Functional significance of neuronal and extraneuronal transmitter uptake in rat salivary glands

Summary The effect of normetanephrine (NM) on effector cell response to noradrenaline (NA) in vivo and on extraneuronal amine uptake in vitro has been investigated in rat submaxillayr glands. Tissue slices were incubated with ³H-NA and the metabolic pattern of the radioactivity in the slices as well as of the efflux from the slices was analyzed. The level of extraneuronally retained radioactive material was found to be markedly reduced at a NM concentration of 20 g/ml after inhibition of neuronal uptake by protriptyline (PTP). In the presence of an intact neuronal membrane pump mechanism 200 g/ml of NM also reduced the level of radioactive material, indicating that NM has an inhibitory effect on extraneuronal and in higher concentration also on neuronal uptake and retention of NA and its metabolites. NM was found to markedly potentiate the secretory response to NA in vivo, when doses of NA larger than 1 g were used. At lower NA doses there was no difference between NM pretreated and control rats. When neuronal uptake was prevented by pretreatment with PTP, NM was found to potentiate the secretory response to NA over the entire dose range. Inhibition by PTP of neuronal uptake only did not affect the dose-response curve for NA and data are presented which indicate that the absence of such a potentiation is due to a decreased salivary gland blood flow induced by NA after PTP pretreatment. The increased responses recorded after NM could not be ascribed to an additive effect of this amine, since NM was found to be a very weak -receptor agonist in the rat submaxillary gland; doses of more than 1000 g i.v. were needed to detect a secretory response. The data indicate that the extraneuronal uptake of the transmitter has a functional significance in the rat submaxillary gland by reducing high transmitter concentrations in the vicinity of the receptors.     

On the role of the adrenergic receptors for extraneuronal amine uptake and retention in rat salivary glands in vitro

Summary The effect of an -receptor blocking agent, phenoxybenzamine, and a series of -receptor blocking agents on extraneuronal uptake and retention of radioactivity after incubation of rat salivary gland slices with ³H-noradrenaline or ³H-isoprenaline has been investigated. In some experiments with ³H-noradrenaline as substrate, neuronal uptake was prevented by adding protriptyline to the incubation medium. Phenoxybenzamine reduced extraneuronal accumulation of radioactivity after both ³H-noradrenaline and ³H-isoprenaline in a concentration-dependent manner, whereas after propranolol the levels of extraneuronally retained radioactive material were markedly increased, the efflux from the slices not being diminished. Some other nonselective and selective -receptor blocking agents with and without intrinsic activity were found to produce the same effect as propranolol upon the retention of radioactivity after incubation with ³H-noradrenaline and ³H-isoprenaline. When both phenoxybenzamine and propranolol were present in the incubation medium the effect of extraneuronally retained radioactivity after ³H-noradrenaline as well as after ³H-isoprenaline was the same as when only phenoxybenzamine was used. The metabolic pattern of the radioactivity in the slices revealed that the extraneuronally retained radioactive compounds consisted mainly of tritiated catabolites, especially 3-O-methylated ones. The relationship between the extraneuronal accumulation and adrenergic receptor mechanisms is discussed.     

Kinetic constants of isoprenaline and corticosterone for extraneuronal uptake in different cell types from various tissues

Extraneuronal uptake of isoprenaline was studied in a number of different, histologically identified, cell types: trachealis smooth muscle cells, vascular smooth muscle cells and myocardial cells. Segments of cat, rat or rabbit trachea, dog coronary artery or cat atria were incubated in isoprenaline, in the presence of U-0521 (100 mumol l-1) to inhibit catechol-O-methyltransferase. The intensities of formaldehyde-induced fluorescence (due to accumulation of isoprenaline) were measured, using microphotometry, in the appropriate cells in histological sections of the tissue. Endogenous fluorescence in adrenergic nerves was removed by pretreatment of rats with 6-hydroxydopamine and of cats and rabbits with reserpine. Segments of dog coronary artery were incubated with 6-hydroxydopamine in vitro to remove neuronal fluorescence. This treatment was also shown to be effective in guinea-pig trachea without any influence on the determination of the kinetic parameters of isoprenaline uptake in the trachealis smooth muscle cells. Fluorescence in all cell types studied was shown to represent isoprenaline which had accumulated by extraneuronal uptake, in that fluorescence was reduced by drugs which inhibit extraneuronal uptake (corticosterone, normetanephrine, metanephrine and/or phenoxybenzamine), by exposure to a K+-Krebs solution and by incubating tissues in isoprenaline at 0 degree C rather than 37 degrees C. In each cell type, isoprenaline uptake obeyed Michaelis-Menten saturation kinetics, both in the absence and presence of corticosterone. Corticosterone was a competitive inhibitor of isoprenaline uptake in all the cell types.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of reserpine treatment on the extraneuronal uptake of [3H]-isoprenaline into rat atria.

Treatment of rats with reserpine (1 mg kg-1 day-1) for up to 7 days resulted in a marked decrease in a corticosterone-sensitive component of the extraneuronal accumulation of [3H]-isoprenaline into their atria. The change in extraneuronal uptake did not appear to be due to a direct action of reserpine on the uptake mechanism, since it was several days before the treatment had a significant effect on the accumulation of [3H]-isoprenaline. Further, reserpine in vitro did not inhibit extraneuronal uptake. The reserpine-induced change in the accumulation of [3H]-isoprenaline was not an artifact due to changes in water balance, ion distribution, extracellular space or tissue atrophy. Nor was the change due to an increase in the efflux of [3H]-isoprenaline from the tissue. These experiments support the suggestion that the extraneuronal uptake is dependent upon a functional adrenergic innervation.     

Effects of blockade of extraneuronal uptake on responses to isoprenaline in perfused rat heart

1. Isolated rat hearts accumulated 102 pmol/g wet wt/min of isoprenaline when perfused for 5 min with 0-6 muM (+/-)-3H-isoprenaline. 2. The 3-methoxy derivative of isoprenaline ('methoxy isoprenaline') (10 muM) significantly inhibited this uptake by 57%, metanephrine (10 muM) by 29% and normetanephrine (10 muM) by 21%. 3. (+/-)-Isoprenaline (0-6 muM) infused into isolated perfused rat hearts for 5 min activated glycogen phosphorylase 2-4-fold. Normetanephrine (10 muM) or metanephrine (10 muM included in the perfusate significantly potentiated this activation, but 3-0-methyl isoprenaline (10 muM significantly reduced it. However, 3-0-methyl isoprenaline potentiated the ability of 4-8 muM isoprenaline to stimulate phosphorylase. 4. Neither metanephrine (10 muM) nor normetanephrine (10 muM) altered peak inotropic responses to injections of (+/-)-isoprenaline into the solution perfusing isolated rat hearts. 3-0-methyl isoprenaline (10 muM) shifted the isoprenaline dose-response curve to the right, but did not affect the inotropic responses to CaCl2, confirming that 3-0-methyl isoprenaline possess beta-adrenoceptor antagonist activity. 5. Inotropic responses to isoprenaline were significantly prolonged by both 3-0-methyl isoprenaline and normetanephrine (10 muM). 6. These results indicate that blockade of extraneuronal accumulation of catecholamines causes potentiation of both metabolic and mechanical beta-adrenoceptor-mediated responses of the heart to isoprenaline. It is suggested that Uptake2 and the cardiac beta-adrenoceptor are separate entities, and that the beta-adrenoceptor is localized in the sarcolemma. The physiological function of Uptake2 may be to help clear the sympathetic synaptic gap of liberated neurotransmitter.     

Further studies on the extraneuronal uptake and metabolism of isoprenaline in the perfused rat heart

Summary To simultaneously determine the kinetics of removal, O-methylation and accumulation of ³H-isoprenaline, isolated rat hearts were perfused for 4 min with various concentrations of ³H-isoprenaline. The apparent K _m for the O-methylation of ³H-isoprenaline (3.3±0.5 M) was more than one order of magnitude lower than the corresponding value for the accumulation of unchanged amine (71.3±7.1 M). The apparent K _m for removal was very similar to that for accumulation (63.2±5.9 M). At perfusion concentrations higher than 25 M, i.e. when O-methylation was saturated, removal virtually equalled accumulation. However, at low substrate concentrations removal of ³H-isoprenaline was overwhelmingly followed by O-methylation; this led to a marked difference between rates of removal and those of accumulation.When initial rates of uptake of ³H-isoprenaline were determined after 1.5 min of perfusion of the hearts by the method of Graefe et al. (1978), the uptake of ³H-isoprenaline consisted of two components: a nonsaturable and a saturable (after subtraction of the nonsaturable component from the total uptake).The kinetic constants of the saturable component of uptake were higher than those obtained after 4 min perfusion (see above) (K _m : 110±19 M; V _max: 80±4 nmoles·g^–1·min^–1).Corticosterone competitively inhibited the saturable component of uptake of ³H-isoprenaline (K _m : 1.2 M).During wash out of accumulated ³H-isoprenaline, O-methylation took place predominantly in one of the two extraneuronal compartments. The efflux of 3-O-methyl-³H-isoprenaline (³H-OMI), the O-methylated metabolite of ³H-isoprenaline, was characterized by a half time of about 1.2 min. O-methylation accelerated the loss of radioactivity from the tissue during wash out.The extraneuronal uptake of ³H-isoprenaline was characterized as a pump and leak system by means of steady-state kinetics of accumulation of ³H-isoprenaline. Half saturation of the steady-state accumulation was observed at a concentration of 104.5 ±18.5 M ³H-isoprenaline; the leak component was characterized by a rate constant of 0.0359 min^–1.This study was supported by the Deutsche Forschungsge-meinschaftA preliminary account was presented at the 6th International Congress of Pharmacology (Graefe et al., 1975)     

The neuronal and extraneuronal uptake and metabolism of 3H-(−)-noradrenaline in the perfused rat heart

1. Hearts were obtained from reserpine-pretreated rats and perfused with 0.95 micron 3H(-)-noradrenaline. The rate of removal of 3H-noradrenaline from the perfusion fluid was measured (from the arterio-venous difference) as well as the rate at which the 3H-metabolites appeared in the venous effluent. 2. When either 30micron corticosterone was added under steady-state conditions during perfusion with 3H-noradrenaline (to inhibit neuronal and extraneuronal uptake, respectively), each inhibitor reduced the removal of noradrenaline by about 50%; in the presence of both inhibitors removal was abolished. 3. Dihydroxymandelic acid (DOMA) was of neuronal, normetanephrine (NMN) of extraneuronal origin; dihydroxyphenylglycol (DOPEG) and the OMDA fraction (containing methoxyhydroxyphenylglycol-MOPEG-and methoxyhydroxymandelic acid-VMA) were formed both neuronally and extra-neuronally. 4. The extraneuronal metabolism of 3H-noradrenaline was in quick equilibrium with the 3H-noradrenaline in the perfusion fluid; most of the total formation of DOPEG, MOPEG and NMN was recovered from the venous effluent. 5. Extraneuronally formed DOPEG, MOPEG and NMN distributed in the tissue with half times corresponding to their half time for efflux. 6. Inhibition of monoamine oxidase (MAO) by pargyline increased the extraneuronal formation of NMN; MAO and catechol-O-methyl transferase (COMT) appear to be contained in the same extraneuronal compartment. 7. The extraneuronal accumulation of 3H-noradrenaline required 30 min or more to reach a steady state; inhibition of one or both enzymes slowed this process. Inhibition of MAO increased the extra-neuronal accumulation of 3H-noradrenaline; inhibition of COMT failed to do so, since the enzyme inhibitor (U-0521) was a weak inhibitor of extra-neuronal uptake. 8. The rate constants for the efflux of the metabolites of noradrenaline decreased in the order of MOPEG greater than DOPEG greater than NMN greater than DOMA greater than VMA.     

Inhibitory effect of azelastine on allergic itch-associated response in mice sensitized with mosquito salivary glands extract

We examined whether azelastine would inhibit itch-associated responses of mice to mosquito allergy. Repeated injections of mosquito salivary gland extract increased scratching and sensory nerve activity. Azelastine inhibited the increased scratching and nerve activity, while terfenadine was without effects. Dexamethasone did not affect the increased scratching. Azelastine suppressed high K(+)-induced increase in intracellular free Ca(2+) in primary cultures of mouse sensory neurons. Direct inhibition by azelastine of sensory neurons may be at least involved in the anti-pruritic effect of azelastine. Histamine, substance P, and leukotriene B(4) may not play a key role in the itching of mosquito allergy.     

Propranolol uptake into the central nervous system and the effect on rat behaviour and amine metabolism

Propranolol readily penetrated all areas of the central nervous system after acute intraperitoneal administration and was cleared within 8–16 hr. Propranolol levels in the heart followed a similar pattern. After chronic oral administration brain levels of propranolol were equal to those 3 hr after intraperitoneal injection. Propranolol caused no significant difference in the brain levels of noradrenaline, normetanephrine, dopamine, 3-methoxytyramine, homovanillic acid, 5-hydroxy-tryptamine or total indoles, nor did it cause measurable changes in rat behaviour. An increase by propranolol in sleeping time after chloral hydrate in rats and mice was observed; this effect could not be correlated with changes in brain amine concentrations.     

Effects of lithium on adrenergic amine uptake in rat brain synaptosomes

The effects of lithium on the uptake and retention of (?)-erythro-metaraminol (MA) and (?)-m-octopamine (OA) by synaptosomes from rat whole brain were examined after (a) addition of lithium chloride (1 mmol/l.) to synaptosomal fractions in vitro and (b) administration of lithium chloride (1 mmol/kg) i.p. to rats twice daily for 10 days prior to isolation of synaptosomes. Under control conditions, apparent K_m concentrations (5 min incubation) for MA and OA were 33 and 3 μM respectively. As incubation times were increased to 15 min, retention of MA increased and OA decreased. Retention of both amines was reduced by low temperature. Short term application of lithium to synaptosomes in vitro did not alter the uptake and retention of either amine during 5 or 10 min incubation periods. Chronic pretreatment of rats with lithium resulted in greater uptake and retention of low concentrations of MA when compared with synaptosomes from control (saline-treated) animals. Chronic lithium pretreatment did not alter retention of OA. The data support the hypothesis that lithium acts to increase synaptosomal membrane uptake of aromatic amines and decrease binding of accumulated amine.     

Effect of prostaglandins on tyrosylprotein sulfotransferase activity in rat submandibular salivary glands

• 1.1. Tyrosylprotein sulfotransferase (TPST) is a key enzyme in the processing of several secretory proteins, including those found in saliva. In this report, the effect of prostaglandins (PG) on TPST activity in submandibular salivary gland was investigated.
• 2.2. The results revealed that PGE₂ exhibited TPST stimulatory activity with a 1.5-fold stimulation at 100 μM concentration and a half maximal stimulation at 50 μM. The PGE₂ stimulation was accompanied by an increase in the affinity of TPST towards sulfate acceptor (Km 1.4 μM → 0.12 μM) with little change in V_max.
• 3.3. The TPST activity was also stimulated by two other major prostaglandins of salivary glands, PGF_2α and 6-Keto-PGF_1α, however to lesser extent, 22 and 23%, respectively. Arachidonic acid, an intermediate prostaglandin precursor, had no effect on TPST activity.
• 4.4. The results suggest that prostaglandins and in particular PGE₂ may play a role in the regulation of TPST catalyzed secretory protein tyrosine sulfation in salivary glands.