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Notes: Summary This study in the anaesthetized rabbit aimed at determining the role of nitric oxide (NO), the putative endothelium-derived relaxing factor, in the regulation of haemodynamics and the release into plasma of noradrenaline and adrenaline. Specific inhibition of NO formation was achieved by i.v. bolus injection of l-NG-monomethyl-arginine (l-NMMA; 3–100 mg kg−1). Phenylephrine was infused i.v. at constant rates (2.5–20 μg kg−1 min−1) in order to assess baroreflex-mediated changes in release due to direct peripheral vasoconstriction. Rates of noradrenaline and adrenaline release into plasma were determined by the radio-tracer technique. l-NMMA, but not d-NMMA, dose-dependently increased mean arterial pressure and total peripheral vasular resistance, whereas both heart rate and cardiac output decreased concomitantly. The corresponding ED50 values for l-NMMA ranged from 11.2 to 18.5 mg kg−1. Inhibition of NO formation by l-NMMA as well as phenylephrine infusion caused decreases in the plasma clearance of noradrenaline and adrenaline which were correlated with the drug-induced decreases in cardiac output. Both l-NMMA and phenylephrine reduced the rate of noradrenaline release into plasma as they increased total peripheral resistance. Moreover, the curvilinear relationship between these two parameters obtained for l-NMMA was virtually identical to that produced by phenylephrine, indicating that the reduction in noradrenaline release by l-NMMA is mediated solely by the baroreflex. From the l-NMMA-induced maximum inhibition of noradrenaline release, it is concluded that the counter-regulation against peripheral vasodilation by NO accounts for 69% of basal noradrenaline release. The baroreflex-sensitive component of noradrenaline release, as determined by the maximum inhibition of release induced by phenylephrine, amounted to 83% of basal release. l-NMMA also reduced the release into plasma of adrenaline; the maximum inhibition of release was 52%. However, when related to total peripheral resistance, this inhibition of adrenaline release was more pronounced than that induced by phenylephrine, suggesting that the formation of endogenous NO facilitates the release of adrenaline.

Notes: Summary 1. Rabbit hearts (with monoamine oxidase and catechol-O-methyl transferase inhibited) were obtained from reserpine-pretreated animals. They were perfused at rates ranging from 1.3–11.3 ml·g−1·min−1 with 0.1 mM 14C-sorbitol and various concentrations of 3H-(−)noradrenaline (NA). From measurements of the arterio-venous concentration difference of 3H and 14C activity the removal of NA and sorbitol from the perfusion fluid was followed for 2–3 min at intervals of 5 s. The uptake of NA into intracellular spaces of the heart (known to be over-whelmingly into sympathetic nerve terminals) was obtained by subtracting the removal of sorbitol from that of NA. If was cumulated and plotted against time. 2. The progress curves of NA uptake were sigmoid in shape: following a lag period, uptake proceeded at first at a constant initial rate and from then on at gradually decreasing rates. Irrespective of the NA concentration used, the lag period became shorter and the initial rate of uptake increased whenever the rate of perfusion was increased. Furthermore, at high rates of perfusion the initial rate was maintained for a shorter time than at low ones. 3. At any given perfusion rate, the initial rates of NA uptake obeyed Michaelis-Menten kinetics. While changes of the rate of flow did not alter the apparent K m (range: 2.2–2.4 μM), a rectangular hyperbolic relationship was found between V max and the perfusion rate. The V max was half-maximal at a rate of flow of 2.7 ml·g−1·min−1 and approached a maximum value of 9.0 nmoles·g−1·min−1. 4. From the lack of change in the K m it can be concluded that the uptake sites of the perfused heart are functionally arranged in parallel. The change in V max, on the other hand, indicate that the accessibility of the sites is limited by the rate of perfusion.

Notes: Summary 1. The uptake and O-methylation of 3H-(±)isoprenaline was studied in slices of the rat submaxillary gland. 2. The initial uptake of 3H-isoprenaline after inhibition of catechol-O-methyl transferase (COMT) was described by a single saturable process with relatively high K m (311 μM) and V max (101 nmoles·g−1·min−1). Both corticosterone and normetanephrine were competitive inhibitors of uptake. 3. When examined at substrate concentrations lower than the K m for uptake (and after block of COMT), 3H-isoprenaline distributed into two compartments in the tissue which approached equilibrium with half times of 2.4 and 15.8 min. The filling of both compartments was inhibited by corticosterone or phenoxybenzamine and also by high-K+ medium (in which 118 mM NaCl of the incubation medium had been replaced by KCl), but remained unaffected on substituting 118 mM NaCl with Tris-HCl. 4. In tissues in which COMT was not inhibited, the metabolism of 3H-isoprenaline to 3H-O-methylisoprenaline proceeded at a constant rate from the beginning of the incubation with the amine. When the substrate concentration was very low, little unchanged 3H-isoprenaline was found in the tissue. On the other hand, at high substrate concentrations the parent amine accumulated in the tissue, and at a time when 0-methylation had reached a steady state, the accumulation of 3H-isoprenaline was continuing. 5. The formation of 3H-O-methylisoprenaline was impaired by the presence of corticosterone, normetanephrine, phenoxybenzamine or 17-β-oestradiol with no indication of inhibition of COMT. While lowering the external Na+ concentration (on replacing 118 mM NaCl by 236 mM sucrose) did not affect the formation of 3H-O-methylisoprenaline, replacement of 118 mM NaCl by KCl reduced it. 6. The dependence of the steady-state rate of formation of 3H-O-methylisoprenaline on the substrate concentration in the incubation medium showed that two saturable components participated in the O-methylation of 3H-isoprenaline (low K m system: K m =7.2 μM and V max=1.2 nmoles·g−1·min−1; high-K m system: K m =339 μM and V max=4.6 nmoles·g−1·min−1). Corticosterone and normetanephrine competitively inhibited both the low-K m and the high-K m O-methylation. 7. The results indicate that the submaxillary gland of the rat resembles other tissues in having a low-K m (high-affinity) “O-methylating system” as well as a high-K m (low-affinity) extraneuronal uptake mechanism for catecholamines.

Notes: Summary 1. Vasa deferentia obtained from reserpine-pretreated rats were incubated (monoamine oxidase and catechol-O-methyltransferase inhibited) in media containing various concentrations of3H-(−)noradrenaline and Na+ and initial rates of the neuronal uptake of3H-noradrenaline measured both in the absence and presence of uptake inhibitors after 1 min of incubation. 2. When rates of uptake were determined at various3H-noradrenaline (1.0–12.2 μmol/l) and two fixed Na+ concentrations (25 and 140 mmol/l), the inhibition of uptake produced by (+)amphetamine, (−)metaraminol, desipramine, nomifensine and cocaine was competitive with respect to3H-noradrenaline at both Na+ concentrations. While theK i for (+)amphetamine, (−)metaraminol desipramine and nomifensine increased when the Na+ concentration was lowered, that for cocaine decreased. 3. When the Na+ concentration was varied (10–140 mmol/l) and the3H-noradrenaline concentration held constant (1.2 μmol/l), (+)amphetamine, (−)metaraminol, nomifensine and desipramine acted as mixed-type inhibitors with respect to Na+, and the inhibition of uptake produced by these drugs was the more pronounced, the higher the Na+ concentration. On the other hand, cocaine was competitive with Na+ and the inhibition produced by this drug was the more pronounced, the lower the Na+ concentration. 4. It is concluded that the inhibitors of neuronal uptake tested here act in dependence on the external Na+ concentration. Desipramine and nomifensine resemble alternative amine substrates in being more potent at high than at low Na+ concentrations. On the other hand cocaine is more potent at low than at high Na+ concentrations.

Notes: Summary After loading of the incubated rat vas deferens with 0.2 μmol/l 3H-noradrenaline (followed by 100 min of wash-out with amine-free solution), the efflux of endogenous and exogenous compounds was determined by HPLC with electrochemical detection and by column chromatography with scintillation counting. Two different types of heterogeneity of labelling were found. The first one is due to the preferential labelling of varicosities close to the surface of the tissue, the second one to the preferential labelling of vesicles close to the surface of loaded varicosities. As diffusion distances within the tissue and within varicosities are then longer for endogenous than for exogenous amine and metabolites, the composition of spontaneous efflux of exogenous compounds differed from that for endogenous compounds. Because of preferential neuronal and vesicular re-uptake of endogenous noradrenaline, the percentage contribution by noradrenaline to overall efflux was: endogenous 〈 exogenous. While 3H-DOPEG was the predominant exogenous metabolite, DOPEG and MOPEG equally contributed to the “endogenous” efflux. Desipramine abolished the consequences of the first heterogeneity of labelling, i.e., it increased the efflux more for endogenous than for exogenous noradrenaline; moreover it decreased the efflux of 3H-DOPEG, but increased that of 3H-MOPEG. The reserpine-like compound Ro 41284, on the other hand, abolished the consequences of the second type of heterogeneity; it reduced the specific activity of “total efflux” (i.e., of the sum of noradrenaline + DOPEG + MOPEG) to the specific activity of the tissue noradrenaline. The degree of heterogeneity of labelling was reduced after inhibition of monoamine oxidase and also when the tissues were loaded with 2 or 20 μmol/l 3H-noradrenaline. It is proposed that the various “compartments” and “pools” of noradrenaline described in the literature reflect the two heterogeneities described here.

Notes: Summary Isolated rabbit hearts were perfused with 20 to 200 ng/ml of (−)-noradrenaline and arterio-venous differences were determined at various times to measure the rate of net removal of the amine from the perfusion fluid. Animals were untreated or pretreated with reserpine and/or pargyline to block vesicular retention and/or intraneuronal monoamine oxidase (MAO). The arterio-venous difference (in percent of the arterial concentration) remained rather constant during prolonged perfusions of untreated hearts with (−)-noradrenaline, the magnitude of the difference being inversely related to the arterial concentration. After block of MAO the rate of net removal declined exponentially with time; the rate of decline increased with increasing arterial concentration of the amine and also after the additional pretreatment with reserpine. The time-dependent decline in the rate of net removal was shown to be due to an increased efflux of the amine from the nerve endings. The net removal of noradrenaline-H3 at the 5th min of perfusion of pargyline-pretreated hearts was mainly due to neuronal net uptake, since a) O-methylation accounted for only 5% of the removal, and b) cocaine (10–30 (μg/ml) virtually abolished net removal. Initial rates of removal were not affected by the various pretreatments. In untreated hearts retention of exogenous (−)-noradrenaline increased linearly with the duration of the perfusion but the increase was exponential after block of MAO. Apparently, the storage capacity becomes exhausted during prolonged perfusions of pargyline-pretreated hearts. The ratio “noradrenaline retained by the heart/noradrenaline removed by the heart” was quite small in untreated (0.16), very small in reserpine-pretreated (0.03) and nearly unity in pargyline-pretreated hearts. It is concluded that any impairment of the intraneuronal mechanisms of inactivation (vesicular storage and MAO) leads to an increase in the axoplasmic concentration of free noradrenaline which causes an increased efflux of the amine, while the influx remains unchanged. The axoplasmic concentration of free noradrenaline seems to rise more after block of MAO than after pretreatment with reserpine and is most pronounced after both. Changes in the sensitivity of the pacemaker to (−)-noradrenaline were found to be correlated with changes in the rate of removal of the amine from the perfusion fluid.

Notes: Summary 1. The carrier-mediated transport of 3H-noradrenaline out of noradrenergic neurones was studied in vasa deferentia obtained from rats after pretreatment with reserpine and pargyline (to inhibit vesicular storage and monoamine oxidase, respectively). The tissue was first preincubated with various concentrations of 3H-noradrenaline (0.3–100 μmol/l; 30 min) and then washed out for 110 min with amine-free medium. During the last 10 min of washout, carrier-mediated neuronal efflux of 3H-noradrenaline was elicited by exposure to either Na+-free medium or 100 μmol/l veratridine; it was measured at 1-min intervals. 2. While the peak rates of carrier-mediated 3H-noradrenaline efflux elicited by Na+-free medium were linearly related to the 3H-noradrenaline content of the tissue (which cannot be raised beyond a certain maximal value, since uptake is saturable), those evoked in response to veratridine approached saturation as the 3H-noradrenaline level in the tissue was raised. Hence, saturation of 3H-noradrenaline outward transport was demonstrated at high (exposure to veratridine), but not at low (exposure to Na+-free medium) intraneuronal Na+ concentrations. 3. The results indicate that the K m for the mediated outward transport of noradrenaline across the plasma membrane of noradrenergic neurones is inversely related to the internal Na+ concentration, just as the K m for the mediated inward transport of noradrenaline (i.e., the neuronal noradrenaline uptake) is inversely related to the external Na+ concentration.

Notes: Summary 1. The effects of choline+ (10–40 mmol/l) on 3H-noradrenaline uptake by, and 3H-noradrenaline efflux from, noradrenergic neurones were studied in vasa deferentia of reserpine-pretreated rats at an external Na+ concentration of 100 mmol/l. Monoamine oxidase and catechol-O-methyltransferase were inhibited. 2. Choline+ (20 and 40 mmol/l) competitively inhibited the neuronal uptake of 3H-noradrenaline. From the choline+-induced changes in the apparent Km for 3H-noradrenaline transport, a Ki of 35 mmol/l was obtained. 3. Choline+ (10, 20 and 40 mmol/l) accelerated the neuronal efflux of 3H-noradrenaline in a concentration-dependent manner. This acceleration of efflux was greatly reduced in the presence of 1 μmol/l desipramine, indicating that choline+ is capable of eliciting “accelerative exchange diffusion”. 4. Choline+ (40 mmol/l) and (−)noradrenaline (4.5 μmol/l) (i.e., concentrations about equivalent to the Ki and Km for choline+ and (−)noradrenaline, respectively) produced virtually identical increases in the neuronal efflux of 3H-noradrenaline. 5. Choline+ (3–300 mmol/l) inhibited the specific binding of 3H-desipramine to plasma membranes derived from cultured rat phaeochromocytoma (PC-12) cells. The Ki for this interaction was 48 mmol/l. 6. This results suggest that choline+ acts as alternative substrate of the neuronal noradrenaline transport system and should, therefore, not be used in transport studies with noradrenaline as substitute for Na+ in Na+-deficient media.

Notes: Summary 1. To examine whether K+ affects the potency of inhibitors of neuronal uptake, experiments were carried out in the rat vas deferens after pretreatment of the animals with reserpine and after inhibition of monoamine oxidase and catechol-O-methyltransferase. Initial rates of the neuronal uptake of 3H-noradrenaline and IC50 values for uptake inhibition by desipramine, cocaine and (−)metaraminol were determined in the presence of various concentrations of external K+ (5–45 mmol/l), both at 100 mmol/l Na+ and 50 mmol/l Na+. 2. When measured at the 3H-noradrenaline concentration used to determine IC50 values (0.024 μmol/l), neuronal uptake was progressively impaired by increasing K+ concentrations at 50, but not at 100 mmol/l Na+. 3. Neither at 100 mmol/l Na+ nor at 50 mmol/l Na+ was there any consistent, concentration-dependent effect of K+ on the IC50 values of desipramine, cocaine and (−)metaraminol. 4. The analysis of the saturation kinetics of 3H-noradrenaline uptake (determined in the presence of 50 mmol/l Na+ at 5 mmol/l K+ or 45 mmol/l K+) showed that high K+ concentrations inhibit neuronal uptake by decreasing V max without any change in K max. 5. The results indicate that K+ does not competitively interact with Na+ at sites on the noradrenaline carrier which mediate the tansport-stimulating properties of Na+ Hence, the inhibition of neuronal uptake produced by high K+ concentrations is probably due to membrane depolarization which simply reduces V max.

Notes: Summary Isolated rat hearts were perfused according to the Langendorff technique and both extraneuronal uptake of noradrenaline and COMT were inhibited. The noradrenergic neurones were first prelabelled with 3H-(−)-noradrenaline (13 nmol/1). Thereafter the hearts were submitted to global ischemia (perfusion rate reduced from 5 up to 0.5 ml/min) for 60 min and subsequently reperfused for 5 min. The coronary effluent was continuously collected and analyzed for the appearance of 3H-noradrenaline and its metabolites. 1. Global ischemia was associated with an early release of 3H-noradrenaline. At reperfusion a brisk increase in the FRL of 3H-noradrenaline was observed which may indicate that, on severe restriction in coronary flow, perfusion of the tissue became heterogenous and thus partially masked the amount of 3H-noradrenaline released from the noradrenergic nerve terminals. Gradual reduction in coronary flow also progressively reduced (but did not abolish) the total formation of 3H-DOPEG. 2. The maximal efflux of 3H-noradrenaline was observed during the 1st min of reperfusion whereafter the efflux declined rapidly, indicating a wash-out of transmitter trapped in the extracellular space. The efflux of the lipophilic metabolite 3H-DOPEG, on the other hand, continuously increased during the reperfusion. This was due to both new formation and “wash-out” of 3H-DOPEG retained and/or distributed into the tissue during the period of restricted flow. 3. Neither a reduction of the extracellular calcium concentration (from 2.6 mmol/l to 0.1 mmol/1) nor the presence of the calcium entry blocker verapamil (250 nmol/l) reduced the efflux of 3H-noradrenaline seen during ischemia and reperfusion. 4. Desipramine (100 nmol/l) markedly reduced the ischemia-induced release of 3H-noradrenaline and simultaneously attenuated the formation of 3H-DOPEG. 5. A moderate reduction in the ischemia-induced mobilization of 3H-noradrenaline was seen in hearts perfused with 1μol/l reserpine, whereas the formation of 3H-DOPEG from such hearts was markedly higher than in corresponding controls. Only minor deviations from this pattern was observed when desipramine was present in addition to reserpine. It is concluded that a severe restriction in myocardial perfusion rate is associated with an enhanced net leakage of vesicular noradrenaline. This results in a rise of the free axoplasmic noradrenaline concentration which, in combination with an altered transmembrane sodium gradient, induces an increased local release of noradrenaline partly mediated by a calcium-independent, carrier-mediated outward transport. Desipramine, which inhibits this transport mechanism, may have, in addition to its effect on the membrane carrier, an additional effect in reducing the net leakage of transmitter from storage vesicles. Furthermore, despite severe restriction in coronary flow, and thus oxygen delivery, DOPEG is still formed, possibly as a consequence of the elevated axoplasmic noradrenaline concentration which may in part compensate for a reduced monoamineoxidase activity.