μM Cocaine Research Articles

Effect of chronic cocaine treatment on D 2 receptors regulating the release of dopamine and acetylcholine in the nucleus accumbens and striatum

The inhibition of electrically stimulated [ 3H]DA and [ 14C]ACh release by a submaximal concentration of quinpirole was measured 1 week after pretreating rats for 9 days with cocaine (15 mg/kg IP, twice per day). Although this pretreatment significantly enhanced behavioral response to a challenge injection of cocaine when compared with rats pretreated with saline only, no significant differences were apparent in the degree of inhibition of electrically evoked [ 3H]DA or [ 14C]ACh release by quinpirole in either the nucleus accumbens or striatum. In addition, the potentiation of electrically evoked [ 3H]DA release and corresponding inhibition of [ 14C]ACh release by 10 μM cocaine, measured in striatal slices only, was not significantly different between the two treatment groups. These results suggest that the enhanced behavioral response resulting from chronic cocaine treatment (behavioral sensitization) is not caused by a subsensitivity of D 2 terminal autoreceptors or by a supersensitivity of postsynaptic D 2 receptors on cholinergic neurons.

Chronic cocaine administration decreases norepinephrine-induced phosphoinositide hydrolysis in rat aorta

The effect of chronic cocaine administration on norepinephrine stimulated hydrolysis of inositol 1,4,5-trisphosphate from the membrane phosphatidylinositol phosphate pool in isolated rat aorta was investigated. Rats received saline (controls), or 10 or 20 mg/kg cocaine once a day for 15 days. This treatment resulted in a dose-dependent reduction in norepinephrine (0.36 μM) stimulated phosphoinositide hydrolysis. The effect of acute cocaine was determined by adding 30 μM cocaine to the in vitro incubation solution. When aortas were exposed to cocaine and norepinephrine simultaneously, in vitro, inositol phosphate formation doubled. By itself, cocaine did not affect phosphoinositide hydrolysis. Contraction of aortic helical strips by norepinephrine decreased in tissues from rats chronically treated with 20 mg/kg cocaine. In vitro cocaine shifted the norepinephrine concentration/response curve to the left and increased the maximum response. Neither acute nor chronic cocaine treatment affected prazosin's apparent dissociation constant, suggesting that cocaine did not affect receptor affinity. These data suggest that chronic, but not acute cocaine administration may interfere with pharmacomechanical coupling in rat aorta.

Amphetamine actions on pre- and postpubertal rat hippocampal dentate granule neurons

Clinical evidence suggests different actions of amphetamine (AMPH) in children and adults. Using intracellular recording techniques, the actions of AMPH at 10 and 40 μM were investigated in granule neurons of hippocampal slices from pre- and postpubertal rats. AMPH (10–40 μM) caused depolarization of most postpubertal neurons, often with increased spontaneous activity, whereas most prepubertal neurons were hyperpolarized. In both age groups, AMPH caused increased neuronal excitability by reducing spike threshold, attenuating the postspike train afterhyperpolarization, reducing spike frequency adaptation, and potentiating excitatory postsynaptic potentials. Changes in cell input resistance were variable and Ca 2+ currents were unaffected. AMPH actions took 10–15 min to appear and became maximal 30–55 min after application. The effects were reversible at 10μM, but at 40 μM, prolonged washout for up to 2 h did not completely reverse these actions. The β-adrenergic blocker, propranolol, partially blocked AMPH actions. The dopamine (D 2) blocker, haloperidol, did not block AMPH actions. Mature neurons were also tested with 2.5 μM AMPH showing similar but more reversible effects as the higher concentrations. Depleting catecholamines by reserpine partly attenuated the effects of 40 μM AMPH in mature neurons. Perfusion of neurons with 10 and 20 μM cocaine did not produce effects similar to those of AMPH. It is suggested that AMPH produces its effects on granule neurons only in part through the release of norepinephrine. The involvement of other neurotransmitters and/or neuromodulators released by AMPH 48,49, or direct postsynaptic actions of AMPH are also possible.

Dopamine uptake inhibition potentiates the effects of l-DOPA on rat substantia nigra zona compacta neurons

Intracellular electrophysiological recordings (current and voltage-clamp mode) were used to asses whether the actions of l-DOPA on rat substantia nigra zona compacta neurons maintained in vitro are potentiated by drugs that block the dopamine uptake system (cocaine and nomifensine). Bath application of l-DOPA (100 μM) hyperpolarized the dopaminergic cells and inhibited the spontaneous occurring action potential discharge. Under voltage-clamp experiments, l-DOPA produced an outward current that was related to the hyperpolarization of the membrane. In the presence of 10 μM cocaine and 10 μM nomifensine, the cellular responses to l-DOPA application were augmented and prolonged. In contrast to this, amphetamine (a dopamine releasing drug), did not augment the membrane response to l-DOPA. Cocaine (10 μM), nomifensine (10 μM) and amphetamine (3 μM), slightly hyperpolarized the membrane potential and inhibited the firing activity of the cells. It is concluded that the dopamine uptake system have an important role in regulating the cellular responses (membrane hyperpolarization or outward current) to l-DOPA application in the substantia nigra zona compacta.

Isolated perfused lungs of guinea-pig, in contrast with rat, lack an uptake process for noradrenaline

The uptake and metabolism of noradrenaline were compared in isolated perfused lungs of guinea-pigs and rats. Lungs were perfused with 3H-(−)-noradrenaline either a) at a concentration of 10 nM for 20 min in experiments to measure the metabolism of the amine or b) at a concentration of 2 nM for 2 min, in the presence or absence of 10 μM cocaine and with MAO and COMT inhibited, in experiments to measure the uptake of noradrenaline. The total formation of metabolites during the 20 min perfusion period was 36.2 ± 2.3 pmol g −1 (n = 6) in guinea-pig lungs, and 526 ± 26 pmol g −1 (n = 6) in rat lungs (14.5-fold greater). In guinea-pig lungs, the rate of uptake of noradrenaline was 0.392 ± 0.044 pmol g −1 min −1 (n = 3) and was unaffected by cocaine, whereas in rat lungs it was 5.63 ± 0.03 pmol g −1 min −1 (n = 5) and was inhibited (88%) by cocaine. It is concluded from these results that the lungs of the guinea-pig lack the specific uptake process that, in rat lungs, allows removal of noradrenaline from the pulmonary circulation.

Mode of Potentiating Action of Cocaine in Morphine Analgesia

The mechanism of antinociceptive interactions among morphine, cocaine and alcohol was studied in mice, guinea pigs and rabbits. In the tail-pressure test in mice, cocaine and alcohol alone showed almost no antinociceptive effects at doses up to 8 mg/kg, s.c, and 4 g/kg, i.g., respectively. Alcohol at 2 g/kg, i.g., also did not influence the effect of morphine, while cocaine at 4 mg/kg, s.c, significantly potentiated the antinociceptive effects of not only morphine but also pentazocine. In an analysis of serum and brain concentration levels of morphine in mice, when morphine and cocaine were simultaneously administered at 2 mg/kg, s.c, and 4 mg/kg, s.c, respectively, both serum and brain levels of morphine showed neither increase nor decrease in comparison with the levels in mice administered morphine alone. In myenteric plexus-longitudinal muscle preparations of isolated guinea pig ileum, 1 μM cocaine enhanced the agonistic effects of both pentazocine and ethylketocyclazocine. Furthermore, cocaine as well as ethylketocyclazocme showed naloxone-reversible agonistic effects in isolated rabbit vas deferens. These results indicate that cocaine may potentiate the antinociceptive effects of morphine and pentazocine by acting on the κ-opioid receptors as an agonist.

Detection of dopamine overflow and diffusion with voltammetry in slices of rat brain

Voltammetric electrodes have been used to monitor extracellular dopamine in rat brain slices. The electrode tips are small enough to be immersed inside the slice. Specificity for dopamine is increased through the use of voltammetry and a cation exchange membrane at the electrode tip. Dopamine overflow is observed in the caudate nucleus following electrical stimulation (60 Hz, 1 s, 3 V) with an adjacent bipolar electrode. The amount of overflow observed is increased when the tissue is perfused with 10 μM cocaine or nomifensine, both recognized inhibitors of dopamine uptake. The ability of dopamine in the perfusion buffer to permeate the slice was monitored with two voltammetric electrodes, one in the cerebral cortex and the other in the caudate nucleus. At a high concentration (100 μM), dopamine rapidly appeared (2.7 ± 0.4 min) in the interior of the cortex, but dopamine was not observed in the caudate until a significantly later time (8.9 ± 1.0 min). To examine whether this difference is a reflection of the presence of different uptake systems in the two regions, pressure ejection was employed. In this experiment a double-barelled pipette was used to eject dopamine or DOPAC at a fixed distance (∼70 μm) from the voltammetric electrode. Ejection of small amounts of both substances could be detected in the cortex. When the ejector-detector assembly was moved to the caudate, dopamine could only be observed following pressure ejection after perfusion of the slice with 10 μM nomifensine. Detection of DOPAC was unaffected. All of these experiments indicate that uptake systems in the caudate keep dopamine concentrations very low in the extracellular fluid of the slice.

Influence of antipsychotics on presynaptic receptors modulating the release of dopamine in synaptosomes of the nucleus accumbens of rats

The release of preloaded [ 3H]dopamine (DA) from superfused synaptosomes stimulated by 30 mM K + was investigated in the nucleus accumbens of rats. Under conditions preventing the uptake of DA (presence of 40 μM cocaine) release of [ 3H]DA was inhibited by DA and apomorphine in a concentration-dependent manner (IC 50s 0.65 and 0.3 μM, respectively). The maximal inhibitory effects of DA, as well as of apomorphine, were about 50% of the controls. The DA-induced inhibition was antagonized by antipsychotics completely; the rank order of antagonistic potencies was haloperidol > clozapine > sulpiride; methiothepine was ineffective. Furthermore, the K +-stimulated release of [ 3H]DA was inhibited by serotonin in a concentration-dependent manner (IC 50 = 0.9 μM). This inhibitory effect was antagonized by methiothepine with a high efficiency, by clozapine and methysergide with moderate efficiencies; haloperidol and sulpiride were ineffective. The experimental system demonstrated appears to be suitable for characterizing the DA- and serotonin-antagonistic potencies of antipsychotics and other drugs on presynaptic autoreceptors as well as receptors modulating release of DA in the nucleus accumbens.

Identification of mechanisms involved in the modulation of release of noradrenaline in the hippocampus of the rabbit in vitro

The modulation of the electrically-evoked release of noradrenaline by various possible neurotransmitters or neuromodulators in the hippocampus was studied in the dorsal part of the hippocampus of the rabbit. Slices of this tissue were preincubated with [ 3H]noradrenaline and superfused with a medium containing 30 μM cocaine. The evoked overflow of tritium was calcium-dependent, tetrodotoxin-sensitive and subject to modulation by presynaptic α 2-autoreceptors. Drugs with affinity for β-adrenoceptors (up to 1 μM), muscarinic (up to 10 μM), nicotinic (up to 100 μM), GABA- (up to 1000 μM), glutamate- (up to 100 μM) and prostaglandin-receptors (up to 1 μM) did not show any modulatory influence on the evoked release of noradrenaline. In contrast, morphine (1 μM) and fentanyl (1 μM) significantly reduced the evoked overflow; this effect was antagonized by naloxone (10 μM), which, given alone, was ineffective. Apomorphine (1 μM) reduced the release of noradrenaline in the absence, and increased it in- the presence, of 0.1 μM haloperidol; haloperidol (0.1 μM), given alone was ineffective. From these results it is concluded that, in addition to the well-known α 2-autoreceptor mechanism, presynaptic opiate, D2- and probably D1-receptors might modulate the release of noradrenaline in the hippocampus.

Pharmacological Properties of Presynaptic β-Adrenoceptors in Guinea-Pig Pulmonary Arteries

Pharmacological properties of the facilitatory presynaptic β-adrenoceptor mechanism were studied in superfused spiral preparations of guinea-pig pulmonary arteries preloaded with 3H-norepinephrine. (-)-lsoproterenol (0.3 μM)-induced increases in total 3H efflux per pulse evoked by transmural field stimulation (1, 5, 10 and 20 Hz, 10 V, 2 msec pulse width, 100 pulses and 30 min intervals) were neither dependent on impulse-frequencies nor selective at lower frequencies. Isoproterenol increased 3H efflux at 5 Hz in a concentration-dependent manner (1 nM to 1 μM): pD2 was 7.7. Salbutamol increased 3H efflux in a similar manner to isoproterenol: pD2 was 7.4. Prenalterol at 3 μM only slightly increased 3H efflux. Tazolol (10 nM to 3 μM) produced no increases. Atenolol (3 μM) and practolol (3 μM) did not antagonize isoproterenol (0.3 μM)-induced increases in 3H efflux. Butoxamine (3 μM) and H 35/25 (3 μM) did antagonize this parameter. (-)-Epinephrine (1 nM to 0.1 μM) decreased 3H efflux at 5 Hz and concentration-dependently increased this parameter in the presence of 10 μM phentolamine. (-)-Norepinephrine (10 nM to 1 μM) concentration-dependently inhibited evoked 3H efflux and did not increase the parameter in the presence of 10 μM phentolamine, 10 μM cocaine and 10 μM normetanephrine. Thus, there exist presynaptic β2-subtype receptors on noradrenergic nerve endings innervating guinea-pig pulmonary arteries.

Functional characterization of central α-adrenoceptors by yohimbine diastereomers

Rat occipital cortex slices were preincubated with [ 3H]noradrenaline and then superfused with medium containing 30 μM cocaine. They were stimulated electrically at 3 Hz. Unlabelled noradrenaline (0.1 μM), α-methylnoradrenaline (0.01–0.1 μM), xylazine (0.1–10 μM) and guanabenz (0.01 μM) decreased, whereas yohimbine (0.01–1 μM), rauwolscine (0.01–1 μM), corynanthine (10 μM), tolazoline (0.1–10 μM) and azapetine (0.1–1 μM) increased the stimulation-evoked overflow of tritium without a change in basal outflow. Pseudoyohimbine and prazosin at up to 0.1 μM did not change the evoked overflow, and at higher concentrations enhanced the basal outflow of tritium. In vivo, yohimbine 10 mg/kg and rauwolschine 10 mg/kg markedly, and corynanthine 10 mg/kg slightly accelerated the α-methyltyrosine-induced disappearance of noradrenaline from rat whole brain. Yohimbine and rauwolscine but not corynanthine also accelerated the α-methyltyrosine-induced depletion of dopamine. The results add four compounds to the list of drugs with α-adrenoceptor affinity which inhibit (agonists) or facilitate (antagonists) action-potential-evoked release of noradrenaline in rat brain cortex. The presynaptic receptors are of the α 2-type. The receptors which control the activity of noradrenaline and dopamine neurons in vivo also appear to be α 2.

Two distinct adrenoceptor-biophases in the vasculature: one for alpha- and the other for beta-agonists.

Strips of two canine vessels with different patterns of sympathetic innervation were used: the mesenteric artery which has an adventitio-medial plexus and the saphenous vein in which nerve terminals are distributed throughout the media. The pD2 of (−)-adrenaline for α-and β-adrenoceptors was determined for both vessels (in the presence of 0.5 μM propranolol and 7 μM phentolamine, respectively; in the latter case the strips were contracted by 0.28 μM prostaglandin F2α) and found to be very similar: 6.96 and 7.01 in the saphenous vein and 6.77 and 6.91 in the mesenteric artery, respectively. The similarity of pD2 for adrenaline acting on α-and β-adrenoceptors in both preparations allowed us to compare the effect of inhibition of neuronal uptake by 12 μM cocaine with that of inhibition of COMT by 50 μM dihydroxy-2-methyl propiophenone (U-0521) on: a) the potency of adrenaline (noradrenaline was used in the saphenous vein only) acting on α-and β-adrenoceptors and b) the time required by the strips to recover 50% in oil (t 50) after contractions or relaxations caused by 0.23 μM adrenaline. In the saphenous vein cocaine increased the potency of both adrenaline and noradrenaline for α-effects more than for β-effects (2.8 times vs. no increase for adrenaline; 7.1 vs. 1.9 times for noradrenaline) and U-0521 increased the potency of both adrenaline and noradrenaline for β-effects more than for α-effects (4.1 vs. 2.6 times for adrenaline; 1.8 times vs. no increase for noradrenaline); regarding the termination of action of adrenaline, cocaine prolonged the t 50 1.6 times after contraction and did not change it after relaxation, whereas U-0521 prolonged the t 50 6.9 times after relaxation and only 1.8 times after contraction. In the mesenteric artery only sensitivity experiments were done. Cocaine increased the potency of adrenaline by a factor of 2.1 for the α-effects while there was no influence on its potency with regard to its β-effects, and U-0521 increased the potency of adrenaline for the β-effects more than for α-effects (3.9 vs. 1.8 times, respectively). These results show that there are two different biophases for sympathomimetic agonists in the vasculature: one for α-adrenoceptors which is more under the influence of the neuronal uptake and one for β-adrenoceptors which is more under the influence of COMT activity. We conclude that α-adrenoceptors are situated close to the nerve endings and β-adrenoceptors close to COMT sites. Since these results do not differ qualitatively in two vessels with different patterns of innervation, we conclude that this asymmetry in the distribution of α-and β-adrenoceptors may be due to either an uneven distribution of cells with only one type of receptors each or due to an uneven distribution of receptors on the same cell.

The neuronal and extraneuronal distribution of 3H(-)-noradrenaline in the perfused rat heart.

1. After inhibition of both COMT and MAO, rat hearts were perfused with 1 μM 3H-(−)-noradrenaline for 30 min. Cocaine-sensitive neuronal and corticosterone-sensitive extraneuronal compartments were of about equal importance for the distribution of the amine into the heart. The absence of calcium from the perfusion fluid increased the axoplasmic and decreased the extraneuronal distribution of noradrenaline. 2. Under the same experimental conditions, hearts were washed out after the initial perfusion, and the efflux curves were subjected to compartmental analysis. For the nerve endings three distribution compartments were identified (the axoplasmic compartments B and C, characterized by half times of 8 and 45 min, respectively, as well as the vesicular “bound fraction”), while there was only one important extraneuronal distribution compartment (compartment C with a half time of about 45 min). 3. Efflux from the axoplasmic compartment C was accelerated by unlabelled noradrenaline, not affected by cocaine, reduced by lowering of the temperature from 37° to 27° C. Moreover, it was greatly accelerated by the omission of sodium from the wash-out solution. 4. Efflux from the extraneuronal compartment C, on the other hand, was not affected by the presence of unlabelled noradrenaline, and impaired by lowering of the temperature as well as by corticosterone. The omission of sodium from the wash-out solution caused an acceleration of the extraneuronal efflux which was significantly smaller than that of axoplasmic efflux. 5. Other hearts were perfused with either 1 or 16.7 μM 3H-(−)-noradrenaline (plus 30 μM cocaine) for 30 min and then washed out with amine-free solution; in these experiments either MAO or COMT was inhibited. When MAO was inhibited, the efflux curves for normetanephrine indicated that the accumulation of the amine in the extraneuronal compartment C can easily exceed that concentration which saturates the intracellular COMT. When MAO was not inhibited, afflux curves for DOPEG failed to provide any evidence for saturation of extraneuronal MAO. These results are in good agreement with earlier determinations of the kinetic constants of the “extraneuronal metabolizing systems” of the rat heart (Fiebig and Trendelenburg, 1978b). 6. The results indicate that extraneuronal mechanisms play an important role in the rat heart, and also that the extraneuronal compartment C (for noradrenaline) corresponds to the compartment III (for isoprenaline described by Bonisch et al. (1974).

Effects of presynaptic modulators on Ca2+-induced noradrenaline release from central noradrenergic neurons. Noradrenaline and enkephalin inhibit release by decreasing depolarization-induced Ca2+ influx.

Slices of rat occipital cortex and hypothalamus were preincubated with 3H-noradrenaline. Tritium overflow was induced by introduction of 0.65 or 1.3 mM CaCl2 for 2–5 min after superfusion of the slices with Ca2+-free solution; Ca2+-induced overflow was promoted either by the presence of 30 mM K+ throughout superfusion or by incubation with the ionophore A 23187 (300 μM; subsequent to preincubation with 3H-noradrenaline) before onset of superfusion. The effects of drugs interfering with presynaptic α-adrenoceptors and opiate receptors on CaCl2-induced tritium overflow and on the metabolism of 3H-noradrenaline released were investigated. 1. The CaCl2-evoked tritium overflow promoted by 30 mM K+ was decreased by methionineenkephalin (1 μM). This effect was antagonized by simultaneous administration of naloxone (10 μM). When given alone, naloxone (10 μM) did not alter the CaCl2-induced overflow of tritium. 2. When cocaine (100 μM) was present in the superfusion fluid throughout the experiments, the CaCl2-evoked tritium overflow promoted by 30 mM K+ was decreased by unlabelled noradrenaline (1 μM) and increased by phentolamine (1 μM); the inhibition of CaCl2-induced tritium overflow caused by unlabelled noradrenaline was reversed by simultaneous administration of phentolamine (1 μM each). 3. The CaCl2-evoked tritium overflow promoted by ionophore A 23187 was not affected by 1 μM methionine-enkephalin or 1 μM unlabelled noradrenaline (the latter in the presence of 100 μM cocaine throughout superfusion). 4. Methionine-enkephalin, unlabelled noradrenaline and phentolamine did not significantly alter the metabolism of 3H-noradrenaline released by 1.3 mM CaCl2 under both conditions (30 mM K+ and 300 μM A 23187, respectively).

Ca2+-induced noradrenaline release from central noradrenergic neurons promoted by high K+ concentration or ionophore A23187

In slices of rat occipital cortex and hypothalamus preincubated with 3H-noradrenaline and superfused with various Ca2+-free solutions, the effect of introduction of CaCl2 on efflux of total tritium, 3H-noradrenaline and 3H-metabolites was studied. 1. When slices of both brain regions were superfused with a Ca2+-free buffer containing 30 mM K+ and 119 mM Na+, introduction of 0.65–2.6 mM CaCl2 for 2 min induced a concentration-dependent overflow of tritium. Cocaine (100 μM) doubled the overflow of tritium evoked by 1.3 mM CaCl2. When slices were superfused with physiological salt solution after 100 min of superfusion with the Ca2+-free buffer and 2 stimulation periods with 1.3 mM CaCl2, electrical field stimulation (3 Hz, 13 mA, 2 ms, 2 min duration) still elicited on overflow of tritium which contained 59% 3H-noradrenaline. The CaCl2-induced overflow was not affected by tetrodotoxin (1 μM), but was inhibited by Mg2+ ions (10 mM). 2. When cortical slices incubated with Ca2+-free solution containing 300 μM ionophore A 23187 (subsequent to preincubation with 3H-noradrenaline) were superfused with Ca2+-free solution containing 0.3 mM Mg2+ (otherwise composed like physiological salt solution), introduction of 1.3 or 2.6 mM CaCl2 for 5 min evoked a concentration-dependent overflow of tritium. The evoked overflow of 3H-noradrenaline was similar to that produced by the same Ca2+ concentrations after superfusion with K+-rich solution. The Ca2+-induced tritium overflow from slices preincubated with A 23187 was completely inhibited by 10 mM MgCl2. 3. The Ca2+-induced overflow of tritium from cortex slices superfused with 30 mM K+ or preincubated with A 23187 was mainly represented by 3H-noradrenaline; the most important degradation products were 3H-dihydroxyphenylglycol and O-methylated deaminated metabolites. Under both conditions 100 μM cocaine increased the proportional 3H-noradrenaline overflow.

The influence of monoamine oxidase and catechol-O-methyl transferase on the distribution of 3H-(+/-)-noradrenaline in rabbit aortic strips.

1. Rabbit aortic strips were incubated with 1.18 μM 3H-(±)-noradrenaline for 30 min, and the accumulation of noradrenaline in the strips was measured as well as the total formation of metabolites. Some strips were exposed to 30 μM cocaine or 87μM corticosterone to inhibit neuronal and extraneuronal uptake, respectively. In some strips monoamine oxidase (MAO) and/or catechol-O-methyl transferase (COMT) was inhibited by pargyline and/or U-0521. Of the extraneuronal enzymes COMT is much more important than MAO; there is virtually no accumulation of noradrenaline in extraneuronal tissue when either COMT or both enzymes are intact. For adrenergic nerve endings, MAO is most important; very little or no COMT activity is associated with the nerve ending. When MAO is intact, there is virtually no axoplasmic accumulation of noradrenaline. Storage vesicles and MAO compete for axoplasmic noradrenaline. Neuronal and extraneuronal uptake and metabolism do not seem to compete for noradrenaline. 2. Other strips were incubated as described above and then washed out with amine-free solution for 240 min. When storage vesicles are intact, the long half time of the late neuronal efflux of radioactivity reflects the long half time for the conversion of “bound” to “free” amine; most of the free amine is then deaminated to dihydroxyphenyl glycol (DOPEG) when MAO is intact. Axoplasmically accumulated noradrenaline generates an efflux with a half time that is shorter than that for vesicular noradrenaline. A small proportion of the neuronal efflux of noradrenaline is taken up extraneuronally and converted to normetanephrine (NMN). When little or no noradrenaline accumulates in the strips during initial incubation, the strips lose some metabolites (DOPEG, NMN) quickly during wash out, while the acid metabolite, dihydroxymandelic acid (DOMA), leaves the tissue with a long half time; late efflux can then consist nearly exclusively of DOMA.

The kinetic constants for the extraneuronal uptake and metabolism of 3H-(-)-noradrenaline in the perfused rat heart.

1. Rat hearts were perfused with 50–2000 μM 3H-(−)-noradrenaline in the presence of 14C-sorbitol (to measure the extracellular distribution of the amine) and of 100 μM cocaine (to inhibit neuronal uptake). Initial rates of removal of noradrenaline were determined. Extraneuronal uptake determined in this way consisted of two components: a saturable and a non-saturable one. 2. When monoamine oxidase (MAO) was inhibited, the kinetic constants for saturable extraneuronal uptake of noradrenaline were: Km 60 μM, Vmax 50 nmoles · g−1 · min−1. U-0521, an inhibitor of catechol-O-methyl transferase (COMT) acted like a competitive inhibitor of extraneuronal uptake: the Km increased, Vmax remained unaffected. 3. In separate experiments either MAO or COMT was intact. Rat hearts were perfused with various concentrations of 3H-(−)-noradrenaline until the metabolites appeared at a constant rate in the venous effluent. These steady-state rates of metabolite formation were used to determine the kinetic parameters of the metabolizing systems. 4. O-methylation of noradrenaline in the intact heart was a saturable process and obeyed Michaelis-Menten kinetics; it had very low Km (1.7 μM) and Vmax (1.2 nmoles · g−1 · min−1). 5. Also the deamination of noradrenaline by the perfused heart was a saturable process, but Km and Vmax were considerably higher than for O-methylation (140 μM and 25 nmoles · g−1 · min−1, respectively). 6. It is concluded that “extraneuronal uptake and subsequent metabolism” of noradrenaline represents a site of loss that is highly effective at very low substrate concentrations (i.e., below the Km of the two metabolizing systems).

Immobilized catecholamine and cocaine effects on contractility of cardiac muscle.

Isoproterenol, norepinephrine, and epinephrine covalently bound to glass beads exert a positive inotropic effect on isometrically contracting papillary muscles from cats. Immobilized isoproterenol maintains increases in force and velocity of contraction for more than 5 hr. 1 muM Cocaine potentiates the action of immobilized norepinephrine, isoproterenol, and epinephrine, but not of isoproterenol in solution. The data presented indicate that the effects of immobilized catecholamines are not due to their coming off the glass. The effects observed with cocaine and immobilized catecholamines are not altered by prior treatment of the muscle with reserpine. These results suggest that the major site of catecholamine action is on receptors located on the extended surface of myocardial cells and a post-junctional site for cocaine potentiation.

Cocaine and procaine on the electrically stimulated metabolism of cerebral tissues

The effects of cocaine and procaine on the metabolism of electrically stimulated slices of cerebral tissues have been examined. Cocaine was taken up by the slices to give distribution ratios of 1.2 (grey matter) and 1.4 to 1.6 (white matter) in favour of the tissue, and did not appear to be hydrolysed. Procaine also became associated with the tissue, giving equal distribution between slice and saline, but approximately one-third was hydrolysed to p-aminobenzoic acid. In tissues to which electrical pulses were not applied, 50 μM cocaine caused a small diminution in respiratory and glycolytic rates. The phosphocreatine level was also approximately 10 per cent lower. Maximally stimulated grey matter was more sensitive, 50 μM cocaine causing a 45 per cent reduction in both respiration and glycolysis. Inhibition of response occurred to the same degree in white matter exposed to localized stimulation. The rate of phosphocreatine breakdown caused by electrical stimulation was retarded by approximately 90–97 per cent in the presence of 20 μM cocaine. Likewise the concomitant rate of formation of inorganic phosphate was reduced to a similar extent. Results obtained using procaine were fundamentally the same as those found for cocaine but it was necessary to use nearly four times the concentrations found effective with cocaine, for the inhibition of stimulated respiration and glycolysis.