receptors on both epithelial and smooth muscle cells.     

Alpha 2-Adrenergic stimulation is protective against ischemia-reperfusion-induced ventricular arrhythmias in vivo

We previously reported that alpha(2)-adrenergic receptor (alpha(2)-AR) stimulation in Purkinje fibers in vitro prolongs action potential duration and suppresses beta-adrenergic-induced delayed afterdepolarizations and sustained triggered activities. We examined the effects of alpha(2)-AR stimulation on reperfusion-induced ventricular arrhythmias [ventricular tachycardia/ventricular fibrillation (VT/VF)] in vivo. Arterial blood pressure, heart rate, surface electrocardiogram, and renal sympathetic nerve activities were recorded simultaneously in Sprague-Dawley rats. The incidence of VT/VF was 87.5% for controls, 50% for the beta-blocker group, 72% for the alpha(1)-blocker group, and 12.5% for the alpha(1) + beta-blockers group (unopposed alpha(2)-adrenergic activation). Direct alpha(2)-AR stimulation with UK-14304 also prevented VT/VF. These effects were reversed by the alpha(2)-adrenergic antagonist yohimbine. Increases in renal sympathetic nerve activity were associated with left anterior descending coronary artery ligation and reperfusion (33 +/- 1.5 and 62 +/- 1.7% over baseline, respectively) in controls. Similar patterns were observed among all experimental groups irrespective of the incidence of VT/VF on reperfusion. We conclude that alpha(2)-AR stimulation has a potent antiarrhythmic effect on ischemia-reperfusion-induced VT/VF in vivo and that this effect is not centrally mediated.     

Beta2- and beta3-adrenergic receptor polymorphisms and exercise hemodynamics in postmenopausal women.

We sought to determine whether common genetic variations at the beta2 (beta2-AR, Gln27Glu) and beta3 (beta3-AR, Trp64Arg) adrenergic receptor gene loci were associated with cardiovascular (CV) hemodynamics during maximal and submaximal exercise. CV hemodynamics were assessed in 62 healthy postmenopausal women (20 sedentary, 22 physically active, and 20 endurance athletes) during treadmill exercise at 40, 60, 80, and 100% maximal O2 uptake using acetylene rebreathing to quantify cardiac output. The beta2-AR genotype and habitual physical activity (PA) levels interacted to significantly associate with arteriovenous O2 difference (a-vDO2) during submaximal exercise (P = 0.05), with the highest submaximal exercise a-vDO2 in sedentary women homozygous for the beta2-AR Gln allele and no genotype-dependent differences in submaximal exercise a-vDO2 in physically active and athletic women. The beta2-AR genotype also was independently associated with a-vDO2 during submaximal (P = 0.004) and approximately 100% maximal O2 uptake exercise (P = 0.006), with a 1.2-2 ml/100 ml greater a-vDO2 in the Gln/Gln than in the Glu/Glu genotype women. The beta3-AR genotype, independently or interacting with habitual PA levels, was not significantly associated with any CV hemodynamic variables during submaximal or maximal exercise. Thus it appears that the beta2-AR genotype, both independently and interacting with habitual PA levels, is significantly associated with a-vDO2 during exercise in postmenopausal women, whereas the beta3-AR genotype does not appear to be associated with any maximal or submaximal exercise CV hemodynamic responses in postmenopausal women.     

Heart rate recovery after maximal exercise is associated with acetylcholine receptor M2 (CHRM2) gene polymorphism

The determinants of heart rate (HR) recovery after exercise are not well known, although attenuated HR recovery is associated with an increased risk of cardiovascular mortality. Because acetylcholine receptor subtype M2 (CHRM2) plays a key role in the cardiac chronotropic response, we tested the hypothesis that, in healthy individuals, the CHRM2 gene polymorphisms might be associated with HR recovery 1 min after the termination of a maximal exercise test, both before and after endurance training. The study population consisted of sedentary men and women (n = 95, 42 +/- 5 yr) assigned to a training (n = 80) or control group (n = 15). The study subjects underwent a 2-wk laboratory-controlled endurance training program, which included five 40-min sessions/wk at 70-80% of maximal HR. HR recovery differed between the intron 5 rs324640 genotypes at baseline (C/C, -33 +/- 10; C/T, -33 +/- 7; and T/T, -40 +/- 11 beats/min, P = 0.008). Endurance training further strengthened the association: the less common C/C homozygotes showed 6 and 12 beats/min lower HR recovery than the C/T heterozygotes or the T/T homozygotes (P = 0.001), respectively. A similar association was found between A/T transversion at the 3'-untranslated region of the CHRM2 gene and HR recovery at baseline (P = 0.025) and after endurance training (P = 0.005). These data suggest that DNA sequence variation at the CHRM2 locus is a potential modifier of HR recovery in the sedentary state and after short-term endurance training in healthy individuals.     

beta2-Adrenergic receptor agonists stimulate L-type calcium current independent of PKA in newborn rabbit ventricular myocytes

Selective stimulation of beta(2)-adrenergic receptors (ARs) in newborn rabbit ventricular myocardium invokes a positive inotropic effect that is lost during postnatal maturation. The underlying mechanisms for this age-related stimulatory response remain unresolved. We examined the effects of beta(2)-AR stimulation on L-type Ca(2+) current (I(Ca,L)) during postnatal development. I(Ca,L) was measured (37 degrees C; either Ca(2+) or Ba(2+) as the charge carrier) using the whole-cell patch-clamp technique in newborn (1 to 5 days old) and adult rabbit ventricular myocytes. Ca(2+) transients were measured concomitantly by dialyzing the cell with indo-1. Activation of beta(2)-ARs (with either 100 nM zinterol or 1 microM isoproterenol in the presence of the beta(1)-AR antagonist, CGP20712A) stimulated I(Ca,L) twofold in newborns but not in adults. The beta(2)-AR-mediated increase in Ca(2+) transient amplitude in newborns was due exclusively to the augmentation of I(Ca,L). Zinterol increased the rate of inactivation of I(Ca,L) and increased the Ca(2+) flux integral. The beta(2)-AR inverse agonist, ICI-118551 (500 nM), but not the beta(1)-AR antagonist, CGP20712A (500 nM), blocked the response to zinterol. Unexpectedly, the PKA blockers, H-89 (10 microM), PKI 6-22 amide (10 microM), and Rp-cAMP (100 microM), all failed to prevent the response to zinterol but completely blocked responses to selective beta(1)-AR stimulation of I(Ca,L) in newborns. Our results demonstrate that in addition to the conventional beta(1)-AR/cAMP/PKA pathway, newborn rabbit myocardium exhibits a novel beta(2)-AR-mediated, PKA-insensitive pathway that stimulates I(Ca,L). This striking developmental difference plays a major role in the age-related differences in inotropic responses to beta(2)-AR agonists.