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.

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.

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.

Regulation of metaraminol efflux from rat heart and salivary gland.

1. The turnover rate of noradrenaline (NA) in heart and submaxillary salivary gland was studied in rats exposed to 4 degrees C or maintained at room temperature (22 degrees C). Cold exposure increased the turnover of the NA store in heart but not in salivary gland.2. In another series of experiments the decline of metaraminol (M) from heart and submaxillary salivary gland was studied in rats exposed to 4 degrees C or maintained at room temperature. Cold exposure accelerated the efflux of M from heart, but not from salivary gland. It is concluded that the accelerated decline of M from heart is the consequence of selective activation of the sympathetic nerves that innervate the heart.3. The turnover of NA was studied in rat heart after the administration of M (100 mug/kg intravenously) or its precursor alpha-methyl-meta-tyrosine (200 mg/kg intraperitoneally). Turnover remained essentially normal after these drugs.4. The administration of desipramine (DMI, 20 mg/kg intraperitoneally) 1 hr after M (100 mug/kg intravenously) induced a rapid sustained efflux of M from heart and salivary gland. The results of this study suggest that the slow decline of M from heart is the result of the great affinity of the amine retrieval mechanism in sympathetic nerve endings for M. DMI inhibits the retrieval mechanism, thus accelerating the efflux of M.

Differences in amine storage in rat heart and brain.

1 The characteristics of storage of amphetamine-releasable amine in rat heart and brain were studied in vitro with labelled and unlabelled metaraminol ([(3)H]-MA and MA).2 In one series of experiments, heart and brain slices were incubated with [(3)H]-MA prior or subsequent to incubation with the same concentration of MA.3 When brain slices, thus treated, were subjected successively to field stimulation and to amphetamine, it was found that the ratio of [(3)H]-MA release by field stimulation to release by amphetamine was dependent upon the order in which brain tissue was exposed to [(3)H]-MA and MA.4 With heart slices, on the other hand, the field stimulation/amphetamine ratio of [(3)H]-MA release remained the same whether the tissue was exposed to [(3)H]-MA before or after MA.5 The (+)-isomer of amphetamine released three to four times more [(3)H]-MA from brain slices than did the (-)-isomer, while the isomers were equipotent with regard to [(3)H]-MA release from heart slices.6 Amphetamine-induced [(3)H]-MA release from heart slices was unaffected by the presence of alpha-methyl-p-tyrosine, while release from brain slices was inhibited.7 On the basis of the foregoing results it appears that amine storage differs in brain and heart, with brain exhibiting more than one functional amine pool.

The effect of narcotic analgesics on the uptake of 5-hydroxytryptamine and (-)-metaraminol by blood platelets.

1. The effects of narcotic analgesic and related drugs were studied on the uptake of 5-hydroxytryptamine (5-HT) and (-)-metaraminol by blood platelets.2. The most potent drug in inhibiting the uptake of 5-HT (10 muM) by human platelets was methadone, followed by pentazocine>piminodine approximately pethidine approximately anileridine approximately cyclazocine approximately thebaine > dextropropoxyphene. Alphaprodine, papaverine, apomorphine, nalorphine, codeine, and morphine were almost without effect. Methadone was slightly less active than desipramine, and had 10% of the activity of imipramine under similar conditions. Naloxone did not antagonize the effect of methadone on 5-HT uptake.3. The most potent inhibitor of metaraminol (3 muM) uptake by human platelets was piminodine, followed by pentazocine>/=anileridine>cyclazocine=methadone > dextropropoxyphene approximately thebaine >/= papaverine approximately alphaprodine >pethidine>morphine. The activity of morphine was 1% of that of piminodine. Piminodine was more potent than desipramine and protriptyline under similar conditions. The order of potency of drugs studied in inhibiting the uptake of metaraminol by rabbit platelets was similar to that obtained with human platelets.4. The effects of the analgesics studied on inhibiting uptake of monoamines did not correlate with their pain-relieving properties.

Effects of Na+ and K+ on the uptake of metaraminol by rabbit ventricular slices.

1. The ionic dependence of [(3)H]-metaraminol transport by rabbit ventricular slices was studied.2. Transport was Na(+) dependent. Choline, Li(+), K(+), Rb(+) or Cs(+) could not be substituted for Na(+).3. Transport was K(+) dependent. Rb(+) and Cs(+), but not Li, could be substituted for K(+), their relative potencies being K(+) = or >Rb(+)>Cs(+)>>Li(+). Higher concentrations of K(+), Rb(+), Cs(+) and Li inhibited [(3)H]-metaraminol transport.4. The inhibitory effects on transport of either a marked reduction in the concentration of Na(+) or of omission of K(+) were rapidly reversible on exposure of slices to Krebs solution.5. The inhibitory effect of ouabain on [(3)H]-metaraminol transport was markedly time dependent, being significantly increased by preincubation with ouabain. The inhibitory action of ouabain was not affected, however, by the Na(+) concentration present during preincubation.6. An inwardly directed Na(+) gradient did not increase [(3)H]-metaraminol transport in slices in which Na(+) pumping had been prevented by 10(-5)M ouabain, 4 degrees C, omission of K(+) or by metabolic inhibition.7. These findings provide additional evidence that the Na(+) and K(+) activated adenosine triphosphatase may participate in the transport of metaraminol and related amines. In the absence of Na(+) pumping, induced Na(+) gradients were unable to produce transport of amine as would be predicted by the co-transport model proposed by Bogdanski & Brodie (1969).

Effects of mianserin, a new antidepressant, on the in vitro and in vivo uptake of monoamines.

1 The effects of mianserin and of selected tricyclic antidepressants were compared in a number of monoamine uptake models. 2 The ability of mianserin to block the noradrenergic neurone membrane amine pump of rabbit brain stem slices was comparable to that of imipramine and amitriptyline and less than that of desipramine and nortriptyline. Both mianserin and desipramine were competitive inhibitors of noradrenaline uptake in vitro. The effect of mianserin on noradrenaline uptake in vivo was studied both peripherally and centrally. The ability of 6-hydroxydopamine to lower rat heart noradrenaline levels was found to be very sensitive to inhibition by tricyclic antidepressants. Mianserin was active in this model. However, its ability to block the 6-hydroxydopamine-induced fall in rat heart noradrenaline concentration was appreciably less than that of the tricyclics studied. 3 Mianserin, like tricyclic antidepressants, was essentially devoid of effect on dopamine uptake both in vitro and in vivo. 4 The ability of mianserin to inhibit [3H]-5-hydroxytryptamine uptake by rat hypothalamic synaptosomes was appreciably less than that of the tricyclic antidepressants studied. Mianserin was essentially devoid of effect on rat brain 5-hydroxytryptamine uptake in vivo. 5 It is concluded that in certain situations large doses of mianserin may block noradrenaline uptake in vivo. However, in no way does mianserin rival tricyclic antidepressants in blocking monoamine uptake in vivo. The clinical efficacy of mianserin cannot be attributed to inhibition of monoamine uptake.

On the mechanism of action of clozapine on the adrenergic neurone.

1 The antipsychotic drug, clozapine, lowered noradrenaline and metaraminol (MA) concentrations in the rat heart. This action was blocked by the presence of a ganglionic blocking drug. 2 Other alpha-adrenoceptor blocking drugs (phenoxybenzamine, phentolamine) did not significantly lower heart amine concentrations. An inhibitor of neuronal amine uptake (desipramine) caused only a slight lowering. The combination of phentolamine and desipramine showed considerable activity, and this action was blocked by ganglionic blockade. 3 Clozapine had little or no action in blocking amine uptake, yet greatly potentiated amine release caused by the phentolamine-desipramine combination. 4 Other antipsychotic drugs (haloperidol, chlorpromazine, thioridazine) or other agents (propranolol, atropine) did not share this action of clozapine. 5 Ganglionic blockade markedly delayed amine release induced by reserpine administration. 6 It is suggested that clozapine may have an incomplete reserpine-like effect specifically on the adrenergic neurone, facilitating impulse-induced amine release.

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.

Distribution of metaraminol and its relation to norepinephrine.

A modified fluorometric procedure of high specificity and sensitivity id described for the analysis of metaraminol (MET) in biological material. With this method the distribution of MET was studied in the rat. Except for brain, peripherally administered MET was well distributed. Organs with a large population of norepinephrine (NE) storage vesicles such as the heart, spleen and adrenal, took up relatively large amounts of MET and retained it longer than those tissues with few vesicles such as the liver, lung and kidney. MET had a longer blood half-life than described for NE in other species. In part, this can be attributed to the lack of metabolism of MET as well as our finding that MET, unlike NE, was concentrated in the cellular fraction of blood. Under the conditions of these experiments there was no close correlation on a concentration or time basis with respect to the appearance and disappearance of MET and NE in various organs. This was particularly evident in the adrenal gland where MET resembled reserpine in that one unit of drug displaced many units of catecholamines. In this case, however, we did not rule out a possible contributing effect via the CNS.

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.

3H-metaraminol releasing action of mescaline from rat hypothalamus in vitro.

The amine releasing action of mescaline was investigated in rat isolated hypothalamus labeled with 3H-metaraminol. Mescaline had no effect on the uptake of 3H-metaraminol but produced its release in a concentration-related manner. 4 x 10(-4) M mescaline, which produced submaximal effects was used for subsequent experiments. 3 x 10(-5) M cocaine had no effect on the 3H-metaraminol releasing action of mescaline. Mescaline was fully effective in Ca2+-free medium while 6 x 10(-2) M KCl was ineffective. 3 x 10(-7) M tetrodotoxin or 6 x 10(-5) M lidocaine partially blocked mescaline-induced release but substantially or completely blocked 3 x 10(-2) M KCl-induced release. Prior exposure of hypothalamus to 3 x 10(-4) M tyramine reduced the releasing action of mescaline. Thus, mescaline appears to release 3H-metaraminol both by Ca2+-independent (tyramine-like) and Ca2+-dependent (lidocaine-sensitive) mechanisms. 3 x 10(-4) M tyramine and 6 x 10(-2) M KCl released 14C from control hypothalamus labelled with 14C-mescaline, but not from reserpinized hypothalamus. The amounts of 14C recovered in 14C-mescaline labeled control and reserpinized hypothalamus at the end of 50 min of efflux were similar suggesting a poor retention of 14C-mescaline by storage particles.

Effect of rubidium on responses of rabbit vas deferens to transmural stimulation and to noradrenaline.

The effects of 2 mM RbCl on adrenergic transmission in rabbit vas deferens were investigated. Rubidium, but not potassium, markedly potentiated the sustained, secondary contractile response to transmural stimulation at 2--16 Hz. This effect was not abolished by indomethacin. Significant spontaneous desensitization to (--)-noradrenaline occurred, but this was prevented by the addition of 2 mM RbCl or KCl. Addition of 2 mM RbCl, but not of 2 mM KCl, significantly increased the amount of [3H] (+/-)-metaraminol released on transmural stimulation at 5 Hz, without affecting the uptake of [3H] (+/-)-metaraminol over a 30 min period. It is suggested that rubidium mainly potentiated responses of rabbit vas deferens to transmural stimulation by increasing transmitter release, possibly as a result of prolongation of the action potential.

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.

The potentiating effects of 4-aminopyridine on adrenergic transmission in the rabbit vas deferens.

The effects of 4-aminipyridine on adrenergic transmission in rabbit vas deferens were investigated. 10(-5)-10(-3) M 4-aminopyridine markedly potentiated the sustained, secondary contractile response to transmural stimulation and significantly increased the amount of (+/-)-|3H]-metaraminol released at 5 Hz, but had little effect on responses to exogenous (-)-noradrenaline. 10(-5)-10(-4) M 4-aminopyridine did not significantly alter the accumulation or efflux of (-)-[3H]-noradrenaline in rabbit heart. It is suggested that 4-aminopyridine potentiated responses of rabbit vas deferens to transmural stimulation mainly by increasing transmitter release, possibly as a result of prolongation of the action potential. Responses to transmural stimulation were also potentiated by by 2- and 3-aminopyridine and by 3,4-diaminopyridine but not by pyridine, aniline, 4-dimethylaminopyridine, 4-aminoethylpyridine or 2-aminopyrimidine.

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.

Relationship between the uptake of 3H-(+/-)metaraminol and the density of adrenergic innervation in isolated rat tissues.

1. The uptake of 3H-(+/-)metaraminol (MA) by tissue slices or pieces was studied in vitro in several peripheral rat organs of varying density of sympathetic innervation (the tissue level of endogenous noradrenaline ranging from 1.7-99.1 nmoles/g). In each individual tissue preparation amine uptake was corrected for entry into the 14C-D-sorbitol space. 2. When the tissues were incubated with 1.4 muM MA, the rate of total amine uptake (i.e., neuronal plus extraneuronal uptake of MA) remained virtually constant for up to 7 min. Therefore, rates of uptake were determined after 2 min of incubation with substrate concentrations ranging from 0.25-12.2 muM. In all tissues the total uptake of MA was saturable. 3. Under the condition of inhibition of neuronal uptake by the presence of 100 muM cocaine, the uptake of MA (considered as extraneuronal amine uptake) was no longer saturable. When tissues were exposed to 1.4 muM MA, the relative contribution by extraneuronal (measured in the presence of cocaine) to total amine uptake (measured in the absence of cocaine) was inversely correlated with the log endogenous noradrenaline content. 4. After correction of the rates of total MA uptake for the cocaine-resistant distribution of the amine, a saturable component of uptake was obtained for each tissue. This uptake was considered to be neuronal; it was subjected to kinetic analysis. 5. Apparent Km values for the neuronal uptake of MA were very similar in all tissues and did not show any dependence on the tissue level of endogenous noradrenaline (average Km = 1.2 muM). 6. V max values for the neuronal uptake of MA were linearly correlated with the endogenous noradrenaline content of the tissues (r = 0.976; P less than 0.001), the V max for the vas deferens being excluded. When related to the content of endogenous noradrenaline, the V max obtained in the vas deferens was lower than that for all other tissues. 7. The results presented here strongly suggest that the membrane site involved in neuronal amine uptake (operationally characterized by the Km of MA) is likely to be identical in all rat tissues and that the number of uptake sites available per nerve terminal does not vary greatly between tissues.

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.

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.