Sympathetic denervation in patients with ischemic cardiomyopathy and risk on ventricular tachy-arrhythmias. A pilot study.

BACKGROUND: Patients with ischemic cardiomyopathy (ICM) are at risk for ventricular arrhythmias and are protected by an implantable cardioverter defibrillator (ICD). Visualization of cardiac sympathetic innervation may play an additional role to left ventricular ejection fraction (LVEF) in identifying those patients who will benefit from ICD therapy. The purpose of this study was to detect the role of sympathetic denervation in the genesis of ventricular arrhythmias in ICM patients. METHODS: Twenty patients with ICM and LVEF <30% were included in this pilot study. Included patients were equally stratified into two groups: no history of arrhythmias (group A) and recurrent arrhythmias (group B). All patients underwent cardiac sympathetic denervation (using carbon-11 labelled meta-hydroxy-ephedrine ([11C]-mHED)), myocardial ischemia and viability detection. Patients were followed up to one year after the imaging studies. RESULTS: Mean age was 63±7.5 years. Mean global retention of [11C]-mHED was 0.055±0,012 min-1, and was not different between the two patient groups: 0.056±0.011 min-1 vs. 0.054±0.013 min-1 for group A vs. group B, respectively. During follow-up, seven patients developed ventricular arrhythmias, and four patients died. No difference in [11C]-mHED retention was found between patients with and without ventricular arrhythmia during follow-up. However, size of denervated area was larger in patients who died during follow-up: 10±1 segments vs. 6±2 segments, P=0.002. CONCLUSIONS: Cardiac sympathetic innervation is impaired in patients with ischemic cardiomyopathy. All-cause mortality occurred in those patients with large areas of [11C]-mHED defect.

Assessment of cardiac sympathetic nerve integrity with positron emission tomography.

The autonomic nervous system plays a critical role in the regulation of cardiac function. Abnormalities of cardiac innervation have been implicated in the pathophysiology of many heart diseases, including sudden cardiac death and congestive heart failure. In an effort to provide clinicians with the ability to regionally map cardiac innervation, several radiotracers for imaging cardiac sympathetic neurons have been developed. This paper reviews the development of neuronal imaging agents and discusses their emerging role in the noninvasive assessment of cardiac sympathetic innervation.

Methanol solvent may cause increased apparent metabolic instability in in vitro assays.

Methanol was widely used as a substrate-delivering solvent in in vitro metabolic stability screenings. Its interaction with enzyme activities, particularly those of cytochrome P450s, has been investigated extensively in the past. Little was known about the interaction of methanol, whether direct or indirect, with substrates. The present study provided data for the first time to show that use of methanol may result in the formation of artifacts, which could mislead the metabolic stability information. The disappearance of LAQ094, metaraminol, and (-)-isoproterenol following 1-h incubation with human liver microsomes was 73, 85, and 66%, respectively, in the presence of 1% methanol, but was only 3, 15, and 24%, respectively, in the absence of organic solvent. The dramatically increased instability in the presence of methanol of these three compounds, each with 1,2-diamino or 1,2-amino hydroxy functional groups, was due to the formation of [M + 12] products resulting from condensation reaction of the substrates with formaldehyde. Formaldehyde was formed from methanol by human liver microsomal enzymes with an apparent K(m) of 35 mM and a V(max) of 7.9 nmol/min/mg of protein. The concentration of formaldehyde reached as high as 600 microM following a 60-min incubation. The [M + 12] products were characterized as five-membered heterocycles by liquid chromatography and tandem mass spectrometry analysis. Inclusion of 10 mM glutathione prevented the formation of such artifacts and is therefore suggested for future in vitro screenings. Our study also documented the novel finding of enzyme-dependent conversion of NADPH to nicotinamide in microsomal incubations.

Sulfation of hypertensive and hypotensive drugs by monkey brain phenol sulfotransferase.

The substrate specificity and affinity of two forms of phenol sulfotransferase (PST) from Rhesus macaque brain cortex were studied. Catecholamines, their methylated metabolites (normetanephrine, metanephrine) and methylated precursor, alpha-methylDOPA, were examined as substrates for both the cationic (PST I) and the anionic (PST II) forms of the enzyme. Sulfation of hypertensive drugs (phenylephrine, octopamine, metaraminol), hypotensive drugs (alpha-methylDOPA, minoxidil), and related agents without a free hydroxy group on the benzene ring were also studied. Results indicated that both PST forms sulfated alpha-methylDOPA and minoxidil, but only PST II transferred the sulfate group to catecholamines and most of the adrenergic agents examined.

Metabolic fate of the heart agent [18F]6-fluorometaraminol.

Studies were performed to determine whether [18F]6-fluorometaraminol (18F-FMR), a new neuronal heart radiopharmaceutical, is metabolized in vivo and if the metabolites are taken up in heart. Rat, dog, baboon and guinea pig were injected with 18F-FMR and tissue samples were analyzed for metabolites by HPLC. Liver contained the most metabolites of the tissues studied with 25-90% of the radioactivity present as metabolites at 1 h in all the species studied. While metabolites of 18F-FMR are found in blood, no significant accumulation of these metabolites is found in heart (less than or equal to 0.3%) 1 h after i.v. administration in any species except rat. These studies suggest that 18F-FMR is a suitable agent for quantitative imaging of the heart by positron emission tomography.

Heterogeneity of alpha 1 receptors associated with vascular smooth muscle: evidence from functional and ligand binding studies.

The nature of the alpha 1 receptor associated with rabbit aorta has been examined in functional and receptor binding studies. In isolated aortic rings the dose-response curve for (-)metaraminol was not parallel to that of (-)epinephrine, (-)norepinephrine or (-)phenylephrine. Following inactivation of a portion of the alpha receptors with phenoxybenzamine, the occupancy versus response relationship for metaraminol, in contrast to the other test agonists, was biphasic. These results suggest the possibility that metaraminol interacts with different functional groups on the alpha 1 receptor than the other test agonists. In microsomes prepared from frozen aorta, metaraminol bound to two classes of sites (KH = 0.41 +/- 0.12 microM, KL = 39.1 +/- 7.1 microM) labelled by the selective alpha 1 antagonist [3H] prazosin. Similar binding characteristics were observed in microsomes prepared from aorta shipped in serum on ice or aorta from animals killed in our laboratory. Norepinephrine also bound to two sites on the alpha receptor in all three preparations tested (KH = 0.06 +/- 0.01 microM, KL = 5.09 +/- 2.4 microM; estimates from frozen aorta). The Scatchard plot of [3H]prazosin binding to microsomes prepared from frozen aorta was curvilinear. Estimates of the affinities and site densities were 49.6 +/- 15.3 pM and 44.8 +/- 11.8 pmol/gm protein and 1.0 +/- 0.2 and 43.8 +/- 17.4 pmol/gm for the high and low affinity sites, respectively. These data are consistent with the idea that there are subtypes of the alpha 1 receptor.

[125I] radioiodinated metaraminol: a new platelet-specific labeling agent.

In our search for a platelet-specific labeling agent, metaraminol (MA), a low-toxic pharmaceutical for the treatment of hypotension and cardiogenic shock, attracted our attention. Its active incorporation and accumulation by platelets have been recognized. At first, the preparation of 125I radioiodinated metaraminol (125I-MA) was carried out using the chloramine-T method. Then, upon the harvest of platelets as platelet-rich plasma (PRP), their labeling with this new radiopharmaceutical was easily performed by incubation for 10 min at 37 degrees C. The cell-labeling efficiency was dependent on cell density, reaching 63.0% +/- 3.1% at 2.4 X 10(9) cells/ml. The specific incorporation of 125I-MA by an active transport system similar to that of 5-hydroxytryptamine (5-HT) as well as by passive diffusion was demonstrated. In in vitro studies, the unaltered state of 125I-MA-labeled platelets with their cellular functions fully retained was estimated. In vivo studies carried out in rabbits with induced thrombi in the femoral artery showed a rather rapid disappearance of the radioactivity from circulating blood, reaching a high thrombus-to-blood activity ratio of 19.8 +/- 4.3 within 30 min of the administration of 125I-MA-labeled autologous platelets. Thus, with the potential availability of 123I, 123I-MA-labeled platelets appear to be a promising agent for thrombus imaging using single-emission computed tomography (CT) studies.

Effects of chronic ethanol feeding on sympathetic innervated organs: temporal sequence of biochemical, functional, and trophic changes.

In noradrenergic nerves as well as in effector organs of the sympathetic nervous system, chronic ethanol feeding induced biochemical, functional, and trophic changes. The time sequence of modifications in the activities of tyrosine hydroxylase, phenylethanolamine-N-methyl transferase, monoamine oxidase, endogenous norepinephrine levels, as well as the neuronal uptake mechanism and secretory responses to norepinephrine in the submaxillary gland of the rat, confirms that the effects of chronic ethanol administration are related to an increase in sympathetic tone.

The effects of photoperiod on the responses of the hamster vas deferens to nerve stimulation.

Hamster vas deferens responds to nerve stimulation with a biphasic contraction which is completely blocked by guanethidine and tetrodotoxin. The second component of the contraction is inhibited by phentolamine, whereas the initial component is slightly potentiated by phentolamine. When hamsters are subjected to short photoperiods or castration, the vas deferens becomes supersensitive to nerve stimulation and shows spontaneous mechanical activity. The supersensitivity is not due to increased postsynaptic adrenergic supersensitivity, decreased neuronal uptake of catecholamine, decreased presynaptic feedback by prostaglandin or adrenergic agonist. The supersensitivity is still demonstrable in the presence of 4-aminopyridine, suggesting that the release of transmitter is also not different in animals kept in short photoperiods. The activity of the Na+/K+ pump is increased in vasa deferentia from hamsters kept in short photoperiods and the postsynaptic response to the ATP receptor agonist, diadenosine pentaphosphate, is also enhanced. It is suggested that the supersensitivity induced in the hamster vas deferens by short photoperiod is caused by testosterone withdrawal and is due to increased responsiveness to ATP, which could be acting as a cotransmitter in this tissue.

Degeneration activity of the pineal gland after sympathetic denervation.

After removal of the superior cervical ganglia the pineal gland of the rat showed a period of transient increase in activity of serotonin-N-acetyltransferase. This degeneration activity was preceded by a decline of the levels of endogenous noradrenaline and an impairment of the 3H-metaraminol uptake. Twenty-seven hours after sympathetic denervation the levels of the endogenous neurotransmitter were almost completely depleted, while either 24 h or 21 days after denervation total uptake of the tritiated amine decreased to only about 50% of the control values. Previous chronic sympathetic decentralization elicited in the pineal gland post-junctional supersensitivity, as judged by the enhanced degeneration activity observed in decentralized glands after sympathetic denervation. The present findings confirm that, in endocrine glands and similarly to other autonomically innervated organs, acute denervation induces degeneration activity.

Mechanisms of accumulation of tyramine, metaraminol, and isoproterenol in isolated chromaffin granules and ghosts.

The effects of the transmembrane pH gradient (delta pH) and the transmembrane potential gradient (delta psi) on the uptake of several sympathomimetic amines were investigated, using bovine adrenal chromaffin granules isolated in isotonic sucrose. As previously described [R. Johnson and A. Scarpa, J. Biol. Chem. 254 3750 (1979)], freshly isolated chromaffin granules maintain an intragranular pH of 5.5 as measured by [14C] methylamine distribution and, in the presence of ATP, generate a delta psi of 80 mV, positive inside, as measured by [14C] methylamine distribution. When tyramine, metaraminol, and isoproterenol (1-50 mM) were added to well-buffered suspensions of granules at pH 7.0, a dose-related alkalinization of the granule interior was observed. Study of the time-resolved influx of the same amines labeled radiochemically (5-21 microM) revealed that all the amines were accumulated against an apparent concentration gradient. However, while accumulation of [14C] serotonin and [3H] isoproterenol was totally inhibited by reserpine, [14C] tryramine accumulation was inhibited by only 60% and [14C[ metaraminol uptake was unaffected. The ATP-dependent generation of a delta psi produced a stimulation of amine uptake in the order: serotonin greater than isoproterenol greater than tyramine; metaraminol accumulation was not enhanced by ATP addition. The relationship between the electrochemical proton gradient (delta micro H+) and the electrochemical gradient for each of the sympathomimetic amines (delta micro A) was investigated utilizing chromaffin ghosts devoid of endogenous matrix gradients or components. All amines were accumulated in the presence of delta pH alone. In the presence of delta psi alone, [14C] serotonin, (14C] tyramine, and [3H] isoproterenol were accumulated, but no [3H] metaraminol uptake was demonstrable. The results indicate that serotonin and isoproterenol accumulated in isolated chromaffin granules and ghosts via a reserpine-sensitive mechanism, driven by the magnitude of the electrochemical proton gradient. Conversely, metaraminol permeated the membrane of the chromaffin granule through the apolar lipid phase and distributed according to the delta pH alone. Tyramine uptake proceeded by both mechanisms. The implications of the mechanism of accumulation of these potent physiologic and pharmacologic agents for their in vivo action are discussed.

Insulin-induced enhancement of uptake of noradrenaline in atrial strips.

1 Addition of insulin to the organ bath increased the force of contraction of guinea-pig left atrial strips driven electrically at 1 Hz. 2 The positive inotropic response to insulin remained unaltered in atria depleted of catecholamine or when beta-adrenoceptors were blocked by addition of propranolol to the organ bath. 3 The response os isolated atria to noradrenaline was significantly reduced in the presence of insulin. 4 Insulin affected neither the calcium accumulating abilities of the heart sarcolemma, mitochondria or microsomes, nor the cyclic adenosine 3',5'-monophosphate (cyclic-AMP)-protein kinase-induced stimulation of microsomal calcium uptake. 5 Addition of insulin to the organ bath enhanced significantly the ability of the cardiac tissue to take up [3H]-noradrenaline as well as [3H]-metaraminol. The activities of monoamine oxidase and catechol-O-methyl transferase were not changed after addition of insulin to homogenates of the heart. 6 The ability of insulin to facilitate uptake of noradrenaline would be expected to cause a decrease in the amount of the amine reaching the receptors, thus leading to a diminished response to this amine. This may explain, at least in part, insulin-induced subsensitivity to noradrenaline. 7 This view is supported by the observation that after blockade of amine uptake by destruction of nerve terminals, insulin failed to reduce the positive inotropic response to noradrenaline.

Inhibitory effect of two steroids on the accumulation of [3H]metaraminol by rat tissues in vivo.

The present experiments were designed to investigate the effect of methylprednisolone and dexamethasone on tritiated metaraminol uptake in the whole animal. Rats received i.p. injections of methylprednisolone (5, 10 and 20 mg kg-1) or dexamethasone (1, 2 and 4 mg kg-1) on each of 3 consecutive days. Under light ether anaesthesia 5 min before death the rats received radioactive metaraminol (3 microgram kg-1) via the femoral vein. Samples of various organs were removed and prepared for scintillation counting. The highest radioactivity was observed in the lungs and heart, followed by the kidney medulla, aorta, kidney cortex and submaxillary gland. Both steroids significantly inhibited uptake of metaraminol in the tissues examined. Dexamethasone seems to be more potent that methylprednisolone. It is concluded that these steroids may modify the uptake mechanism responsible for inactivation of the sympathomimetic neurotransmitter.

Tyramine antagonistic properties of AGN 1135, an irreversible inhibitor of monoamine oxidase type B.

1 The effects of the irreversible monoamine oxidase (MAO) inhibitors, AGN 1133, AGN 1135 and (-)-deprenyl, on tyramine and noradrenaline responses and uptake of [3H]-metaraminol were investigated in the isolated vas deferens of the rat. Uptake of [3H]-metaraminol and [3H]-octopamine was compared in mouse vas deferens. The modification of tyramine and noradrenaline-induced pressor responses by AGN 1133 and AGN 1135 was examined in anaesthetized rats and cats. 2 AGN 1133 (7.5 x 10(-6)M) greatly potentiated responses to tyramine in the rat isolated vas deferens. Both AGN 1135 and (-)-deprenyl inhibited tyramine responses selectively at concentrations above 10(-5)M (which caused almost complete inhibition of MAO types A and B) but tyramine responses were potentiated on washing out the inhibitors. 3 AGN 1135 (10(-4)M) and (-)-deprenyl (10-5)M) inhibited [3H]-metaraminol uptake by about 20% in rat and mouse vas deferens; neither inhibitor affected [3H]-octopamine uptake in mouse vas deferens. Desmethylimipramine (10(-6)M) inhibited amine uptake by more than 70%. 4 AGN 1133 (1.5 mg/kg) potentiated pressor responses to tyramine in rats and cats whereas AGN 1135 (1.5 mg/kg) did not. 5 AGN 1135 possesses tyramine antagonistic activity which is qualitatively similar to that of (-)-deprenyl but which cannot satisfactorily be explained by inhibition of neuronal or granula amine uptake.

Comparison of 3H-norepinephrine and 3H-metaraminol accumulation as indices of adrenergic nerve density in rabbit blood vessels.

The possibility that accumulation of 3H-metaraminol, which is not metabolized, would provide a better estimate of adrenergic nerve density than 3H-norepinephrine accumulation was examined in a series of rabbit blood vessels. Accumulation of 3H-metaraminol was linear for up to 10 min. Norepinephrine accumulation was correlated with metaraminol acumulation in the vessels studied, but the relationship was not a simple one. The findings suggest that, even with short incubation times, metabolism may significantly alter the amount of norepinephrine retained by the tissue. Nevertheless, accumulation of metaraminol was still not well-correlated with endogenous norepinephrine content. Norepinephrine contents of the basilar artery, mesenteric artery and mesenteric vein were quite similar: 2.7, 2.8 and 2.8 micrograms/g respectively. However, values for metaraminol accumulation in these vessels ranged from 8.7 to 1.78 ml cleared/g. These findings suggest that norepinephrine content of a single adrenergic varicosity may vary from tissue to tissue. The relationship between norepinephrine content, storage capacity, uptake activity and transmitter release needs to be more carefully examined. No single parameter can provide an adequate estimate of adrenergic nerve density.

Delay by bretylium of adrenergic nerve degeneration after sympathectomy of the submaxillary gland.

Administration of 24 mumol/kg of bretylium 10 h after ganglionectomy delayed the loss of endogenous norepinephrine and the impairment of neuronal uptake of 3H-metaraminol (3H-MA) that follow sympathetic denervation. This delay was evident 16 and 20 h after denervation. Twenty four h after ganglionectomy, when NE stores and uptake of 3H-MA were reduced to their lowest values in untreated rats, in bretylium-treated ones these values were approximately 40% of those in normal glands. The onset of degeneration secretion in treated rats was delayed by about 9 h. The development of prejunctional supersensitivity was also delayed. The subcellular distribution of NE in normal and 16 h denervated glands showed that denervation reduced the neurotransmitter to the same extent in the 3 fractions: coarse, supernatant and microsomal. Treatment with bretylium and pargyline prevented the loss of NE from the microsomal fraction. Previous administration of pargyline antagonized the protection of 3H-MA uptake seen in 28 h denervated rats treated with bretylium. However, this drug combination induced a greater retention of endogenous NE 24 h after denervation. Bretylium inhibited intraneuronal MAO by 40%. It is concluded that bretylium treatment can delay the degeneration of adrenergic nerve terminals separated from the cell bodies by a pharmacological effect probably not related to MAO inhibition or to its neurone blocking action.