Interstitial Norepinephrine Concentrations Research Articles

PS-B02-5: NICORANDIL SUPPRESSES ISCHEMIA-INDUCED CARDIAC NOREPINEPHRINE ENHANCEMENT AND VENTRICULAR ARRHYTHMIAS IN PRESSURE OVERLOAD-INDUCED HYPERTROPHIC HEARTS.

Objective: Ventricular arrhythmias are a common cause of sudden death in acute myocardial infarction (MI) and hypertension is a major risk factor of MI. Nicorandil, an ATP sensitive potassium channel (KATP) opener, is usually used in the treatment of acute MI. KATP expresses in nerve terminals as well as cardiomyocytes and KATP in nerve terminals regulates norepinephrine release. The effects of nicorandil on ischemic myocyte are fully defined. On the other hand, the roles of nicorandil on ischemic norepinephrine release in hypertrophic cardiac tissue has remained unexplored. We examined whether nicorandil suppressed interstitial norepinephrine level and ventricular arrhythmia during acute ischemia in chronic pressure overload-induced hypertrophic hearts. Design and Methods: Rats were divided into two groups: abdominal aortic constriction (AAC) group and sham-operated (Sham) group. Four weeks after constriction, cardiac geometry and function were examined using echocardiography and hemodynamic measurements, and cardiac tissues were stained with hematoxylin and eosin and Sirius Red for histological examinations. In other rats, 4 weeks after constriction myocardial ischemia was induced by the left anterior descending artery occlusion for 100 minutes in the presence or absence of intravenous infusion of nicorandil. Cardiac interstitial norepinephrine concentration in ischemic region was measured using the microdialysis method. Ventricular arrhythmias were monitered by ECG during whole ischemic period. Results: Four weeks after constriction, remarkable left ventricular wall thickening and diastolic dysfunction were observed in AAC group. In histological examination, remarkable myocyte hypertrophy and interstitial fibrosis were observed in AAC group. Before ischemia, ventricular arrhythmia was not found in both groups. Number of ventricular arrhythmias, including ventricular tachycardia and ventricular fibrillation, was increased in early ischemic period (−20 min) in both groups, and was grater in AAC group. Before ischemia, interstitial norepinephrine concentration in cardiac tissue was higher level in AAC group than in Sham group, and ischemia increased norepinephrine concentration in both groups. Nicorandil administration in ischemic period significantly suppressed the number of ventricular arrhythmias and abolished the ventricular tachycardia and fibrillation without hemodynamic alterations in hypertrophic hearts. Nicorandil also attenuated epinephrine enhancement in acute ischemic period in hypertrophic hearts. Conclusion: Ischemia-induced ventricular arrhythmias were more frequent and severe in hypertrophic hearts and associated with high interstitial norepinephrine concentration. Nicorandil suppressed ischemia-induced interstitial norepinephrine release by neuronal KATP opening, which attenuated ventricular arrhythmias in pressure overload-induced hypertrophic hearts.

Nicorandil Suppresses Ischemia-Induced Norepinephrine Release and Ventricular Arrhythmias in Hypertrophic Hearts.

Ventricular arrhythmias (VAs) are a common cause of sudden death in acute myocardial infarction (MI), for which hypertension is a major risk factor. Nicorandil opens ATP-sensitive potassium (KATP) channels, which are expressed by nerve terminals and cardiomyocytes and regulate the release of norepinephrine (NE). However, the effects of nicorandil on ischemic NE release in cardiac tissue remain unclear. Therefore, we herein investigated whether nicorandil suppressed interstitial NE concentrations and VAs during acute MI in pressure overload-induced hypertrophic hearts. Rats were divided into two groups: an abdominal aortic constriction (AAC) group and sham-operated (Sham) group. Four weeks after constriction, cardiac geometry and functions were examined using echocardiography and hemodynamic analyses. Myocardial ischemia was induced by coronary artery occlusion for 100min with or without the administration of nicorandil. VAs were assessed by electrocardiography, and NE concentrations in the ischemic region were measured using a micro-dialysis method. AAC induced left ventricular hypertrophy with diastolic dysfunction. VAs markedly increased in the early phase (0-20min) of ischemia in both groups and were more frequent in the AAC group. Cardiac interstitial NE concentrations were higher in the AAC group before ischemia and significantly increased during ischemia in both groups. Nicorandil significantly suppressed ischemia-induced VAs and NE increases in the AAC group. Ischemia-induced VAs were more frequent in hypertrophic hearts and associated with high interstitial concentrations of NE. The attenuation of ischemia-induced increases in NE through neuronal KATP opening by nicorandil may suppress ischemia-induced VAs in hypertrophic hearts.

P1603Nicorandil suppresses ischemia-induced cardiac interstitial norepinephrine enhancement and ventricular arrhythmia in hypertrophic hearts

Abstract Background Myocardial infarction (MI) is a major cause of death in western countries and Japan, and hypertension is a major risk factor of MI. In hypertensive heart, acute myocardial infarction often leads to lethal ventricular arrhythmia. Nicorandil, an ATP sensitive potassium channel (KATP) opener, is usually used in the treatment of acute myocardial infarction. The effects of nicorandil on ischemic myocyte are fully defined. On the other hand, KATP in neuroterminals is known to regulate norepinephrine release, but the effect of nicorandil on ischemic norepinephrine release in cardiac tissue has remained unexplored. Purpose We examined whether nicorandil suppressed norepinephrine release via neuronal KATP and ventricular arrhythmia during acute ischemia in pressure overload-induced hypertrophic hearts. Methods SD Rats were divided into two groups; abdominal aortic constriction (AAC) group and sham-operated (Sham) group. Four weeks after constriction, cardiac geometry and function were examined using echocardiography. Then, myocardial ischemia was induced by the left anterior descending artery occlusion for 100 minutes in the presence or absence of intravenous infusion of nicorandil. Cardiac interstitial norepinephrine concentration in ischemic region was measured using the microdialysis method and concentration of cyclic AMP, a second messenger of norepinephrine, in cardiac tissue was measured by ELISA. Ventricular arrhythmias were monitered by ECG during whole ischemic period. Results Four weeks after constriction, remarkable left ventricular wall thickening was observed in AAC group. Before ischemia, ventricular arrhythmia was not found in both groups. Number of ventricular arrhythmia, including ventricular tachycardia and ventricular fibrillation, was increased in early ischemic period (- 40 min) in both groups, and was grater in AAC group. Before ischemia, interstitial norepinephrine concentration in cardiac tissue was higher level in AAC group than in Sham group. Ischemia obviously increased norepinephrine concentration in both groups time dependently and AAC further increased norepinephrine than Sham group. Concentration of cyclic AMP in cardiac tissue was raised in early ischemic period (- 40 min) and then gradually decreased. Nicorandil significantly suppressed the number of ventricular arrhythmias, and abolished the ventricular tachycardia and fibrillation without hemodynamic alterations. Nicorandil also attenuated norepinephrine and cAMP enhancement in acute ischemic period in both groups. Conclusion Ischemia-induced ventricular arrhythmia was more frequent and severe in hypertrophic hearts and interstitial norepinephrine enhancement may play a role in this ischemic arrhythmia. Nicorandil suppressed ischemia-induced interstitial norepinephrine release by neuronal KATP opening, which attenuated ventricular arrhythmias in normal and hypertrophic hearts.

Open-loop (feed-forward) and feedback control of coronary blood flow during exercise, cardiac pacing, and pressure changes.

A control system model was developed to analyze data on in vivo coronary blood flow regulation and to probe how different mechanisms work together to control coronary flow from rest to exercise, and under a variety of experimental conditions, including cardiac pacing and with changes in coronary arterial pressure (autoregulation). In the model coronary flow is determined by the combined action of a feedback pathway signal that is determined by the level of plasma ATP in coronary venous blood, an adrenergic open-loop (feed-forward) signal that increases with exercise, and a contribution of pressure-mediated myogenic control. The model was identified based on data from exercise experiments where myocardial oxygen extraction, coronary flow, cardiac interstitial norepinephrine concentration, and arterial and coronary venous plasma ATP concentrations were measured during control and during adrenergic and purinergic receptor blockade conditions. The identified model was used to quantify the relative contributions of open-loop and feedback pathways and to illustrate the degree of redundancy in the control of coronary flow. The results indicate that the adrenergic open-loop control component is responsible for most of the increase in coronary blood flow that occurs during high levels of exercise. However, the adenine nucleotide-mediated metabolic feedback control component is essential. The model was evaluated by predicting coronary flow in cardiac pacing and autoregulation experiments with reasonable fits to the data. The analysis shows that a model in which coronary venous plasma adenine nucleotides are a signal in local metabolic feedback control of coronary flow is consistent with the available data.

Interstitial norepinephrine levels and local electrophysiological properties of the myocardium during sympathetic nerve activation

Cardiac sympathetic innervation originates mainly in the left and right stellate ganglia. The efferent and afferent fibers of the left stellate ganglion (LSG) predominantly innervate the left ventricle (LV), whereas the fibers of the right stellate ganglion (RSG) innervate the right ventricle. The major neurotransmitter, norepinephrine, shortens ventricular action potential duration (APD) and increases contractility. Activation recovery interval (ARI) can serve as a surrogate for APD in both animal models and patients. However, the linkage between myocardial interstitial norepinephrine levels and ARI during stimulation of left and right stellate ganglion remains unclear. In six anesthetized pigs, interstitial norepinephrine concentrations and ARIs were measured in the perfusion areas of the left anterior descending coronary artery. LV pressure and volume were recorded by a 5Fr Millar conductance catheter. LSG stimulation significantly shortened ARI (28%) and increased systemic blood pressure (SBP, 43%), LV dP/dtmax (200%), and interstitial norepinephrine (110%) with a mild change in heart rate (13%). RSG stimulation also significantly shortened ARI (100%) and increased SBP (63%), heart rate (80%), dP/dtmax (300%), and interstitial norepinephrine (120%). We conclude that both LSG and RSG play an important role in regulating LV function and local sympathetic nerve activity.

Muscle interstitial ATP and norepinephrine concentrations in the human leg during exercise and ATP infusion

ATP has been proposed to play multiple roles in local skeletal muscle blood flow regulation by inducing vasodilation and modulating sympathetic vasoconstrictor activity, but the mechanisms remain unclear. Here we evaluated the effects of arterial ATP infusion and exercise on leg muscle interstitial ATP and norepinephrine (NE) concentrations to gain insight into the interstitial and intravascular mechanisms by which ATP causes muscle vasodilation and sympatholysis. Leg hemodynamics and muscle interstitial nucleotide and NE concentrations were measured during 1) femoral arterial ATP infusion (0.42 +/- 0.04 and 2.26 +/- 0.52 micromol/min; mean +/- SE) and 2) one-leg knee-extensor exercise (18 +/- 0 and 37 +/- 2 W) in 10 healthy men. Arterial ATP infusion and exercise increased leg blood flow (LBF) in the experimental leg from approximately 0.3 l/min at baseline to 4.2 +/- 0.3 and 4.6 +/- 0.5 l/min, respectively, whereas it was reduced or unchanged in the control leg. During arterial ATP infusion, muscle interstitial ATP, ADP, AMP, and adenosine concentrations remained unchanged in both legs, but muscle interstitial NE increased from approximately 5.9 nmol/l at baseline to 8.3 +/- 1.2 and 8.7 +/- 0.7 nmol/l in the experimental and control leg, respectively (P < 0.05), in parallel to a reduction in arterial pressure (P < 0.05). During exercise, however, interstitial ATP, ADP, AMP, and adenosine concentrations increased in the contracting muscle (P < 0.05), but not in inactive muscle, whereas interstitial NE concentrations increased similarly in both active and inactive muscles. These results suggest that the vasodilatory and sympatholytic effects of intraluminal ATP are mainly mediated via endothelial purinergic receptors. Intraluminal ATP and muscle contractions appear to modulate sympathetic nerve activity by inhibiting the effect of NE rather than blunting its local concentration.

Interstitial norepinephrine concentrations in skeletal muscle of ischemic heart failure

During exercise, sympathetic nerve responses are accentuated in heart failure (HF), and this enhances norepinephrine (NE) release and evokes vasoconstriction. Two key pathophysiological responses could contribute to the greater NE release: 1) increased sympathetic nerve discharge and 2) increased NE in the neurovascular junction for a given level of sympathetic discharge. In this report, we focus on the second of these two general issues and test the following hypotheses: 1) in HF for a given level of sympathetic nerve stimulation, NE concentration in the interstitium (an index of neurovascular NE) would be greater, and 2) the greater interstitial NE concentration would be linked to reduced NE uptake. Studies were performed in rats 8-10 wk after induction of myocardial infarction (MI). Interstitial NE samples were collected from microdialysis probes inserted into the hindlimb muscle. Dialysate concentration of NE was determined by the HPLC method. First, interstitial NE concentration increased during electrical stimulation of the lumbar sympathetic nerves in eight control rats. An increase in interstitial NE concentration was significantly greater in 10 rats with severe MI. Additionally, an NE uptake-1 inhibitor (desipramine, 1 microM) was injected into the arterial blood supply of the muscle in six control and eight MI rats. Desipramine increased interstitial NE concentration by 24% in control and by only 3% (P < 0.05 vs. control) in MI rats. In conclusion, given levels of electrical stimulation of the lumbar sympathetic nerve lead to higher interstitial NE concentration in HF. This effect is due, in part, to reduced NE uptake-1 in HF.

Ibotenic acid lesions reduce noradrenergic activation in ventromedial hypothalamus during hypoglycemia

Noradrenergic and GABAergic systems in the ventromedial hypothalamus (VMH) are activated during hypoglycemia and initiate part of the compensatory counterregulatory response. Norepinephrine (NE) terminals innervating the VMH originate in glucosensing hindbrain areas, but whether NE activity in the VMH is under local control or in the hindbrain is unclear. To elucidate the role of neurons intrinsic to the VMH on NE release in the VMH during hypoglycemia, ibotenic acid (IBO), an NMDA receptor agonist that selectively destroys cell bodies, was used. In a 2 × 2 factorial study, IBO (3–5 μg/0.5 μL) or vehicle was stereotaxically administered into the VMH of male Sprague–Dawley rats. One week later, NE concentration in the VMH was measured by microdialysis during insulin-induced hypoglycemia (2.0 U/kg) or euglycemia (saline control). Baseline levels of NE were not statistically different ( p = 0.10) in IBO-treated compared with vehicle-treated rats (13.3 ± 2.8 nM vs. 7.9 ± 1.1 nM). The initial increase in interstitial NE concentration during hypoglycemia in control rats was absent in IBO-treated rats ( p < 0.01). In IBO-treated hypoglycemic rats, NE concentrations increased after 45 min to a similar level observed in control rats during the first 20 min of hypoglycemia. These results are consistent with the suggestion that local neurons in the VMH respond to hypoglycemia and modify NE activation in the VMH during hypoglycemia.

Anaphylactic Shock

The pathophysiology of anaphylactic shock during anesthesia is incompletely characterized. It is described as distributive by analogy with septic shock (anaerobic metabolism, high tissue oxygen pressure [Ptio2] values). The Ptio2 profile and its metabolic consequences during anaphylaxis are not known. Ovalbumin-sensitized anaphylactic shock rats (n = 11) were compared to nicardipine-induced hypotension rats (n = 12) for systemic hemodynamics, Ptio2, sympathetic nervous system activation, skeletal muscle blood flow, and interstitial lactate and pyruvate concentrations using combined microdialysis and polarographic Clark-type oxygen probes. In both groups, the time course and the magnitude of arterial hypotension were similar. The ovalbumin group but not the nicardipine group displayed decreased skeletal muscle blood flow (from 45 +/- 6.2 ml x 100 g(-1) x min(-1) to 24.3 +/- 5 ml x 100 g(-1) x min(-1); P < 0.0001) and Ptio2 values (from 42 +/- 5 to 5 +/- 2; P < 0.0001). The ovalbumin group had more intense sympathetic nervous system activation with higher plasma epinephrine and interstitial norepinephrine concentrations. For the ovalbumin group, there was skeletal muscle anaerobic metabolism (lactate concentration increased from 0.446 +/- 0.105 to 1.741 +/- 0.459 mm; P < 0.05) and substrate depletion (pyruvate concentration decreased from 0.034 +/- 0.01 mm to 0.006 +/- 0.002 mm; P < 0.05) leading to increased interstitial lactate/pyruvate ratios (from 17 +/- 6 to 311 +/- 115; P < 0.05). This profile suggests decreased skeletal muscle blood flow and oxygen delivery. Persistent energy consumption results in decreased Ptio2 and substrate depletion through anaerobic glycolysis leading to complete failure of cellular energy production. This could explain rapid organ dysfunction and resuscitation difficulties.

Interstitial ATP and Norepinephrine Concentrations in Active Muscle

Sympathetic nervous system activity increases with exercise in normal subjects. Heightened peripheral sympathetic nervous activity and the resultant increased neurovascular levels of norepinephrine (NE) evoke vasoconstriction and serve to maintain blood pressure and perfusion to vital organs. Previous work demonstrated that the interstitial ATP concentrations ([ATP]i) rise in contracting skeletal muscle, and it is known that sympathetic nerves have purinergic P2X receptors. Thus, in this report we tested the hypothesis that elevated ATP would stimulate these receptors and increase interstitial NE concentrations ([NE]i). Muscle interstitial samples were collected from microdialysis probes inserted in the skeletal muscle of rats, and dialysate concentrations of ATP and NE were determined by the high-performance liquid chromatography method. Stretch (0.5 kg of tension) increased [ATP]i by 68% (P<0.05) and [NE]i by 45% (P<0.05) in active muscle. The rise in NEi was linearly linked to the elevated ATPi (r=0.878, P<0.001). [NE]i was also elevated by 76% (P<0.05) after ATP (3 micromol/L) was injected into the arterial blood supply of the hindlimb muscles. The [NE]i response to muscle stretch was blunted after the P2X receptor antagonist pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) was given. Finally, this response was potentiated by the nucleotidase inhibitor 6-N,N-diethyl-beta-gamma-dibromomethylene-D-adenosine-5'-triphosphate (ARL67156). ATPi released by skeletal muscle during stretch stimulates P2X receptors on the sympathetic nerves and increases the concentration of NEi in the muscle interstitium.

Differential pre- and postsynaptic effects of desipramine on cardiac sympathetic nerve terminals in RHF.

Right heart failure (RHF) is characterized by chamber-specific reductions of myocardial norepinephrine (NE) reuptake, beta-receptor density, and profiles of cardiac sympathetic nerve ending neurotransmitters. To study the functional linkage between NE uptake and the pre- and postsynaptic changes, we administered desipramine (225 mg/day), a NE uptake inhibitor, to dogs with RHF produced by tricuspid avulsion and progressive pulmonary constriction or sham-operated dogs for 6 wk. Animals receiving no desipramine were studied as controls. We measured myocardial NE uptake activity using [(3)H]NE, beta-receptor density by [(125)I]iodocyanopindolol, inotropic responses to dobutamine, and noradrenergic terminal neurotransmitter profiles by glyoxylic acid-induced histofluorescence for catecholamines, and immunocytochemical staining for tyrosine hydroxylase and neuropeptide Y. Desipramine decreased myocardial NE uptake activity and had no effect on the resting hemodynamics in both RHF and sham animals but decreased myocardial beta-adrenoceptor density and beta-adrenergic inotropic responses in both ventricles of the RHF animals. However, desipramine treatment prevented the reduction of sympathetic neurotransmitter profiles in the failing heart. Our results indicate that NE uptake inhibition facilitates the reduction of myocardial beta-adrenoceptor density and beta-adrenergic subsensitivity in RHF, probably by increasing interstitial NE concentrations, but protects the cardiac noradrenergic nerve endings from damage, probably via blockade of NE-derived neurotoxic metabolites into the nerve endings.

Interaction between beta-adrenergic receptor stimulation and nitric oxide release on tissue perfusion and metabolism.

Nitric oxide (NO) may be an important modulator of sympathetic tone. We used im and sc microdialysis in humans to characterize the interaction of NO synthase inhibition and adrenoreceptor stimulation on tissue perfusion, metabolism, and norepinephrine release. Microdialysis probes were perfused with L- or D-nitro-L-arginine-methyl-ester (100 micromol/L) followed by incremental doses of isoproterenol, epinephrine, or nitroprusside. Blood flow was estimated based on the ethanol dilution technique. In muscle, the increase in blood flow with isoproterenol was abolished by L-NAME. The ethanol ratio was 0.03 +/- 0.011 with D-NAME and 0.075 +/- 0.014 with L-NAME during isoproterenol treatment (1 micromol/L). The effect was less pronounced in adipose tissue. The vasodilatory effect of nitroprusside was similar with D- and L-NAME. L-NAME augmented isoproterenol- and epinephrine-induced glycerol release. Dialysate glycerol during 1 micromol/L isoproterenol was 47 +/- 6.7 micromol/L with D-NAME and 72 +/- 15 micromol/L with L-NAME. In skeletal muscle, dialysate norepinephrine during 1 micromol/L isoproterenol treatment was 0.73 +/- 0.17 and 1.3 +/- 0.15 nmol/L with D- and L-NAME, respectively. We conclude that NO synthase inhibition attenuates beta(2)-adrenoreceptor-mediated vasodilation and enhances beta-adrenoreceptor-mediated lipolysis. These effects are in part mediated through an increase in interstitial norepinephrine concentrations. The data are consistent with the idea that in humans, NO is important in modulating and ameliorating sympathetic effects in peripheral tissues.

Quantitative analysis of feedforward sympathetic coronary vasodilation in exercising dogs.

Recent experiments demonstrate that feedforward sympathetic beta-adrenoceptor coronary vasodilation occurs during exercise. The present study quantitatively examined the contributions of epinephrine and norepinephrine to exercise coronary hyperemia and tested the hypothesis that circulating epinephrine causes feedforward beta-receptor-mediated coronary dilation. Dogs (n = 10) were chronically instrumented with a circumflex coronary artery flow transducer and catheters in the aorta and coronary sinus. During strenuous treadmill exercise, myocardial oxygen consumption increased by approximately 3.9-fold, coronary blood flow increased by approximately 3.6-fold, and arterial plasma epinephrine concentration increased by approximately 2.4-fold over resting levels. At arterial concentrations matching those during strenuous exercise, epinephrine infused at rest (n = 6) produced modest increases (18%) in flow and myocardial oxygen consumption but no evidence of direct beta-adrenoceptor-mediated coronary vasodilation. Arterial norepinephrine concentration increased by approximately 5. 4-fold during exercise, and coronary venous norepinephrine was always higher than arterial, indicating norepinephrine release from cardiac sympathetic nerves. With the use of a mathematical model of cardiac capillary norepinephrine transport, these norepinephrine concentrations predict an average interstitial norepinephrine concentration of approximately 12 nM during strenuous exercise. Published dose-response data indicate that this norepinephrine concentration increases isolated coronary arteriolar conductance by approximately 67%, which can account for approximately 25% of the increase in flow observed during exercise. It is concluded that a significant portion of coronary exercise hyperemia ( approximately 25%) can be accounted for by direct feedforward beta-adrenoceptor coronary vascular effects of norepinephrine, with little effect from circulating epinephrine.

Impaired Norepinephrine Reuptake in Skeletal Muscle But not in Adipose Tissue After the Development of Hypertension in Rats: an in Vivo Microdialysis Study

P50 Presynaptic reuptake of norepinephrine (NE) represents the main mechanism to end its effects, once released from sympathetic nerves. This study evaluated the effect of locally administered neuronal uptake inhibitor (desipramine, 10 pmol/min through microdialysis probes) on interstitial NE concentration in the skeletal muscle and subcutaneous adipose tissue from awake and freely moving Wistar-Kyoto (WKY)and spontaneously hypertensive rats (SHR) before and after the development of arterial hypertension. Systolic arterial pressure and plasma NE levels in SHR were similar to WKY at 5 weeks of age, but significantly increased at the age of 16 weeks. Basal interstitial NE was increased in both tissues of young and old SHR as compared to age-matched WKY. Desipramine perfusion induced a higher rise of interstitial NE dialysate from skeletal muscle and adipose tissue of SHR than WKY with a difference in the skeletal muscle between young and old SHR (see graphic). These results indicates an increased interstitial NE turnover in pre-hypertensive SHR, not shown by plasma NE level. Once hypertension is established, NE turnover is always increased in SHR as compared to WKY in both tissues. In addition in the skeletal muscle, where sympathetic efferences are baroreflex-dependent, old SHRs show a reduced interstitial NE reuptake contributing to a higher availability of interstitial NE for postsynaptic vascular effects.

Norepinephrine Concentrations in the Epicardial Transudate Reflect Early Changes in Adrenergic Activity in the Isolated Perfused Heart

M. Lorbar, K. Skalova, A. Nabi, E. S. Chung, R. A. Fenton, J. G. Dobson, Jr and T. E. Meyer. Norepinephrine Concentrations in the Epicardial Transudate Reflect Early Changes in Adrenergic Activity in the Isolated Perfused Heart. Journal of Molecular and Cellular Cardiology (2000) 32, 1695–1701. The aim of this study was to establish whether epicardial transudates could be used to uncover small, but physiologically important changes in interstitial NE concentrations under normal and pathological conditions. Norepinephrine (NE) concentrations measured in epicardial transudate fluid were compared to NE levels in the coronary effluent in normal and pressure overload hypertrophied (POH) rat hearts. Hearts were isolated together with the stellate ganglion and perfused in the inverted position. Epicardial surface transudates, representative fluid of the interstitial myocardial compartment, and coronary effluents were collected for determination of NE levels in the presence and absence of stellate ganglion stimulation. The same protocol was repeated in the presence and absence of nisoxetine, a NE uptake blocker. NE concentrations in epicardial transudates were 16- and 19-fold higher than in the coronary effluent in both sham and POH groups, respectively. NE concentrations in the transudates but not in the coronary effluents were significantly higher (1.6-fold) in hearts with POH when compared to normal hearts. Likewise, nisoxetine (10−5m) increased (1.3-fold) NE concentrations in the transudates but not in the effluents of sham animals. As expected, stellate ganglion stimulation increased NE concentrations in both transudates and effluents in sham and POH hearts. In conclusion, determination of NE concentrations in epicardial transudates represents a simple, rapid and sensitive method to detect increases in adrenergic activity in normal and abnormal hearts.

Direct interaction between the sympathetic and renin–angiotensin system in myocardial tissue: a microdialysis study in anaesthetised rats

It has been suggested that local activation of the renin–angiotensin system is involved in early stages of myocardial pathophysiology. To date, there is increasing evidence for interactions between the renin–angiotensin system and the sympathetic nervous system; consequently, local sympathetic activation may also be involved in this. Microdialysis has great potential in the direct investigation of neurohormonal interactions. Therefore, the present study employs microdialysis to study the local effects of exogenous angiotensin II on the interstitial norepinephrine concentration of the normally innervated left ventricle of the anaesthetised rat. The present study investigates the effect of increasing dosages of exogenous angiotensin II on local interstitial norepinephrine. Furthermore, a single dose of losartan was infused on top of the highest dose of angiotensin II, in order to study possible involvement of angiotensin II type 1 (AT 1) receptors. Both infusion and sampling were carried out locally, via the microdialysis probes. Concomitantly, circulating norepinephrine levels, heart rate and respiratory rate were monitored to evaluate physiologic stability of the preparation throughout the experiment. Time controls consisted of rats that were perfused with only a Ringer's solution. Angiotensin II induced a dose dependent increase in norepinephrine that was significantly reduced by losartan. Norepinephrine levels in both plasma (infusion experiment and time controls) and the left ventricular wall (time controls) remained stable throughout the experiment, just as heart rate and respiratory rate did. This study for the first time employs microdialysis to demonstrate direct interaction between the sympathetic nervous system and the renin–angiotensin system in the rat left ventricle. The data strongly suggest that AT 1 receptors are involved in this interaction, since selective AT 1 receptor blockade with losartan significantly reduced the angiotensin II induced norepinephrine concentration.

Myocardial β-adrenoceptor down-regulation by norepinephrine is linked to reduced norepinephrine uptake activity

Chronic administration of norepinephrine for 8 weeks has been shown to reduce neuronal norepinephrine uptake activity and increase interstitial norepinephrine concentration in the heart. To determine whether the changes could lead to myocardial β-adrenoceptor down-regulation or β-adrenergic subsensitivity, we measured left ventricular contractile responses to dobutamine, myocardial β-adrenoceptor density, β subtype distribution, competitive inhibition agonist binding, and adenylyl cyclase activity activation by isoproterenol, 5′-guanylylimidodiphosphate, and forskolin in dogs after a norepinephrine or saline infusion for 8 weeks. We found that norepinephrine infusion reduced myocardial β-adrenoceptor density, β 1-adrenoceptor subtype density, and high-affinity site for isoproterenol. Left ventricular contractile responses to dobutamine were reduced in the norepinephrine-infused animals. In addition, norepinephrine infusion decreased the basal adenylyl cyclase activity and the adenylyl cyclase responses to isoproterenol, 5′-guanylylimidodiphosphate, and forskolin. The findings indicate that a decrease in cardiac norepinephrine uptake predisposes the heart to norepinephrine-induced myocardial β-adrenoceptor down-regulation, and that norepinephrine, when present in a sufficient amount over a long period as it is in chronic heart failure, can reduce myocardial β-adrenergic responsiveness by both homologous and heterologous desensitization.

Effects of locally administered desipramine on myocardial interstitial norepinephrine levels

We investigated whether locally administered neuronal uptake blockade with desipramine (DMI) modified basal myocardial interstitial norepinephrine (NE) levels and stimulation-induced NE responses. Using the dialysis technique, dialysates were sampled from the midwall of the left ventricle in anesthetized cats. Dialysate NE concentrations, as an index of myocardial interstitial NE level, were measured by high performance liquid chromatography. Through the dialysis probe, local administration of DMI (10 −4 mol/l) increased the dialysate NE level from 24.6±12.2 to 127.8±37.4 and from 11.9±5.4 to 64.9±28.7 pg/ml regardless of whether stellate ganglia were intact or transected. The arterial pressure and heart rate responses to electrical stimulation of the stellate ganglia were not altered by DMI. Control nerve stimulation increased dialysate NE concentrations to 214.5±100.0 and 57.5±22.0 pg/ml at 5 Hz and 2 Hz, respectively. During local administration of DMI, nerve stimulation-induced NE response increased to 997.5±202.0 at 5 Hz and to 436.0±68.5 pg/ml at 2 Hz. We conclude that local administration of DMI caused marked rises in basal myocardial interstitial NE concentrations and responses to cardiac sympathetic activation.

Elevated myocardial interstitial norepinephrine concentration contributes to the regulation of Na +,K +-ATPase in heart failure

Myocardial Na +,K −-ATPase is reduced in congestive heart failure. To study the regulation of Na +,K +-ATPase in congestive heart failure, we performed Western and Northern blot analyses of ventricular myocardium of dogs with pacing-induced congestive heart failure and chronic norepinephrine infusion, using isoform-specific antibodies and cDNA probes. Congestive heart failure and norepinephrine infusion caused similar increases in myocardial interstitial norepinephrine concentration and reductions of myocardial Na +,K +-ATPase α 3-subunit protein, but differed in their effects on myocardial Na +,K +-ATPase α 3-subunit gene expression. Chronic norepinephrine infusion produced no changes in the steady-state mRNA level for the α 3-subunit of Na +,K +-ATPase, suggesting that the changes in Na +,K +-ATPase protein were induced via a post-transcriptional mechanism. In contrast, down-regulation of the Na +,K +-ATPase α 3-subunit in the failing heart was accompanied by a decreased α 3-subunit mRNA level, indicating the presence of a transcriptional event. The α 1-subunit protein content and mRNA level were not affected by either norepinephrine infusion or rapid ventricular pacing. We conclude that, while elevated myocardial interstitial norepinephrine levels may contribute substantially to the down-regulation of the Na +,K +-ATPase α 3-subunit in the failing myocardium, additional regulatory factors are responsible for the decreased myocardial α 3-subunit mRNA expression in congestive heart failure.

Beta-adrenoceptor downregulation in pacing-induced heart failure is associated with increased interstitial NE content.

We used the rapid ventricular pacing model to examine myocardial norepinephrine (NE) uptake kinetics in congestive heart failure. Dogs subjected to pacing at 225 beats/min for 8 wk developed heart failure as evidenced by elevated left atrial pressure, depressed first derivative of left ventricular pressure with respect to time, and depressed cardiac output compared with dogs paced at 100 beats/min for 8 wk. Fast-paced dogs also exhibited an elevated plasma NE and reduced myocardial NE content. Myocardial NE uptake kinetics and interstitial NE concentration were measured in vivo using a triple-isotope intracoronary tracer technique. The rate constant of neuronal uptake of NE was significantly depressed in the fast-paced animals (0.224 +/- 0.027 vs. 0.725 +/- 0.097 s-1, P < 0.001), while the interstitial NE concentration was significantly increased in the heart (1.12 +/- 0.15 vs. 0.17 +/- 0.07 ng/ml, P < 0.001). Myocardial beta-adrenoceptor density was significantly reduced in the fast-paced animals (49 +/- 7 vs. 86 +/- 6 fmol/mg, P < 0.001), and there was a significant inverse correlation between beta-adrenoceptor density and interstitial NE concentration. Thus we conclude that excess myocardial interstitial NE content contributes to the abnormalities in the beta-adrenoceptor system.