Unequal effects of renin-angiotensin system inhibitors in acute cardiac dysfunction induced by isoproterenol

Several clinical trials have demonstrated that angiotensin-converting enzyme inhibitor (ACEI) and angiotensin II type 1 receptor blocker (ARB) are equally effective in the treatment of chronic heart failure. However, this has not been confirmed for acute cardiac dysfunction. We examined whether ACEI or ARB prevents isoproterenol-induced acute left ventricular (LV) dysfunction in dogs. LV dysfunction induced by a large dose of isoproterenol (1 microg.kg(-1).min(-1), 3-h infusion) was compared in dogs treated with ACEI (temocaprilat) or ARB (olmesartan). Atrial pacing induced a constant heart rate and use of adjustable aortic banding provided a nearly constant afterload. LV systolic function (LV dP/dt, fractional shortening, and ejection fraction) and diastolic function (tau and LV end-diastolic pressure) were significantly deteriorated after isoproterenol infusion. The LV dysfunction was almost totally prevented by ARB but was only partially prevented by ACEI. The partial effect of ACEI was complemented by cotreatment with HOE-140, a bradykinin B2 receptor antagonist. At baseline, the response to low doses of isoproterenol was significantly attenuated by ACEI but not by ARB, and the ACEI-induced attenuation was totally abolished by cotreatment with HOE-140. The response to isoproterenol was significantly attenuated after 3 h of excess isoproterenol loading, and it was almost completely preserved by ARB but not by ACEI. In conclusion, acute LV dysfunction and beta-adrenergic desensitization induced by excess isoproterenol administration were almost totally prevented by ARB but only partially prevented by ACEI. These differences were attributable at least in part to bradykinin pathways activated by ACEI administration in acute LV dysfunction.     

Exercise-induced functional desensitization of canine cardiac beta-adrenergic receptors

To test the hypothesis that the high levels of endogenous catecholamines associated with strenuous exercise produce functional desensitization of cardiac beta-adrenergic receptors, we measured the bolus chronotropic dose of isoproterenol necessary to produce a 25-beats/min increase in heart rate (CD25) in the resting state and after the return of heart rate to resting levels after 60 min of treadmill running in 13 normal dogs. Immediately after exercise, 12 of 13 dogs were less sensitive to the chronotropic effects of beta-adrenergic receptor stimulation: mean CD25 increased from 1.16 +/- 0.17 to 3.50 +/- 0.98 micrograms (P less than 0.02). A similar reduction in isoproterenol sensitivity was evident regardless of whether testing was performed in the presence or absence of vagal blockade with atropine. By 3 h after exercise, CD25 had returned to the preexercise level, with no further change noted 24 h after exercise. There was no change in the CD25 when measured serially in three unexercised dogs. We conclude that a single bout of dynamic exercise is sufficient to produce a significantly decreased chronotropic responsiveness to isoproterenol. This phenomenon may represent an acute but transient desensitization of cardiac beta-adrenergic receptors.     

Effects of adrenomedullin on load and myocardial performance in normal and heart-failure dogs

Myocardial actions of the vasodilator peptide adrenomedullin (ADM) in the intact animal are unknown. Negative and positive inotropic actions have been reported in ex vivo experiments. Myocardial and load-altering actions of ADM in dogs before and after development of heart failure were studied. With controlled heart rate (atrial pacing) and after beta-blockade, ADM was administered to five normal dogs in doses of 20 ng. kg(-1). min(-1) iv, 100 ng. kg(-1). min(-1) iv, and 200 ng. kg(-1). min(-1) into the left ventricle (LV). LV peak systolic pressure and end-systolic volume decreased with each dose of ADM. End-systolic pressure decreased with the two higher doses. At the highest dose, arterial elastance and the time constant of LV isovolumic relaxation (tau) decreased, and LV end-systolic elastance (E(es)) increased. LV end-diastolic pressure and volume were unchanged. In five additional normal dogs receiving only the highest dose of ADM (200 ng. kg(-1). min(-1) intra-LV), to control for increased heart rate and sympathetic activation observed with the cumulative infusion, ADM produced arterial vasodilation but no change in E(es) or tau. In four dogs with pacing-induced heart failure, ADM (200 ng. kg(-1). min(-1) intra-LV) was without effect on tau, E(es), and systolic or diastolic pressure and volume. In vivo, ADM appears to be a selective arterial dilator without inotropic or lusitropic effects. The vasodilatory actions are attenuated in heart failure.     

Mediation of isoproterenol-induced thirst in rats by beta2-adrenergic receptors.

Isoproterenol-induced thirst in rats has been attributed to the activation of beta-adrenergic receptors. Since these receptors can be further differentiated pharmacologically into beta1 and beta2 types, experiments were performed using several beta-adrenergic agonists and antagonists to determine the receptor type initiating the isoproterenol-induced thirst. The beta1- and beta2-adrenergic antagonist, d,l-propranolol (1 mg/kg, ip), blocked the increase in water intake usually accompanying acute subcutaneous administration of isoproterenol (25 microgram/kg) to female rats. Since l-propranolol is known to stabilize membranes and to possess anesthetic-like properties, d-propranolol was also used. This isomer has little beta-adrenergic-blocking activity but possesses anesthetic-like activity. Administration of d-propranolol (1 mg/kg, ip) failed to affect the drinking response to acute administration of isoproterenol (25 microgram/kg). Practolol (125 mg/kg), a beta1-adrenergic antagonist with little anesthetic properties, also had no effect on water intake of isoproterenol-treated rats. Butoxamine, a selective beta2-adrenergic antagonist, attenuated the drinking response to isoproterenol. Salbutamol (150 microgram/kg), a beta2-adrenergic agonist, mimicked the effect of isoproterenol on water intake. These results are consistent with the suggestion that beta2-adrenergic receptors mediate the isoproterenol-induced thirst in rats.     

Adaptations in beta-adrenergic cardiovascular responses to training in older women.

To determine whether endurance exercise training can alter the beta-adrenergic-stimulated inotropic response in older women, we studied 10 postmenopausal healthy women (65.4 +/- 0.9 yr old) who exercised for 11 mo. Left ventricular (LV) function was evaluated with two-dimensional echocardiography during infusion of isoproterenol after atropine. Maximal O(2) consumption increased 23% in response to training (from 1.35 +/- 0.06 to 1.66 +/- 0.07 l/min; P = 0.004). Training had no effect on baseline LV function, end-diastolic diameter, LV wall thickness, or LV mass. The increase in LV systolic function in response to isoproterenol was unaffected by training. Furthermore, neither the systolic shortening-to-end-systolic wall stress relationship nor the end-systolic wall stress-to-end-systolic diameter relationship during isoproterenol infusion changed with training. We conclude that older postmenopausal women can increase their maximal O(2) consumption with exercise training without eccentric LV hypertrophy or enhancement of beta-adrenergic-mediated LV contractile function. These observations provide an explanation for the finding that maximal cardiac output and stroke volume are not increased in older women in response to training.     

beta-adrenergic responsiveness of rats treated chronically with isoproterenol

The effect of chronic administration of isoproterenol on isoproterenol-induced thirst and isoproterenol-induced changes in heart rate and selected organ weights of male rats was studied. Administration of 25 micrograms isoproterenol/kg, s.c., in saline daily for 10 days was accompanied by a significant attenuation of the characteristic increase in water intake following a challenging dose of isoproterenol (25 micrograms/kg, s.c.) on the 11th day. Administration of 25 micrograms isoproterenol/kg, s.c., every 2nd, 3rd or 4th day for 10 days was without significant effect on water intake following isoproterenol (25 micrograms/kg, s.c.) on the 11th day. Administration of 25 micrograms isoproterenol/kg, s.c., every day for 10 days led to a slight increase in cardiac responsiveness to a challenging dose of isoproterenol (25 micrograms/kg) on the 11th day. Chronic treatment with this low dose of isoproterenol for 10 days was also accompanied by a significant increase in the ratio of heart weight to body weight but no significant changes in the ratio of kidney, adrenal, thyroid, spleen, or interscapular brown fat to body weight. Thus, daily administration of the beta-adrenergic agonist isoproterenol for 10 days can alter beta-adrenergic responsiveness in the rat with beta 1 (heart rate) and beta 2 (thirst) mediated responses showing opposite effects. In addition, the results suggest that tests of beta-adrenergic responsiveness must be assessed in terms of the frequency of administration of the agonist.     

Decreased cardiac response to isoproterenol infusion in acute and chronic hypoxia

The hypothesis of a blunted chronotropic response of cardiac beta-adrenergic receptors in altitude hypoxia was tested in nine subjects at sea level (SL) by infusion of isoproterenol. Observations were made at SL, in acute hypoxia (2 days at 4,350 m, condition H1), in more prolonged hypoxia [13 days between 850 and 4,800 m, condition H2] and in chronic hypoxia [21 days at 4,800 m, condition H3]. Resting heart rate was higher in all hypoxic conditions. Resting norepinephrine concentrations were found to be significantly higher in conditions H2 (1.64 +/- 0.59) and H3 (1.74 +/- 0.76) than at SL (0.77 +/- 0.18 ng/ml). Isoproterenol, diluted in saline, was infused at increasing doses of 0.0, 0.02, 0.04, and 0.06 micrograms.kg-1.min-1. For the highest dose, there was a significantly smaller increase in heart rate in conditions H1 (35 +/- 9), H2 (33 +/- 11), and H3 (31 +/- 11) than at SL (45 +/- 8 min-1). The increase in pulse (systolic/diastolic) pressure, considered as the vascular response to isoproterenol infusion, was smaller in condition H3 (29 +/- 16) than at SL (51 +/- 24 mmHg). There was a significant increase in the dose of isoproterenol required to increase heart rate by 25 min-1 and decrease in slope of heart rate increase vs. log(dose) relationship in conditions H2 and H3. Thus an hypoxia-related attenuated response of beta-adrenergic receptors to exogenous stimulation was found in humans.(ABSTRACT TRUNCATED AT 250 WORDS)     

Desensitization of the turkey erythrocyte beta-adrenergic receptor in a cell-free system. Evidence that multiple protein kinases can phosphorylate and desensitize the receptor

We have used a recently developed cell-free system (cell lysate) derived from turkey erythrocytes to explore the potential role of cAMP-activated and other protein kinase systems in desensitizing the adenylate cyclase-coupled beta-adrenergic receptor. Desensitization by the agonist isoproterenol required more than simple occupancy of the receptor by the agonist since under conditions where adenylate cyclase was not activated, no desensitization occurred. As in whole cells, addition of cyclic nucleotides to the cell lysate produced only approximately 50% of the maximal isoproterenol-induced desensitization obtainable. Addition of the purified cAMP-dependent protein kinase holoenzyme plus isoproterenol to isolated turkey erythrocyte plasma membranes mimicked the submaximal desensitization induced in lysates by cAMP. This effect was entirely blocked by the specific inhibitor of the cAMP-dependent protein kinase. By contrast, maximal desensitization induced in lysates by isoproterenol was only approximately 50% attenuated by the protein kinase inhibitor. In the lysate preparations, isoproterenol was also shown to induce, in a stereospecific fashion, phosphorylation of the beta-adrenergic receptor. Phosphorylation promoted by isoproterenol was attenuated by cAMP-dependent protein kinase inhibitor to the same extent as desensitization (i.e. approximately 50%). Phorbol diesters also promoted receptor desensitization and phosphorylation in cell lysates. The desensitization was mimicked by incubation of isolated turkey erythrocyte membranes with partially purified preparations of protein kinase C plus phorbol diesters. In the cell lysate, calmodulin also promoted receptor phosphorylation and desensitization which was blocked by EGTA. Desensitization of adenylate cyclase by isoproterenol, phorbol diesters, and calmodulin was not observed to be additive. These findings suggest that: (a) multiple protein kinase systems, including cAMP-dependent, protein kinase C-dependent, and Ca2+/calmodulin-dependent kinases, are capable of regulating beta-adrenergic receptor function via phosphorylation reactions and that (b) cAMP may not be the sole mediator of isoproterenol-induced phosphorylation and desensitization in these cells.     

Differential alterations in cardiac adrenergic signaling in chronic hypoxia or norepinephrine infusion

Norepinephrine (NE)-induced desensitization of the adrenergic receptor pathway may mimic the effects of hypoxia on cardiac adrenoceptors. The mechanisms involved in this desensitization were evaluated in male Wistar rats kept in a hypobaric chamber (380 Torr) and in rats infused with NE (0.3 mg. kg(-1). h(-1)) for 21 days. Because NE treatment resulted in left ventricular (LV) hypertrophy, whereas hypoxia resulted in right (RV) hypertrophy, the selective hypertrophic response of hypoxia and NE was also evaluated. In hypoxia, alpha(1)-adrenergic receptors (AR) density increased by 35%, only in the LV. In NE, alpha(1)-AR density decreased by 43% in the RV. Both hypoxia and NE decreased beta-AR density. No difference was found in receptor apparent affinity. Stimulated maximal activity of adenylate cyclase decreased in both ventricles with hypoxia (LV, 41%; RV, 36%) but only in LV with NE infusion (42%). The functional activities of G(i) and G(s) proteins in cardiac membranes were assessed by incubation with pertussis toxin (PT) and cholera toxin (CT). PT had an important effect in abolishing the decrease in isoproterenol-induced stimulation of adenylate cyclase in hypoxia; however, pretreatment of the NE ventricle cells with PT failed to restore this stimulation. Although CT attenuates the basal activity of adenylate cyclase in the RV and the isoproterenol-stimulated activity in the LV, pretreatment of NE or hypoxic cardiac membranes with CT has a less clear effect on the adenylate cyclase pathway. The present study has demonstrated that 1) NE does not mimic the effects of hypoxia at the cellular level, i.e., hypoxia has specific effects on cardiac adrenergic signaling, and 2) changes in alpha- and beta-adrenergic pathways are chamber specific and may depend on the type of stimulation (hypoxia or adrenergic).     

Effect of U50,488H, a κ-opioid receptor agonist on myocardial α-and β-myosin heavy chain expression and oxidative stress associated with isoproterenol-induced cardiac hypertrophy in rat

Both oxidative stress and β-MHC expression are associated with pathological cardiac hypertrophy. β-adrenergic receptor stimulation plays an important role in cardiac hypertrophy. Recent studies have reported a negative interplay between opioid receptors and adrenoceptors in heart. This study investigated the effect of U50,488H (a selective κ-opioid receptor agonist) on myocardial oxidative stress and α- and β-MHC expression in isoproterenol-induced cardiac hypertrophy. Male Wistar rats were administered normal saline (control), isoproterenol (ISO) (5 mg/kg BW s.c. OD), and isoproterenol with U50,488H (0.4 and 0.6 mg/kg BW, i.p. OD) for 14 days. In a separate group, nor-binaltorphimine (nor-BNI) (0.5 mg/kg, BW, i.p.) (κ-receptor antagonist) was administered along with ISO and U50,488H. ISO administration caused significant increase in left ventricular (LV) wall thicknesses, LV mass in echocardiography, heart weight to body weight ratio, and myocyte size as compared to control. Both the doses of U50,488H offered significant protection against these changes. The higher dose of U50,488H significantly prevented ISO-induced increase in myocardial lipid peroxidation and depletion of myocardial antioxidants (glutathione, superoxide dismutase, and catalase), while a similar trend (although not significant) was observed with the lower dose also. ISO-induced myocardial fibrosis was also significantly attenuated by both the doses of U50,488H. Isoproterenol-induced β-MHC expression in the hypertrophied heart was not altered by either doses of U50,488H, however, the latter prevented the loss of myocardial α-MHC expression. All these effects of U50,488H were blocked by nor-BNI. This study provides the evidence that U50,488H reduced oxidative stress and preserved expression of α-MHC in isoproterenol-induced cardiac hypertrophy.     

Active metabolite of GLP-1 mediates myocardial glucose uptake and improves left ventricular performance in conscious dogs with dilated cardiomyopathy

We have shown previously that the glucagon-like peptide-1 (GLP-1)-(7-36) amide increases myocardial glucose uptake and improves left ventricular (LV) and systemic hemodynamics in both conscious dogs with pacing-induced dilated cardiomyopathy (DCM) and humans with LV systolic dysfunction after acute myocardial infarction. However, GLP-1-(7-36) is rapidly degraded in the plasma to GLP-1-(9-36) by dipeptidyl peptidase IV (DPP IV), raising the issue of which peptide is the active moiety. By way of methodology, we compared the efficacy of a 48-h continuous intravenous infusion of GLP-1-(7-36) (1.5 pmol.kg(-1).min(-1)) to GLP-1-(9-36) (1.5 pmol.kg(-1).min(-1)) in 28 conscious, chronically instrumented dogs with pacing-induced DCM by measuring LV function and transmyocardial substrate uptake under basal and insulin-stimulated conditions using hyperinsulinemic-euglycemic clamps. As a result, dogs with DCM demonstrated myocardial insulin resistance under basal and insulin-stimulated conditions. Both GLP-1-(7-36) and GLP-1-(9-36) significantly reduced (P < 0.01) LV end-diastolic pressure [GLP-1-(7-36), 28 +/- 1 to 15 +/- 2 mmHg; GLP-1-(9-36), 29 +/- 2 to 16 +/- 1 mmHg] and significantly increased (P < 0.01) the first derivative of LV pressure [GLP-1-(7-36), 1,315 +/- 81 to 2,195 +/- 102 mmHg/s; GLP-1-(9-36), 1,336 +/- 77 to 2,208 +/- 68 mmHg] and cardiac output [GLP-1-(7-36), 1.5 +/- 0.1 to 1.9 +/- 0.1 l/min; GLP-1-(9-36), 2.0 +/- 0.1 to 2.4 +/- 0.05 l/min], whereas an equivolume infusion of saline had no effect. Both peptides increased myocardial glucose uptake but without a significant increase in plasma insulin. During the GLP-1-(9-36) infusion, negligible active (NH2-terminal) peptide was measured in the plasma. In conclusion, in DCM, GLP-1-(9-36) mimics the effects of GLP-1-(7-36) in stimulating myocardial glucose uptake and improving LV and systemic hemodynamics through insulinomimetic as opposed to insulinotropic effects. These data suggest that GLP-1-(9-36) amide is an active peptide.     

Effects of long-term pretreatment with isoproterenol on bromocriptine-induced tachycardia in conscious rats

It has been shown that bromocriptine-induced tachycardia, which persisted after adrenalectomy, is (i) mediated by central dopamine D2 receptor activation and (ii) reduced by 5-day isoproterenol pretreatment, supporting therefore the hypothesis that this effect is dependent on sympathetic outflow to the heart. This study was conducted to examine whether prolonged pretreatment with isoproterenol could abolish bromocriptine-induced tachycardia in conscious rats. Isoproterenol pretreatment for 15 days caused cardiac hypertrophy without affecting baseline blood pressure and heart rate. In control rats, intravenous bromocriptine (150 microg/kg) induced significant hypotension and tachycardia. Bromocriptine-induced hypotension was unaffected by isoproterenol pretreatment, while tachycardia was reversed to significant bradycardia, an effect that was partly reduced by i.v. domperidone (0.5 mg/kg). Neither cardiac vagal nor sympathetic tone was altered by isoproterenol pretreatment. In isolated perfused heart preparations from isoproterenol-pretreated rats, the isoproterenol-induced maximal increase in left ventricular systolic pressure was significantly reduced, compared with saline-pretreated rats (the EC50 of the isoproterenol-induced increase in left ventricular systolic pressure was enhanced approximately 22-fold). These results show that 15-day isoproterenol pretreatment not only abolished but reversed bromocriptine-induced tachycardia to bradycardia, an effect that is mainly related to further cardiac beta-adrenoceptor desensitization rather than to impairment of autonomic regulation of the heart. They suggest that, in normal conscious rats, the central tachycardia of bromocriptine appears to predominate and to mask the bradycardia of this agonist at peripheral dopamine D2 receptors.     

Electrocardiography in two models of isoproterenol-induced left ventricular remodeling

We studied the ability of the ECG to detect pathological changes in isoproterenol-induced remodeling of rat heart. Myocardial hypertrophy in rats was induced by repeated injections of isoproterenol (5 mg/kg s.c. 7 days, Iso5, n=7). Single overdose of isoproterenol (150 mg/kg s.c., Iso150, n=7) evoked myocardial infarction followed with ventricular remodeling. The electrocardiograms were recorded in anesthetized animals (thiopenthal 45 mg/kg i.p.) and myocardial contractile performance was analyzed in isolated hearts perfused according to Langendorff. The hypertrophic hearts were characterized by increased heart and left ventricular (LV) weight as well as by thicker LV free wall and interventricular septum. Mean values of LV contraction did not significantly differ from controls. Longer QT interval, QRS complex, negative Q and S waves, higher R amplitude were typical characteristics for Iso5 rats. Iso150 animals showed tendency to decreased systolic blood pressure and heart frequency. Decrease in the thickness of LV compared to Iso5 as well as impaired LV function were related to the dilated left ventricle. Iso150 ECG showed longer QRS and QT, deepened negativity of S wave and mild decrease of R(II) compared to Iso5. Voltage criteria showed that Sokolow-Lyon index is a good predictor of left ventricular hypertrophy in isoproterenol-induced cardiac remodeling without systemic hypertension.     

Reversible effects of isoproterenol-induced hypertrophy on in situ left ventricular function in rat hearts

The aim of the present study was to evaluate specifically left ventricular (LV) function in rat hearts as they transition from the normal to hypertrophic state and back to normal. Either isoproterenol (1.2 and 2.4 mg.kg(-1).day(-1) for 3 days; Iso group) or vehicle (saline 24 microl.day(-1) for 3 days; Sa group) was infused by subcutaneous implantation of an osmotic minipump. After verifying the development of cardiac hypertrophy, we recorded continuous LV pressure-volume (P-V) loops of in situ ejecting hypertrophied rat hearts. The curved LV end-systolic P-V relation (ESPVR) and systolic P-V area (PVA) were obtained from a series of LV P-V loops in the Sa and Iso groups 1 h or 2 days after the removal of the osmotic minipump. PVA at midrange LV volume (PVA(mLVV)) was taken as a good index for LV work capability (13, 15, 20, 21). However, in rat hearts during remodeling, whether PVA(mLVV) is a good index for LV work capability has not been determined yet. In the present study, in contrast to unchanged end-systolic pressure at midrange LV volume, PVA(mLVV) was significantly decreased by isoproterenol treatment relative to saline; however, these measurements were the same 2 days after pump removal. Simultaneous treatment with a beta(1)-blocker, metoprolol (24 mg.kg(-1).day(-1)), blocked the formation of cardiac hypertrophy and thus PVA(mLVV) did not decrease. The reversible changes in PVA(mLVV) reflect precisely the changes in LV work capability in isoproterenol-induced hypertrophied rat hearts mediated by beta(1)-receptors. These results indicate that the present approach may be an appropriate strategy for evaluating the effects of antihypertrophic and antifibrotic modalities.     

Functional antagonism of airway constriction in the canine lung periphery.

We studied the ability of a beta-adrenergic agonist (albuterol) to attenuate calcium chelator- and acetylcholine-induced airway constriction in the lung periphery of anesthetized mongrel and Basenji-Greyhound (BG) dogs. A wedged bronchoscope technique was used to measure collateral system resistance before and after challenges with aerosolized Na2EDTA and acetylcholine. Time course of the response to Na2EDTA differed significantly between mongrel and BG dogs. Peak response to challenge with 4% Na2EDTA occurred within 2 min for mongrel dogs and at 5 min for BG dogs. Albuterol (1 microgram/kg iv) significantly attenuated Na2EDTA-induced bronchoconstriction in both groups of animals (P less than 0.01, each group). Albuterol (1 microgram/kg iv) significantly attenuated acetylcholine-induced bronchoconstriction in mongrel (P less than 0.01) but not in BG dogs. We conclude that a qualitative difference exists in the mechanism of Na2EDTA-induced constriction in the lung periphery of BG compared with mongrel dogs. In addition, the lung periphery of BG dogs demonstrates reduced beta-adrenergic sensitivity with respect to a cholinergic challenge compared with mongrels, suggesting enhanced cholinergic inhibition of the beta-adrenergic system.     

Regulation of adenylyl cyclase activity by beta-adrenergic agonists in a desensitization-resistant mutant cell line.

Mutant clones resistant to ACTH-induced desensitization of adenylyl cyclase (Y1DR) were previously isolated from the Y1 mouse adrenocortical tumor cell line. In this study, both parental Y1 cells (Y1DS) and a Y1DR mutant were transfected with a gene encoding the mouse beta 2-adrenergic receptor, and transfectants isolated from both Y1DS and Y1DR cells were shown to express beta 2-adrenergic receptors. These transfectants responded to the beta-adrenergic agonist isoproterenol with increases in adenylyl cyclase activity and steroidogenesis and changes in cell shape. The transfectants were analyzed to determine whether the Y1DR mutation was specific for ACTH-induced desensitization of adenylyl cyclase or also affected desensitization of adenylyl cyclase via the beta 2-adrenergic receptor. Treatment of intact Y1DS transfectants with isoproterenol caused a rapid desensitization of the adenylyl cyclase system to further stimulation by the beta-adrenergic agonist. Treatment of intact cells with isoproterenol did not affect ACTH-stimulated adenylyl cyclase activity, indicating that desensitization was agonist specific or homologous. Y1DR transfectants were resistant to the desensitizing effects of isoproterenol in intact cells as well as in cell homogenates. These results indicate that the mutation in Y1DR transfectants affects a component that is common to the pathways of isoproterenol-induced desensitization and ACTH-induced desensitization of adenylyl cyclase. As determined using the hydrophilic beta-receptor antagonist CGP-12177, isoproterenol caused a rapid sequestration of cell surface receptors in both Y1DS and Y1DR transfectants. From these results we infer that the DR phenotype does not arise from mutations affecting receptor sequestration and that receptor number does not limit the response to isoproterenol in these transfectants.     

Dissociation between cardiomyocyte function and remodeling with beta-adrenergic receptor blockade in isolated canine mitral regurgitation

The low-pressure volume overload of isolated mitral regurgitation (MR) is associated with increased adrenergic drive, left ventricular (LV) dilatation, and loss of interstitial collagen. We tested the hypothesis that beta1-adrenergic receptor blockade (beta1-RB) would attenuate LV remodeling after 4 mo of MR in the dog. beta1-RB did not attenuate collagen loss or the increase in LV mass in MR dogs. Using MRI and three-dimensional (3-D) analysis, there was a 70% increase in the LV end-diastolic (LVED) volume-to-LV mass ratio, a 23% decrease in LVED midwall circumferential curvature, and a >50% increase in LVED 3-D radius/wall thickness in MR dogs that was not attenuated by beta1-RB. However, beta1-RB caused a significant increase in LVED length from the base to apex compared with untreated MR dogs. This was associated with an increase in isolated cardiomyocyte length (171+/-5 microm, P<0.05) compared with normal (156+/-3 microm) and MR (165+/-4 microm) dogs. Isolated cardiomyocyte fractional shortening was significantly depressed in MR dogs compared with normal dogs (3.73+/-0.31 vs. 5.02+/-0.26%, P<0.05) and normalized with beta1-RB (4.73+/-0.48%). In addition, stimulation with the beta-adrenergic receptor agonist isoproterenol (25 nM) increased cardiomyocyte fractional shortening by 215% (P<0.05) in beta1-RB dogs compared with normal (56%) and MR (50%) dogs. In summary, beta1-RB improved LV cardiomyocyte function and beta-adrenergic receptor responsiveness despite further cell elongation. The failure to attenuate LV remodeling associated with MR could be due to a failure to improve ultrastructural changes in extracellular matrix organization.     

Cardiac dilatation and pump dysfunction without intrinsic myocardial systolic failure following chronic beta-adrenoreceptor activation

There is no direct evidence to indicate that pump dysfunction in a dilated chamber reflects the impact of chamber dilatation rather than the degree of intrinsic systolic failure resulting from myocardial damage. In the present study, we explored the relative roles of intrinsic myocardial systolic dysfunction and chamber dilatation as mediators of left ventricular (LV) pump dysfunction. Administration of isoproterenol, a beta-adrenoreceptor agonist, for 3 mo to rats (0.1 mg.kg(-1).day(-1)) resulted in LV pump dysfunction as evidenced by a reduced LV endocardial fractional shortening (echocardiography) and a decrease in the slope of the LV systolic pressure-volume relation (isolated heart preparations). Although chronic beta-adrenoreceptor activation induced cardiomyocyte damage (deoxynucleotidyl transferase-mediated dUTP nick-end labeling) as well as beta(1)- and beta(2)-adrenoreceptor inotropic downregulation (attenuated contractile responses to dobutamine and salbutamol), these changes failed to translate into alterations in intrinsic myocardial contractility. Indeed, LV midwall fractional shortening (echocardiography) and the slope of the LV systolic stress-strain relation (isolated heart preparations) were unchanged. A normal intrinsic myocardial systolic function, despite the presence of cardiomyocyte damage and beta-adrenoreceptor inotropic downregulation, was ascribed to marked increases in myocardial norepinephrine release, to upregulation of alpha-adrenoreceptor-mediated contractile effects as determined by phenylephrine responsiveness, and to compensatory LV hypertrophy. LV pump failure was attributed to LV dilatation, as evidenced by increased LV internal dimensions (echocardiography), and a right shift and increased volume intercept of the LV diastolic pressure-volume relation. In conclusion, chronic sympathetic stimulation, despite reducing beta-adrenoreceptor-mediated inotropic responses and promoting myocyte apoptosis, may nevertheless induce pump dysfunction primarily through LV dilatation, rather than intrinsic myocardial systolic failure.     

Role of inducible nitric oxide synthase in cardiac function and remodeling in mice with heart failure due to myocardial infarction

Using inducible nitric oxide (NO) synthase (iNOS) knockout mice (iNOS-/-), we tested the hypotheses that 1) lack of iNOS attenuates cardiac remodeling and dysfunction and improves cardiac reserve postmyocardial infarction (MI), an effect that is partially mediated by reduction of oxidative stress due to reduced interaction between NO and reactive oxygen species (ROS); and 2) the cardioprotection afforded by iNOS deletion is eliminated by Nomega-nitro-L-arginine methyl ester (L-NAME) due to inhibition of endothelial NOS (eNOS) and neuronal NOS (nNOS). MI was induced by ligating the left anterior descending coronary artery. Male iNOS-/- mice and wild-type controls (WT, C57BL/6J) were divided into sham MI, MI+vehicle, and MI+l-NAME (100 mg.kg(-1).day(-1) in drinking water for 8 wk). Cardiac function was evaluated by echocardiography. Left ventricular (LV) maximum rate of rise of ventricular pressure divided by pressure at the moment such maximum occurs (dP/dt/instant pressure) in response to isoproterenol (100 ng.kg(-1).min(-1) iv) was measured with a Millar catheter. Collagen deposition, myocyte cross-sectional area, and expression of nitrotyrosine and 4-hydroxy-2-nonenal (4-HNE), markers for ROS, were determined by histopathological and immunohistochemical staining. We found that the MI-induced increase in LV chamber dimension and the decrease in ejection fraction, an index of systolic function, were less severe in iNOS-/- compared with WT mice. L-NAME worsened LV remodeling and dysfunction further, and these detrimental effects were also attenuated in iNOS-/- mice, associated with better preservation of cardiac function. Lack of iNOS also reduced nitrotyrosine and 4-HNE expression after MI, indicating reduced oxidative stress. We conclude that iNOS does not seem to be a pathological mediator of heart failure; however, the lack of iNOS improves cardiac reserve post-MI, particularly when constitutive NOS isoforms are blocked. Decreased oxidative stress and other adaptive mechanisms independent of NOS may be partially responsible for such an effect, which needs to be studied further.     

Alteration of sensitivity of adrenergic vascular responses after prolonged exposure to agonists via osmotic minipump

A study was performed to determine whether a constant 1-week exposure to either alpha or beta agonists in vivo would allow alteration or manipulation of the responses of rat aortic alpha- and beta-adrenergic receptors. Osmotic minipumps delivering either phenylephrine, isoproterenol, or propranolol for 7 days at a dose of 3.2, 4.2, or 5.2 mg/kg/day, respectively, were implanted in male Holtzman rats under halothane anesthesia. Seven days later, rats were killed and aortic ring preparations were used to measure alpha- and beta-adrenergic responses. In phenylephrine-pretreated rats, alpha-adrenergic responses, as measured by contractions induced by phenylephrine, were markedly reduced (P less than 0.05) across a dose range of 10(-9) to 10(-6) M. In contrast, in these same phenylephrine-pretreated preparations, the beta-adrenergic responses involving isoproterenol-induced relaxation were significantly increased (P less than 0.05) across a dose range of 10(-7) to 10(-5) M. Isoproterenol pretreatment for 7 days resulted in a statistically significant reduction of beta-adrenergic aortic relaxation, whereas the alpha-adrenergic responses to phenylephrine remained unchanged compared with controls. Propranolol pretreatment had no effect on either alpha- or beta-adrenergic responses. These findings indicate that the alpha agonist-induced response after in vivo pretreatment induces reciprocal changes in the functionally related beta-adrenergic apparatus, and also suggest linkage between these two receptors. In contrast, the beta response appears to desensitize or downregulate in response to beta agonist exposure in a manner that seems to be independent of or to operate in the absence of an alteration of the alpha response.