F. J. E. Stefano

Different roles of D-1 and D-2 dopamine receptors involved in locomotor activity of supersensitive mice

Simultaneous stimulation of both D-1 and D-2 receptors is necessary to reverse reserpine-induced akinesia in mice. The effect of supersensitivity on locomotor function was studied in mice after treatment with reserpine for five days. The response of these animals to a mixed D-1/D-2 agonist, pergolide, or to a presynaptic dopamine (DA) releaser, amphetamine, was increased 3-fold, indicating behavioural supersensitivity. Under these conditions, both selective D-1 and D-2 dopamine receptor agonist (SKF 38393 and LY 171555, respectively), given separately, induced locomotor activity. The D-1 antagonist, SCH 23390, inhibited the effect of both SKF 38393 and LY 171555, whereas the DA synthesis inhibitor, alpha-methyl-p-tyrosine (AMPT), and the D-2 antagonist, sulpiride, only abolished the effect of LY 171555. Moreover, AMPT increased the response to SKF 38393 by 80%. The amphetamine-mediated responses were abolished by SCH 23390 whereas sulpiride did not block them. Thus, stimulation of the D-1 receptor seems crucial in supersensitive animals. In another set of experiments, AMPT was administered to mice pretreated with reserpine for five days in order to fully deplete DA stores. Low doses of LY 171555 reduced the response of these animals to SKF 38393 by 60% whereas higher doses potentiated it. This bimodal effect of LY 171555 was blocked by sulpiride. Since amphetamine was unable to reverse the reserpine-induced akinesia in these mice, we can conclude that the inhibitory effect of LY 171555 is not related to presynaptic inhibition of DA release.

Clonidine inhibits salivary secretion by activation of postsynaptic α2-receptors

SummaryThe effects of clonidine on the submaxillary gland of the rat were studied. Doses ranging between 100 to 3.000 μg/kg produced a sustained secretory response which was blocked by 0.1 mg/kg of prazosin but not by 1 mg/kg of yohimbine. Clonidine 10 μg/kg markedly inhibited the salivation induced by noradrenaline, methacholine and substance P but not that induced by isoproterenol. The inhibition caused by the α2-agonist was greater for noradrenaline than for either methacholine or substance P. Blockade of α2 adrenoceptors with yohimbine (0.3–1 mg/kg) prevented the inhibition by clonidine of noradrenaline, methacholine and substance P induced salivation. On the other hand, prazosin 0.1 mg/kg did not modify the inhibition by clonidine of methacholine induced secretion. The results obtained indicate that clonidine exerts a dual effect on salivary secretion: at high doses it elicits salivation through activation of α1-adrenoreceptors; at the dose of 10 μg/kg clonidine activates α2-adrenoreceptors which inhibit the secretory response evoked through either muscarine, substance P and α1-adrenorecptors agonists.

Evidence for the presynaptic location of the alpha-adrenoceptors which regulate noradrenaline release in the rat submaxillary gland

SummaryFifteen days after duct ligation, the wet weight of the rat submaxillary gland was reduced to 40% of the contralateral control. Under these experimental conditions, the noradrenaline (NA) content expressed as μg/g was 1.2±0.1 in the control glands and 1.9±0.2 in the atrophied glands.The accumulation of 3H-NA in the tissue expressed as μCi/gland, did not differ when the atrophied glands were compared with the corresponding controls. Consequently, the uptake and retention of 3H-NA was not modified by the atrophy of the secretory cells of the gland.The spontaneous efflux of radioactivity from normal and atrophied submaxillary glands prelabelled with 3H-NA was similar. The analysis of the metabolic pattern in both experimental groups revealed that in the spontaneous outflow and also during potassium-induced depolarization, the formation of the O-methylated metabolite, 3H-normetanephrine (NMN) was reduced by more than 50% in the atrophied glands. During depolarization induced by K+, a 2-fold increase in the outflow of the deaminated glycol 3H-3,4-dihydroxyphenylglycol (DOPEG) was observed.The effect of phentolamine on the release of radioactivity induced by 60 mM K+ in normal and in atrophied submaxillary gland slices prelabelled with 3H-NA was also investigated. In both experimental groups, the fractional release of total radioactivity induced by K+ was similar. Phentolamine, 3.1 μM, produced a 3-fold increase in the fractional release of radioactivity both in the control and the atrophied glands. These results indicate that the increase of K+-induced release of 3H-NA induced by phentolamine was independent of the presence or absence of the postsynaptic structures. It is concluded that phentolamine increases transmitter release by blocking alpha-adrenoceptors located in the noradrenergic nerve endings of the rat submaxillary gland.

Effects of 6-hydroxydopamine treatment at birth on the submaxillary gland of the rat

SummarySix and 18 months after neonatal administration of 6-hydroxydopamine or surgical sympathetic denervation the submaxillary gland of the rat showed a marked depletion of noradrenaline stores. Six months after removal of the superior cervical ganglion the glands endogenous noradrenaline was lowered to 0.032±0.004 μg/g while after neonatal 6-hydroxydopamine the values were 0.228±0.023 μg/g (controls 2.145±0.382 μg/g). Eightheen months after either type of sympathetic denervation the neurotransmitter was still depleted. In rats treated with 6-hydroxydopamine the sialagogue effect of injected noradrenaline was potentiated 2.7-fold while the potentiation of the effect of noradrenaline was 3.6 times after surgical denervation. The magnitude of the supersensitivity developed to isoprenaline did not differ between both types of denervation. No supersensitivity to the cholinomimetic agent, methacholine, was observed. Cocaine administration or removal of the superior cervical ganglion slightly increased the supersensitivity to noradrenaline in rats treated with 6-hydroxydopamine. Eighteen months after surgical or chemical denervation, the activity of choline-acetyltransferase in the submaxillary gland was increased by about 50%. Of the respiratory enzymes studied, succinic dehydrogenase, fumarase and cytochrome oxidase, the activity of only the latter was markedly reduced by chronic sympathetic denervation. From the results obtained it is concluded that neonatal treatment with 6-hydroxydopamine causes a permanent and almost complete sympathectomy of the submaxillary gland of the rat.

Effects of sympathectomy on the in vitro α and β-responses of the parotid gland

SummarySalivary secretion in response to noradrenaline and isoprenaline was measured in innervated and chronically sympathectomized parotid glands of the rat. In innervated glands, the responses to isoprenaline lasted longer than those to noradrenaline. Chronic sympathetic denervation enhanced the responses to both noradrenaline and isoprenaline. The magnitude of the supersensitivity to isoprenaline was related to the dose and the time at which the responses were analyzed. Supersensitivity was greater for the initial than for the total secretion elicited by isoprenaline. Propranolol (1 mg/kg) and phenolamine (5 mg/kg) were used in order to determine the relative participation of α and β-adrenoceptors in the enhanced responses to isoprenaline. The results suggest that postjunctional supersensitivity for the secretory responses of this organ to isoprenaline is mainly mediated through β-adrenoceptors of the secretory cells and α-adrenoceptors of the myoepithelial cells.

Postsynaptic bimodal effect of sulpiride on locomotor activity induced by pergolide in catecholamine-depleted mice.

SummaryIn reserpinized (5 mg/kg, s.c.) mice treated with alpha-methyl-p-tyrosine (200+100 mg/kg, i.p.), increasing doses of the D-2 antagonist sulpiride had varying effects on locomotor activity induced by the mixed D-1/D-2 agonist pergolide (2 mg/kg, s.c.). Low doses of sulpiride (1 mg/kg, i.p.) significantly enhanced this activity whereas at higher doses (50 mg/kg) an inhibitory effect was observed. Amphetamine (3 mg/kg, i.p.) failed to reverse akinesia in this animal model, precluding the possibility of a presynaptically mediated phenomenon; in contrast, mice receiving reserpine alone showed a high degree of locomotor activity when challenged with amphetamine. The bimodal effect of sulpiride is thought to be mediated either by two different D-2 receptors located on the same cell or by the same receptor with different topographical localization on postsynaptic neurons mediating opposite functions.

Amphetamine-clonidine interaction on neurotransmission in the vas deferens of the rat.

SummaryThe interaction between amphetamine and clonidine on neurotransmission in the rat vas deferens was studied.1.In the whole vas deferens, clonidine 0.037 μmol/l displaced to the right the frequency-response curve evoked by either hypogastric or field stimulation. The frequency of stimulation that produced 50% of the maximal response (EF 50) was: control 4.0 Hz, clonidine 18.3 Hz (P<0.001 n=4), for hypogastric nerve stimulation; and 2.1 Hz in controls and 17.1 Hz in clonidine-treated preparations, for field stimulation (P<0.001 n=5). Preincubation with 5.4 μmol/l amphetamine antagonized the effect of clonidine (EF 50 amphetamine alone 6.2 Hz, amphetamine + clonidine 7.3 Hz; P>0.5).2.After 12 min of incubation with clonidine 0.037 μmol/l the responses to 6.4 Hz (3 s, 0.5 ms) were decreased by 77±2.2%. Both yohimbine and amphetamine, in a concentration-dependent manner, attenuated the inhibition. Washout of clonidine produced a slow recovery of the responses.3.Inhibition of the motor response to nerve stimulation (6.4 Hz, 3 s) by 30 μmol/l 2′,3′-cAMP was increased by 10 μmol/l dipirydamole and impaired by 100 μmol/l teophylline. Amphetamine, in a concentration that markedly reduced clonidine inhibition of neurotransmission failed to antagonize 2′,3′-cAMP.4.In the bisected vas deferens clonidine inhibited the peak motor response to short trains of field stimuli in the prostatic portion (“non-adrenergic”) and the sustained response in the epididymal portion (“adrenergic”). Yohimbine potentiated both types of responses and fully prevented the effect of clonidine. In the prostatic portion amphetamine slightly inhibited the peak motor response and attenuated the inhibitory effect of clonidine in both portions of the vas. Cocaine 10 μmol/l failed to prevent the effect of clonidine in the prostatic portion.5.Clonidine inhibited by 90±2.7% the responses to a single stimulus of the prostatic portion. Amphetamine attenuated markedly this inhibition.Our results suggest a specific interaction between amphetamine and clonidine at the α2 receptors, in both the prostatic and epididymal protions of the vas deferens of the rat.

Threshold of dopamine content and D1 receptor stimulation necessary for the expression of rotational behavior induced by D2 receptor stimulation under normo and supersensitive conditions

Abstract We measured the minimum amount of endogenous dopamine (EDA), necessary for the expression of rotational behavior induced by D2 receptor stimulation in striatal or medial forebrain bundle (MFB) lesioned rats. We correlated these results with the minimum dose of D1 receptor agonists needed to substitute EDA in its permissive role for D2 motor effects to take place. Rats with unilateral quinolinic acid (QA) striatal or 6-hydroxydopamine (6-OHDA) MFB lesions were given increasing doses of the tyrosine hydroxylase inhibitor alpha-methyl-para-tyrosine (AMPT) in combination with a fixed dose of the D2 receptor agonist quinpirole (trans-(–)-4aR-4,4a,5,6,7,8,8a,9-Octahydro-5-propyl-1H-pyrazolo(3,4-g)quinoline hydrochloride) and tested for rotational behavior. The animals were later sacrificed and striata removed; EDA was measured by high performance liquid chromatography (HPLC). Rotational responses were abolished by increasing doses of AMPT inducing a stepwise depletion of EDA. EDA content and rotational behavior to D2 stimulation showed a high degree of correlation. There was an abrupt reduction in rotational behavior at dopamine levels of 50–60% of controls in both animal models. In addition, striatal or MFB lesioned rats which were maximally depleted of dopamine by AMPT pretreatment received a fixed dose of quinpirole and then challenged with increasing doses of a D1 receptor agonist SKF 38393 ((±)-1-Phenyl-2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,8-diol hydrochloride). Rotational behavior was restored by SKF 38393 in both animal models in a dose-dependent fashion. Our results confirm the need for simultaneous D1/D2 stimulation in the generation of rotational behavior in both animal models. Moreover, they demonstrate the existence of a threshold level of D1 stimulation necessary to exert its permissive role on D2 mediated responses.

MAO-A and MAO-B in the superior cervical ganglion and in the nictitating membrane of the cat

Abstract 1. 1. The two forms of MAO, A and B, are present in the nictitating membrane and superior cervical ganglion of the cat. 2. 2. The K M for the substrate tyramine differed between both tissues, the affinity of the ganglionar enzyme was almost three times lower than that of the nictitating membrane. 3. 3. The potency of clorgyline to inhibit the form A of MAO, using tyramine as substrate, was lower in ganglia than in the nictitating membrane. 4. 4. Sympathetic denervation decreased type A to a greater extent than type B in the nictitating membrane. 5. 5. In superior cervical ganglion, clorgyline was a more potent inhibitor of noradrenaline metabolism than in the nictitating membrane.

Potassium-evoked efflux of [3H]purines from the rat submaxillary gland

1. Normal, atrophied and denervated submaxillary glands were incubated with [3H]adenine for 1 h. The accumulation of [3H]adenine, expressed as microCi/g tissue, did not differ significantly when the sympathetically denervated glands were compared with the control group. The radioactivity retained in both control and denervated tissues was also similar. 2. In atrophied glands 3H-accumulation as well as 3H-retention were 2-fold higher than these obtained in controls per unit weight, but 30% lower when expressed per gland. 3. The spontaneous efflux of radioactivity, expressed as fractional release, from normal, atrophied and denervated glands prelabelled with [3H]adenine was similar. 4. The outflow of radioactivity was enhanced by exposure of the tissues to 60 mM K+ during 2.5 min. 5. In all three groups, the purine release induced by K+ was the same. 6. Phentolamine 3.1 microM enhanced the K+-induced release of [3H]purine compounds in control and atrophied glands but not in denervated glands. 7. Propranolol 0.3 microM produced no changes among the three experimental groups. 8. Atropine 1 microM and phentolamine 3.1 microM plus atropine 1 microM did not modify the release of tritiated purine compounds in control and denervated glands. 9. Our results cannot discriminate between neuronal or non-neuronal elements as the source of purines released by depolarization but suggest that classical pharmacological tools such as phentolamine and atropine may affect purine metabolism in a complex fashion.