Rat salivary glands as a model for the study of adrenergic receptor modulation.

The properties (kD and Bmax) of alpha-1 and alpha-2 adrenoceptors in rat submaxillary gland have been determined by the binding process of radioactive ligands. Data of dissociation constants compiled from several tissues by this technique closely agree with physiological measurements. The radioactive ligand binding method has afforded evidence for changes in receptor number elicited by procedures that induce either hyper or hyposensitivity of the response. Chemical sympathectomy with reserpine increases 10 fold the number of alpha-2 receptors in rat submaxillary gland, as determined by the binding of [3H] clonidine. Sympathetic surgical denervation also increases the number of alpha-2 adrenoceptors by about 50%. Alpha-1 adrenoceptors were studied using [3H] prazosin or [3H] WB 4101. In gangliectomized submaxillary glands, we did not find an increase in the number of binding sites. Alpha-1 and alpha-2 adrenoceptors are also modulated by the parasympathetic system. Sectioning of the chordalingual nerve 21 days before the experiment produces an increase of about 50% in both types of alpha adrenoceptors in the submaxillary gland. These results indicate a major modulatory role of the cholinergic system on postsynaptic adrenoceptors. This type of regulation has also been found in other tissues such as brown adipose tissue and in complex systems like the brain. The results show that the major salivary glands of the rat are a useful model for the study of adrenoceptor regulation and that the data obtained can be extrapolated to more complex systems.

Rat salivary glands as a model for the study of adrenergic receptor modulation

The properties (kD and Bmax) of alpha-1 and alpha-2 adrenoceptors in rat submaxillary gland have been determined by the binding process of radioactive ligands. Data of dissociation constants compiled from several tissues by this technique closely agree with physiological measurements. The radioactive ligand binding method has afforded evidence for changes in receptor number elicited by procedures that induce either hyper or hyposensitivity of the response. Chemical sympathectomy with reserpine increases 10 fold the number of alpha-2 receptors in rat submaxillary gland, as determined by the binding of [3H] clonidine. Sympathetic surgical denervation also increases the number of alpha-2 adrenoceptors by about 50

. Alpha-1 adrenoceptors were studied using [3H] prazosin or [3H] WB 4101. In gangliectomized submaxillary glands, we did not find an increase in the number of binding sites. Alpha-1 and alpha-2 adrenoceptors are also modulated by the parasympathetic system. Sectioning of the chordalingual nerve 21 days before the experiment produces an increase of about 50

in both types of alpha adrenoceptors in the submaxillary gland. These results indicate a major modulatory role of the cholinergic system on postsynaptic adrenoceptors. This type of regulation has also been found in other tissues such as brown adipose tissue and in complex systems like the brain. The results show that the major salivary glands of the rat are a useful model for the study of adrenoceptor regulation and that the data obtained can be extrapolated to more complex systems.

Rat salivary glands as a model for the study of adrenergic receptor modulation.

The properties (kD and Bmax) of alpha-1 and alpha-2 adrenoceptors in rat submaxillary gland have been determined by the binding process of radioactive ligands. Data of dissociation constants compiled from several tissues by this technique closely agree with physiological measurements. The radioactive ligand binding method has afforded evidence for changes in receptor number elicited by procedures that induce either hyper or hyposensitivity of the response. Chemical sympathectomy with reserpine increases 10 fold the number of alpha-2 receptors in rat submaxillary gland, as determined by the binding of [3H] clonidine. Sympathetic surgical denervation also increases the number of alpha-2 adrenoceptors by about 50

. Alpha-1 adrenoceptors were studied using [3H] prazosin or [3H] WB 4101. In gangliectomized submaxillary glands, we did not find an increase in the number of binding sites. Alpha-1 and alpha-2 adrenoceptors are also modulated by the parasympathetic system. Sectioning of the chordalingual nerve 21 days before the experiment produces an increase of about 50

in both types of alpha adrenoceptors in the submaxillary gland. These results indicate a major modulatory role of the cholinergic system on postsynaptic adrenoceptors. This type of regulation has also been found in other tissues such as brown adipose tissue and in complex systems like the brain. The results show that the major salivary glands of the rat are a useful model for the study of adrenoceptor regulation and that the data obtained can be extrapolated to more complex systems.

Amphetamine antagonizes the presynaptic inhibitory effect of clonidine through an interaction at the level of the alpha 2-adrenoceptors.

1. In the whole rat vas deferens 20 microM noradrenaline (NA) and 0.011 microM clonidine decreased (36 +/- 7.4% and 80 +/- 6.0% respectively) the motor response induced by hypogastric nerve stimulation. These effects were reverted by 1 microM yohimbine. Amphetamine 5.4 microM failed to antagonize the inhibitory effect of NA and attenuated clonidine effect. 2. The effect of amphetamine was not altered by preincubation with either cocaine 1 microM, (-)-propranolol 0.3 microM or cocaine plus prazosin 0.028 microM. 3. In reserpine pretreated animals amphetamine 5.4 microM shifted to the right the concentration-response curve (CRC) to clonidine 0.62 +/- 0.05 log units with a KB value of 1.83 +/- 0.30 microM. 4. Binding of [3H]clonidine and [3H]prazosin were inhibited by amphetamine. Amphetamine was 90 times more potent to inhibit [3H]clonidine binding. 5. The results obtained suggest a possible direct interaction between clonidine and amphetamine on alpha-adrenoceptor.

Sympathetic and parasympathetic nerves regulate postsynaptic alpha-2 adrenoceptor in salivary glands.

The effects of sympathetic denervation or parasympathetic decentralization on the inhibitory effects of postsynaptic alpha-2 adrenoceptors were studied in the submaxillary and the sublingual gland of the rat. Chronic sympathetic denervation enhanced by a factor of 10 the potency of clonidine to inhibit the secretory responses of the submaxillary gland to either norepinephrine or methacholine. In denervated glands, clonidine (1 microgram/kg), reduced markedly the response to norepinephrine, but potentiated this response in control glands. Blockade of postsynaptic alpha-2 adrenoceptors with idazoxan (3 micrograms/kg) enhanced the secretory responses of denervated glands to norepinephrine. Parasympathetic decentralization also potentiated the inhibitory effects of the alpha-2 agonists. In the submaxillary gland the potency of guanabenz to decrease the secretory response to methacholine was increased by a factor of 30. Supersensitivity to the inhibitory effects of clonidine was also observed in parasympathetically decentralized sublingual glands. Parasympathetic decentralization increased the maximum binding site of [3H]clonidine binding by about 50% in both the submaxillary and sublingual glands. No changes in KD were detected. This surgical procedure also increased the maximum binding site of [3H]prazosin binding in submaxillary glands. The present findings show clearly that interruption of either branch of the autonomic nervous system induces supersensitivity of the inhibitory response mediated through postsynaptic alpha-2 adrenoceptors. The enhanced inhibitory activity could mask alpha-1 adrenoceptor supersensitivity after postganglionic sympathetic denervation.

Alpha-1 adrenoceptors mediate secretory responses to norepinephrine in innervated and denervated rat submaxillary glands.

The alpha adrenoceptor that mediates the secretory responses to norepinephrine in submaxillary glands was characterized. All three alpha antagonists employed produced in innervated glands parallel displacements of the dose-response curves to norepinephrine, prazosin being 30 and 100 times more potent than phentolamine and yohimbine, respectively. Chronic sympathetic denervation produced supersensitivity to norepinephrine. Under these conditions prazosin also was more potent than yohimbine but the shifts of the dose-response curves to norepinephrine were nonparallel. Potentiation of norepinephrine by cocaine did not modify the effects of prazosin in innervated glands. The nonparallel displacements observed in denervated glands were related to the development of postjunctional supersensitivity of the beta adrenoceptor-mediated responses because after administration of propranolol the displacements produced by prazosin were parallel. Responses to methoxamine were not potentiated by denervation and were competitively antagonized by prazosin in both innervated and denervated glands. Specific [3H]clonidine binding to membranes of submaxillary glands showed at least two independent sites with a KD1 of 0.37 nM and a KD2 of 65 nM. The number of sites of each component was doubled after chronic sympathetic denervation. Binding of [3H]prazosin (KD, 0.43 nM; maximum binding sites, 78.5 fmol/mg of protein) was not affected by denervation. The present findings show that sialagogue responses to sympathomimetic agents are mediated by alpha-1 adrenoceptor activation in both innervated and denervated submaxillary glands. Chronic sympathetic denervation does not produce postjunctional supersensitivity of the alpha-1-mediated responses and the supersensitivity of the beta-mediated responses interferes with determinations of dose-response curves to the sympathetic neurotransmitter.

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.

Release of radioactive purines from cat nictitating membrane labeled with 3H-adenine.

Cat nictitating membranes were incubated with 1-2 x 10(-7) M 3H-adenine or 3H-adenosine for 1 h. A tissuebath ratio of about 15 was found for both compounds in intact and denervated membranes. In intact nictitating membranes sympathetic nerve stimulation (4 Hz, 5 min) caused a net release of purines (0.66 +/- 17% of the tissue content), which was reduced by alpha-blockade. Noradrenaline (1-3 microM) or tyramine 60 microM), which produced the same contractile response as did nerve stimulation, increased purine release to the same extent as did nerve stimulation. The effect of either agent was reduced or abolished by phentolamine. Purine release could also be induced by acetylcholine and ATP. This release was not altered after surgical denervation. There was an excellent correlation between the contractile response and the purine release induced by nerve stimulation, noradrenaline, tyramine and acetylcholine. However, ATP caused a larger release of 3H-purines than expected from the contractile responses, possibly indicating displacement. The results indicate that most if not all of the 3H-purines released by nerve stimulation in the cat nictitating membrane are derived from postjunctional elements.

Selective inhibition by hydrocortisone of 3H-normetanephrine formation during 3H-transmitter release elicited by nerve stimulation in the isolated nerve-muscle preparation of the cat nictitating membrane.

The metabolism of 3H-noradrenaline released by nerve stimulation in the isolated nerve-muscle preparation of the cat nictitating membrane was determined under control conditions and in the presence of hydrocortisone, 28 muM, a concentration which inhibits the high affinity extraneuronal uptake of noradrenaline in this tissue. In the controls the main fraction in the overflow elicited by stimulation at 10 Hz during 2 min was the deaminated glycol, 3H-DOPEG (3,4-dihydroxyphenylglycol), which accounted for 45.2 +/- 2.96% of the total radioactivity. Under these conditions, 3H-noradrenaline represented 30.8 +/- 1.92%, while 3H-normetanephrine accounted for 14.5 +/- 0.94% of the total overflow of radioactivity. During exposure to hydrocortisone there was a selective inhibition in 3H-normetanephrine formation from 3H-noradrenaline released by stimulation while the other fractions were not affected significantly. In contrast to these results, there were no changes in the spontaneous outflow of 3H-normetanephrine during exposure to hydrocortisone. The results obtained support the view that 3H-normetanephrine in spontaneous release originates from the activity of prejunctional catechol-O-methyltransferase. On the other hand, 3H-normetanephrine formed during transmitter release elicited by nerve stimulation is due to the activity of extraneuronal catechol-O-methyltransferase. Access of 3H-noradrenaline released by nerve stimulation to extraneuronal catechol-O-methyltransferase is mediated through the high-affinity, hydrocortisone-sensitive extraneuronal uptake mechanism.