Andreas Deussen

Quantification of Extracellular and Intracellular Adenosine Production Understanding the Transmembranous Concentration Gradient

BACKGROUND Inhibitors of adenosine membrane transport cause vasodilation and enhance the plasma adenosine concentration. However, it is unclear why the plasma adenosine concentration rises rather than falls when membrane transport is inhibited. We tested the hypothesis that the cytosolic adenosine concentration exceeds the interstitial concentration under well-oxygenated conditions. METHODS AND RESULTS In isolated, isovolumically working guinea pig hearts (n=50), the release rate of adenosine and accumulation of S-adenosylhomocysteine (after 20 minutes of 200 micromol/L homocysteine), a measure of the free cytosolic adenosine concentration, were determined in the absence and presence of specific and powerful blockers of adenosine membrane transport (nitrobenzylthioinosine 1 micromol/L), adenosine deaminase (erythro-9-hydroxy-nonyl-adenine 5 micromol/L), and adenosine kinase (iodotubericidine 10 micromol/L). Data analysis with a distributed multicompartment model revealed a total cardiac adenosine production rate of 2294 pmol. min-1. g-1, of which 8% was produced in the extracellular region. Because of a high rate of intracellular metabolism, however, 70.3% of extracellularly produced adenosine was taken up into cellular regions, an effect that was effectively eliminated by membrane transport block. The resulting approximately 2.8-fold increase of the interstitial adenosine concentration evoked near-maximal coronary dilation. CONCLUSIONS We rejected the hypothesis that the cytosolic adenosine concentration exceeds the interstitial. Rather, there is significant extracellular production, and the parenchymal cell represents a sink, not a source, for adenosine under well-oxygenated conditions.

Spatial heterogeneity of blood flow in the dog heart. I. Glucose uptake, free adenosine and oxidative/glycolytic enzyme activity.

The spatial heterogeneity of myocardial perfusion and metabolism was studied in 11 anaesthetized dogs under resting conditions. In each heart local myocardial blood flow was assessed using the tracer microsphere technique in 256 samples (mean mass: 83.1 mg) taken from the left anterior ventricular wall. In the same samples, the following biochemical parameters were determined: accumulation of [3H]-deoxyglucose (a measure of glucose uptake), free cytosolic adenosine (S-adenosylhomocysteine accumulation technique, a measure of tissue oxygenation and a possible mediator of blood flow regulation), and the specific activities of oxidative (citrate synthase, cytochrome-c-oxidase) and glycolytic (hexokinase, phosphoglycerate kinase) enzymes. Capillary density and mitochondrial and myofibril volume densities were determined by morphometry. Myocardial perfusion in each sample (average 0.77 ml min−1 g−1) varied between 0.1 and 2.5 times the mean (coefficient of variation 0.30±0.02). [3H]-deoxyglucose was deposited locally in proportion to perfusion. Samples showing low flow (< 0.2 ml min−1 g−1) did not exhibit increased levels of cytosolic adenosine. The specific activities of the oxidative and glycolytic enzymes, however, were uniformly distributed between low and high flow areas. Furthermore, capillary density and mitochondrial and myofibril densities were similar in high and low flow regions. The results show firstly that local glucose metabolism in the heart occurs in proportion to local blood flow, suggesting that high flow regions have a higher than average metabolic rate. Secondly, regions of low flow are not compromized by critical oxygenation and most likely have a lower than average oxygen demand and finally, the homogeneous distribution of oxidative and glycolytic enzymes, as well as the homogeneous myocardial ultrastructure, suggest that areas with high and low blood flow under resting conditions may increase their metabolic rate to similar levels when required.

Role of nitric oxide in local blood flow control in the anaesthetized dog

Intravenous infusion of NG-nitro-L-arginine methyl ester (L-NAME), a potent inhibitor of nitric oxide (NO) formation from L-arginine, provokes marked rises in arterial blood pressure by increasing peripheral resistance. In order to further evaluate the contribution of basal NO-formation to control of organ blood flow, regional blood flow distribution within the myocardium, kidney and brain areas was assessed using the tracermicrosphere technique in anaesthetized dogs. After L-NAME (20 mg kg−1 i.v.) kidney perfusion was homogeneously reduced by 55% in the entire cortex and the outer medulla. Within the left ventricular myocardium regional blood flow significantly decreased only in sub epicardial layers (−12%), whereas within the entire right ventricle regional blood flow was reduced by 19–24%. A close inverse relationship was found between all changes in regional myocardial blood flows observed after L-NAME and the respective control values. No significant changes in regional blood flow in different areas of the brain were detectable after L-NAME. It is concluded that the contribution of basal NO formation varies greatly between different organs and exhibits significant regional differences within the heart. It is possible that local metabolic mechanisms may compensate functionally for the inhibition of NO synthesis.

Glutamate degradation in the ischemic dog heart: contribution to anaerobic energy production.

The present study investigated the conversion of amino acids to succinate and the contribution of this pathway to anaerobic energy production during regional ischemia in the dog heart in situ. The relation between regional myocardial blood flow, estimated by the tracer microsphere technique, and myocardial contents of metabolites (glutamate, alanine, succinate, lactate) as well as their local arterio-venous differences (A-V) were determined. During 30 min of coronary artery occlusion, myocardial glutamate decreased from 2.3 mumol/g wet wt in control tissue to 1.2 mumol/g wet wt in severely ischemic areas, while aspartate was unaffected. Myocardial alanine increased in a 1: 1 stoichiometry compared to glutamate, and succinate accumulated. During control perfusion (118 mmHg), A-V of lactate, succinate and glutamate were +470, -0.7 and -3.9 nmol/ml, respectively. Stepwise reduction of perfusion pressure led to the release of lactate and succinate from the underperfused area; extraction of glutamate occurred at the lowest perfusion pressure investigated (34 mmHg; A-V: -500, -10.4 and +4.2 nmol/ml, respectively). The data indicate that during regional ischemia in vivo, succinate is synthetized exclusively from glutamate via 2-oxo-glutarate, following transamination with glycolytic pyruvate yielding alanine, while the contribution of aspartate is negligible. Using tissue levels of glutamate and lactate together with the local arterio-venous concentration differences of these compounds, it can be estimated that degradation of glutamate delivers 20% of the ATP generated by substrate level phosphorylation reactions. Thus energy production by the glutamate degradation pathway is significant in vivo under conditions of flow deprivation.

Coronary Reserve of High- and Low-Flow Regions in the Dog Heart Left Ventricle

BACKGROUND Left ventricular myocardial blood flow is spatially heterogeneous. The hypothesis we tested was whether myocardial areas with a steady-state flow <0.5 times mean flow are underperfused and areas with flow > 1.5 times mean flow are overperfused. METHODS AND RESULTS In anesthetized beagle dogs (n=10), the relationship between local blood flow versus S-adenosylhomocysteine (SAH) concentration, a measure of the free intracellular adenosine concentration, and lactate, a measure of the myocardial NADH/NAD+ ratio, were determined under control conditions and after coronary constriction. Control local myocardial blood flow was 0.99+/-0.46 mL x min(-1) x g(-1), with a coefficient of variation of 0.36+/-0.12 (n=256 per heart; sample wet mass, 125+/-30 mg). Tissue concentrations of SAH (3.4+/-2.5 nmol/g) and lactate (1.88+/-0.80 micromol/g) were not elevated in low-flow samples. However, after coronary artery constriction, poststenotic blood flow decreased from 1.00+/-0.27 to 0.49+/-0.22 mL x min(-1) x g(-1) (P<0.04), with significant correlation between local SAH and flow (r=-0.59) and lactate and flow (r = -0.50). Although nearly all samples from control high-flow regions showed increased SAH concentrations if relative flow after stenosis was <1.0, control low-flow samples frequently displayed low SAH concentrations. The percent reduction in flow determined the changes in the local SAH and lactate concentration, independent of the local control blood flow. CONCLUSIONS When the coronary inflow is unrestricted, the oxygen supply to control low-flow regions meets metabolic demand. Flow to control high-flow regions reflects a higher local demand rather than overperfusion. Thus, blood flow heterogeneity most likely reflects differences in aerobic metabolism.

SPATIAL HETEROGENEITY OF BLOOD FLOW IN THE DOG HEART. II. TEMPORAL STABILITY IN RESPONSE TO ADRENERGIC STIMULATION

The effects of adrenergic stimulation on local myocardial blood flow in the left ventricle were studied in 13 anaesthetized Beagle dogs using the tracer microsphere technique. Adrenergic stimulation was induced by intravenous infusion of orciprenaline (1–2 μg kg−1 min−1) over 15 min or by electrical stimulation of the left ansa subclavia (10 Hz, 1 ms, 4–8 V) over 5 min. Local myocardial blood flow was analysed in 256 samples with an average (±SD) mass of 318±49 mg from the left ventricular myocardium using a standardized dissection procedure. Orciprenaline increased the average myocardial blood flow from 0.85±0.18 to 1.73±0.27 ml min−1 g−1, while oxygen consumption and the pressure-rate product increased by 129 and 119% respectively. The coefficients of variation of local myocardial blood flow, a measure of spatial blood flow heterogeneity, were 0.21 and 0.18 under control and orciprenaline respectively. Except for a slight transmural gradient (endomyocardium/epimyocardium flow ratio 1.19) myocardial blood flow did not exhibit significant spatial gradients. Stimulation with orciprenaline increased the average blood flow in all regions of the left ventricle by comparable extents. However, local blood flow during orciprenaline was significantly lower in samples from regions which had a lower blood flow under resting control conditions. A significant positive relationship was obtained between local myocardial blood flow under resting conditions and orciprenaline (r=0.45±0.18). Moreover, after recovery from orciprenaline stimulation (i.e. 40–112 min after the end of orciprenaline infusion) local myocardial blood flow exhibited a high degree of correlation with local flow before orciprenaline (r=0.71±0.08). Comparable results were obtained with electrical stimulation of the left ansa subclavia. For the comparison stimulation vs. control, the correlation coefficient of local blood flow was 0.52±0.04 and for recovery vs. control 0.77±0.06. From these results it is concluded firstly that local myocardial blood flow under resting conditions is an important determinant of local flow during adrenergic stimulation. Secondly, the anatomical region does not have any predictive value for the blood flow change during adrenergic stimulation and finally, the close relationship between local blood flow before and after cardiac stimulation indicates that the spatial blood flow heterogeneity is temporally stable over hours.

Cardiac adenosine production is linked to myocardial pO2.

Experiments were performed on isolated perfused guinea-pig hearts (n = 45) to further evaluate the stimulus that triggers cardiac adenosine production. Stimulation of hearts with isoproterenol (4 nM, 20 min) enhanced left ventricular dP/dtmax, heart rate and myocardial oxygen consumption within 1 min to new steady state values, whereas coronary venous adenosine concentration only transiently increased reaching its maximum between 1 and 3 min of stimulation. Rate of accumulation of S-adenosylhomocysteine (SAH), a measure of the free cytosolic adenosine concentration, was steepest immediately following onset of stimulation and then progressively declined. Similar to adenosine, changes in coronary venous pO2 were phasic and adenosine release and pO2 closely correlated. Norepinephrine (20 nM) which increased myocardial oxygen consumption to a comparable extent as isoproterenol (4 nM) further decreased coronary venous pO2 and increased coronary venous adenosine. When myocardial oxygen supply was systematically varied by changing coronary perfusion pressure from 60 to 90 and 35 cmH2O, respectively, the adenosine release during isoproterenol (2 nM) was markedly enhanced at 35 cmH2O but blunted at 90 cmH2O. Similarly SAH accumulation was greatest at 35 cmH2O and smallest at 90 cmH2O. It is concluded that changing myocardial oxygen consumption is not a sufficient cause to enhance adenosine formation. Myocardial oxygenation as reflected by changes in coronary venous pO2 closely correlates with changes in free cardiac adenosine as evidenced by two independent indices: tissues SAH and coronary venous adenosine concentration. The stimulus triggering cardiac adenosine formation is most likely the imbalance of oxygen supply and oxygen demand.

Minimal effects of nitric oxide on spatial blood flow heterogeneity of the dog heart

Abstract Eleven Beagle dogs were studied to elucidate the possible role of L-arginine-derived nitric oxide on local blood flow distribution in left and right ventricular myocardium. Local blood flow was determined in 256 samples from the left and 64 samples from the right ventricle per heart using the tracer microsphere technique (mean sample mass 319 ± 131 mg). Nitric oxide production was effectively inhibited by intravenous infusion of 20 mg/kg nitro-L-arginine methylester (L-NAME) as evidenced by a shift of the dose/response curve for the effect of intracoronary administration of bradykinin (0.004–4.0 nmol/min) on coronary blood flow. L-NAME enhanced left and right ventricular systolic pressures from 132 ± 18 to 155 ± 15 mm Hg and from 26 ± 3 to 29 ± 3 mm Hg respectively (both P = 0.043). Mean left ventricular blood flow was 1.14 ± 0.38 before and 0.99 ± 0.28 ml min–1 g–1 after L-NAME (P = 0.068), while right ventricular blood flow fell from 0.72 ± 0.28 to 0.53 ± 0.20 ml min–1 g–1 (P = 0.043). Coronary conductance of left and right ventricular myocardium fell by 31 and 43% respectively (both P = 0.043). The coefficient of variation of left ventricular blood flow was 0.26 ± 0.07 before and 0.29 ± 0.07 after L-NAME (P = 0.068), that of right ventricular blood flow was 0.27 before and after L-NAME. Skewness (0.51) and kurtosis (4.23) of left ventricular blood flow distribution were unchanged after L-NAME, while in the right ventricle skewness decreased from 0.54 to 0.09 (P = 0.043) and kurtosis (3.68) tended to decrease after L-NAME (P = 0.080). The fractal dimension (D = 1.20–1.27) and the corresponding nearest-neighbor correlation coefficient (rn = 0.37–0.53) of left and right ventricular myocardium remained unchanged after infusion of L-NAME. From these results it is concluded that firstly, local nitric oxide release does not explain the higher perfusion of physiological high flow samples and secondly, that spatial myocardial blood flow coordination is not dependent on nitric oxide.

Observation of reorientationally hindered water in biological tissue using 3riple quantum filtered17O-NMR

Water dynamics in aqueous biopolymer solutions often display a two-phase character, resembling water-water and water-protein interactions. Rotationally hindered water molecules in crowded protein environments display triple exponential magnetic relaxation out of the extreme narrowing limit. Because water-protein interactions retard the water dynamics, H2O(17) magnetization passes through a NMR multiple quantum coherence filter, allowing the visualization of reorientationally hindered water without the disturbing resonance of the bulk. In vitro experiments performed on selected biological materials (lens, vitreous body and serum albumin solutions) clearly demonstrate the potential of this technique.

Characterisation of left ventricular relaxation in the isolated guinea pig heart.

The time constant of left ventricular pressure fall, τ, has frequently been used as a measure of myocardial relaxation in the blood-perfused, ejecting heart. The aim of the present study was to characterise τ in relation to β-adrenergic activation, coronary perfusion pressure and flow as well as cardiac oxygen supply and demand in the isolated, isovolumically beating heart. Therefore, τ was analysed from digitised left ventricular pressure data in a total of 23 guinea pig hearts perfused with saline at constant pressure (60 cmH2O). The coronary venous adenosine concentration ([ADO]) served as an index of myocardial oxygenation. Isoprenaline (0.4–3.2 nmol l−1) decreased and propranolol (3–9 μmol l−1) increased τ dose-dependently (linear regression τ vs lg ([isoprenaline]),r=0.74; τ vs. lg([propranolol]),r=0.66, bothP<0.05). During graded reductions in cardiac oxygen supply from 96.1±12.6(SEM) to 44.4±4.4 μl min−1 g−1, τ was prolonged from 61.5±12.7 to 109.9±22.6 ms while left ventricular developed pressure (LVDP) decreased from 90.7±7.2 to 40.7±5.1 mmHg. In parallel, [ADO] increased from 23.7±9.1 to 58.0±19.1 pmol ml−1 (P<0.05). Increasing oxygen supply to 165.4±32.4 μl min−1 g−1 augmented LVDP to 102.7±7.3 mmHg but did not change τ or [ADO]. There was a dual response of τ to changes in cardiac oxygen supply or demand. As long as oxygen supply and demand matched, τ remained constant. However, when the oxygen supply was less than 100 μl min−1g−1, left ventricular relaxation was prolonged in parallel to the reduction in oxygen supply. In addition, a close relationship was observed between [ADO] as an indicator of myocardial oxygenation and τ (Spearman correlation,r=0.99,P<0.005). We conclude that the time constant of left ventricular pressure fall, τ, sensitively reflects myocardial relaxation in the isolated, isovolumically beating guinea pig heart. Moreover, in this model left ventricular relaxation is not influenced by alterations in coronary perfusion pressure or flow as long as cardiac oxygen demand is matched by an adequate supply. Rather, relaxation is strictly coupled to myocardial oxygenation as reflected by coronary venous adenosine concentrations.